Sig-1R knockout cells: selection on HeLa cells

HeLa human cervical cancer cells and mouse neuroblastoma × rat glioma cells were purchased from American Type Cell Collection (ATCC). HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (GIBCO) supplemented with penicillin (100 units/mL), streptomycin (100 μg/mL), and 10% Fetalgro bovine growth serum (RMBIO). Human Sig-1R CRISPR/Cas9 knockout (KO) and Sig-1R HDR plasmids (Santa Cruz) were transiently co-transfected into HeLa cells with the Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) and were cultured in Dulbecco’s modified Eagle’s medium (GIBCO), penicillin (100 units/mL), streptomycin (100 μg/mL), and 10% Fetalgro bovine growth serum (RMBIO). For the selection of stably transfected Sig-1R CRISPR/Cas9-KO cells, cells were maintained in culture media supplemented with puromycin (100 μg/mL, GIBCO) for stable cell lines selection to generate permanent HeLa-Sig-1R-KO cells. NG-108 cells were cultured in Eagle’s minimum essential medium (GIBCO) supplemented with penicillin (100 units/mL), streptomycin (100 μg/mL), and 10% Fetalgro bovine growth serum (RMBIO). All cell lines were kept at 37 °C in a humidified 5% CO₂ incubator (Thermo Fisher Scientific).

NSC-34 cell culture

The NSC-34 cell line was a kind gift from Yijuang Chern’s Laboratory of Taiwan. NSC-34 cells were grown in complete culture Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. To ensure high quality, after passage 30, the cells were no longer used. For differentiation, NSC-34 cells were induced to differentiate into a motoneuron-like phenotype by differentiation medium (1:1 DMEM/Ham’s F12 plus 1% of FBS, 1% of non-essential amino acids, and 1% of penicillin/streptomycin) for 4 days. All materials used in NSC-34 cell culture were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cell monolayers at 50% confluency were used for transfection with plasmids using PolyJet reagent according to the manufacturer’s protocol (SignaGen Laboratories, Gaithersburg, MD, USA). In all, 3 µl of PolyJet was incubated with 1 µg of different plasmids in 0.2 ml of serum-free medium for 30 min at room temperature. Subsequently, the cells in a 3.5-cm dish were transfected by the DNA- PolyJet complexes in 2 ml of differentiation medium, and then incubated at 37 °C in 5% CO2 for 6 h. The cells were then incubated for an additional 36 h using 2 ml of fresh medium. The transfection efficiency was mostly >50%.

Immunostaining

HeLa cells: Cells were seeded on glass slide with coverslip overnight at 37 °C in an incubator followed by fixation with 4% paraformaldehyde in PBS at 4 °C for 20 min. After washing with PBS three times, cells were incubated with permeabilization buffer (0.4% Triton X-100 in PBS) for 15 min. After washing three times with PBS, slides were incubated with SuperBlock blocking buffer (Thermo Fisher Scientific) at room temperature for 30 min and incubated thereafter with indicated primary antibodies in SuperBlock blocking buffer at proper dilution overnight at 4 °C. Cells were then washed three times with wash buffer (0.1% Triton X-100 in PBS) and incubated in SuperBlock blocking buffer with Alexa488- or Alexa594-conjugated secondary antibodies for 1 h. Cells were then washed three times with PBS and mounted with Prolong gold antifade mountant with DAPI. Images of cells were captured by confocal microscopy (Perkin-Elmer Modular laser system 2.0 with Nikon Eclipse TE2000E microscope and Volocity version 6.3 software).

NSC-34 cells: Cells were fixed with 4% (vol/vol) paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS and permeabilized with 0.05% Triton-X 100 in PBS for 3 min. Immunostaining was conducted with primary antibodies such as anti-HA (Proteintech,Chicago, IL, USA), anti-GFP/YFP (Takara Bio/Clontech, Mountain view, CA, USA), anti-Nucleoporin p62/Nup62 (Abcam, Cambridge, UK), or anti-RanGAP1 antibodies (Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 h. Cells were then treated with Alexa Fluor 488- or Alexa Fluor 568-conjugated second antibodies (Thermo Fisher Scientific). Finally, cells were mounted in 90% (vol/vol) glycerol containing 4′-6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific), and examined by using the DeltaVision Spectris Imaging System (Applied Precision) that includes an Olympus X71 microscope and the softWoRx software (version 6). The software deconvolves images to improve contrast, by relocating signal scatter and out-of-focus data, to generate images in the 3D rendering.

Plasmid constructs and gene silencing

The primer pairs for specific gene amplification in the polymerase chain reaction (PCR) are listed in Supplemental Information Table S1. Those sequences were designed based on the nucleotide database of the National Center for Biotechnology Information (NCBI) and were purchased from the Integrated DNA Technologies (IDT). The coding sequence (CDS) of human Sig-1R (NCBI accession: NM_005866) was amplified by using PCR from the complementary DNA (cDNA) of HeLa cells. The CDS of mouse Sig-1R (NCBI accession: NM_011014) was amplified from cDNA of neuro-2a cells. To generate the expressing construct of Sig-1R in mammalian cells and the recombinant protein of Sig-1R from E. coli, the PCR products of the human Sig-1R containing two restriction enzyme sites EcoRI and XhoI (New England Biolabs) were purified by using the Wizard SV Gel and PCR Clean-Up System (Promega). Purified PCR products were then used to perform the pGEM-T Easy vector ligations (Promega), by using the NEB-5 alpha Competent E. coli. (New England Biolabs), and sub-cloned from the pGEM-T Easy vector either into pcDNA3-HA to produce the HA-human Sig-1R or into pGEX-6p3 (GE Healthcare) to produce the pGEX-6p3-human Sig-1R constructs. The purified PCR products of the mouse Sig-1R containing two restriction enzyme sites EcoRI and XhoI were used to perform the T&A cloning (Yeastern Biotech Co. Ltd.) and then sub-cloned from T&A plasmid into pGEX-6p3 to produce the pGEX-6p3-mouse Sig-1R construct.

Site-directed mutagenesis was performed by using the QuickChange site-directed mutagenesis kit (Agilent) to generate human and mouse Sig-1R-E102Q/pGEX-6p3 mutants, respectively. Gene knockdown techniques was performed to downregulate expression levels of the Sig-1R in the cell. The short hairpin RNA (shRNA) against the CDS of Sig-1R, which was cloned into Green Flourscence Protein (GFP)-expressing vector (PLKO.1-hGPK-Puro-CMV-tGFP), and the non-targeting shRNA negative control (MISSION PLKO.1-hGPK-Puro Non-Mammalian shRNA control plasmid DNA) were purchased from Sigma-Aldrich. For the sequence of the Sig-1R shRNA (i.e., shSig-1R), two complementary oligonucleotides were chosen: 5′-GATCCACACGTGGATGGTGGAGTATTCAAGAGATACTCCACCATCCACGTGTTTTTTTGCTAGCG-3′ and 5′-AATTCGCTAGCAAAAAAACACGTGGATGGTGGAGTATCTCTTGAATACTCCACCATCCACGTGTG-3′, where bold letters stand for rat Sig-1R gene 516-534, italics stand for either BamHI or EcoRI overhangs, and the bold italics stand for hairpin loop sequences (Hayashi and Su^73,74; Tsai and Chuang et al.³⁹). Transfections of the shSig-1R were performed following manufacturer’s recommendations by using Lipofetamine 2000 (Thermo Fisher Scientific). In some case, the transfection was performed twice (e.g., Fig. 6c). The first transfection was in Opti-MEM (reduced MEM). Six hours later, the culture medium was changed to complete medium. Twenty-four hours later, the transfection was performed one more time.

Immunoprecipitation

HeLa cells were harvested in 0.3 mL of IP lysis buffer (50 mM NaCl, 0.5% Nonidet P-40, 10 mM Tris-HCl pH 8.0, and 1× protease inhibitor) for 30 min. Protein amounts were measured (Pierce bicinchoninic acid protein assay kit, Thermo Fisher Scientific, Rockford, IL) after centrifugation (18,407xg for 10 min at 4 °C). Protein lysates (500 µg) were incubated with the target antibody (2 µg) and IP lysis buffer in a total volume of 1000 µl and rotated overnight at 4 °C. The Protein-A/G magnetic beads were pre-washed three times with IP lysis buffer and then added into the lysate/antibody mix and rotated for 1 h at 4 °C. After incubation, beads were washed three times with IP lysis buffer containing protease inhibitors (Roche Diagnostics, Indianapolis, IN) for 5 min at 4 °C. After third washing, beads were eluted with 50 µl SDS 2X sample buffer containing dithiothreitol and heated at 95 °C for 10 min. After elution, proteins samples in the mixture were immediately fractionated by using SDS/PAGE as described below in the western blot section to examine the potential protein interaction.

In the Sig-1R-Nup50 co-IP experiments (Fig. 1d), HeLa cell protein lysates (200 µg) were incubated with Nup50 or control IgG antibody (3 μg) and IP lysis buffer in a total amount of 1000 µl and rotated for 2 h at 4 °C. The pre-cleared protein-A/G Agarose (50 µl; Santa Cruz Biotechnology) was added into the protein lysate/antibody mixture and rotated overnight at 4 °C. Beads were washed three times with IP lysis buffer containing protease inhibitors (Roche Diagnostics, Indianapolis, IN) for 5 min at 4 °C. Each wash was accompanied with a 9391×g centrifugation for 1 min at 4 °C to remove the supernatant. After the third wash, the pellet is eluted with 50 µl SDS 2X sample buffer and heated at 95 °C for 10 min. After elution, proteins samples in the mixture were immediately fractionated by using SDS/PAGE and immunoblotted with Nup50 or Sig-1R antibody overnight at 4 °C to examine for their potential interactions. Membranes were washed three times for 15 min followed by probing with secondary antibody of “peroxidase-conjugated Affinipure goat anti-mouse IgG” for Sig-1R or “peroxidase-conjugated IgG fraction monoclonal mouse anti-rabbit IgG, light-chain specific” for Nup50. Blots were washed three times for 15 min with TBST and developed by using the Azure Biosystem C600.

Western blot: HeLa cell, NG-108 cells

Total proteins were extracted from HeLa (ATCC) and NG-108 cells (ATCC). Briefly, collected cells were lysed with RIPA lysis buffer (10 mM Tris-HCl pH 8.0, 140 mM sodium chloride, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 1 mM ethylenediaminetetraacetic acid, and 0.5 mM ethylene glycol tetraacetic acid) containing protease inhibitors (Roche Diagnostics, Indianapolis, IN) and the protein amount was measured (Pierce bicinchoninic acid protein assay kit, Thermo Fisher Scientific, Rockford, IL). Equal amount (30 µg) of protein samples were fractionated by using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride membrane. After incubation with 5% (wt/vol) nonfat milk in TBST (10 mM Tris. pH 8.0, 150 mM NaCl, and 0.5% (vol/vol) Tween 20) for 1 h, membranes were incubated with various primary antibodies (see Supplementary Information Table 1) overnight at 4 °C. Alpha-tubulin or actin was used as loading control. Membranes were washed three times with TBST for 15 min followed by probing with secondary antibody of goat anti-rabbit or goat anti-mouse antibody. Blots were washed three times for 15 min with TBST and developed by using the LiCor system (LiCor CLx). Expression bands were analyzed by Image Studio Lite (LiCor 5.2.5) according to the manufacturer’s manual.

Nuclear/cytoplasmic fractionation of HeLa cells

HeLa (or HeLa-Sig-1R-KO) cells were grown to 80% confluency and transiently transfected with indicated vectors including pcDNA5(C₄G₂)₃₁ and pEGFP-N3.31-(C₄G₂)₃₁ (gifts from Mauro Cozzolino). Twenty-four hours after transfection, cells were harvested for subcellular fractionation. The subcellular fractionation was performed by using the Subcellular Protein Fraction Kit for Cultured Cells (Thermo Fisher Scientific). The procedure is briefly described per manufacturer’s instructions as follows. HeLa cells were rinsed once with phosphate-buffered saline (PBS) and gently lysed with cytoplasmic extraction buffer that was supplemented with protease inhibitor cocktail at 4 °C for 10 min. The cytoplasmic fraction (supernatant) were collected by centrifugation at 500×g for 5 min at 4 °C. The pellets were then incubated with membrane extraction buffer at 4 °C for 10 min and the membrane fraction (supernatant) was prepared by centrifugation at 3000×g for 5 min at 4 °C. For the isolation of the nuclear extract, the resultant pellets were subsequently incubated with nuclear extraction buffer at 4 °C for 10 min and the soluble nuclear extract (supernatant) were collected by centrifugation at 5000×g for 5 min at 4 °C.

Biotin pull-down assay

The commercially synthesized biotinylated RNAs were purchased from Integrated DNA Technologies (Table S2; Supplementary Information). The purified recombinant proteins, HeLa cell lysates, or extracts from rat liver microsomes (BIOIVT) were incubated with 20 nM biotin-labeled RNAs in binding buffer (10 mM HEPES pH 8.0, 40 mM KCl, 3 mM MgCl₂, 5% glycerol, 2 mM DTT, 0.5% Nonidet P-40, and 1% tween-20) supplemented with protease inhibitor cocktail (Roche) and were rotated overnight at 4 °C. The reaction mixture was incubated with NeutrAvidin agarose resin (Thermo Fisher Scientific) at 4 °C for 24 h. The agarose resin was washed three times with binding buffer. The RNA-protein complex was analyzed by using western blot with indicated antibodies. The blots in Fig. 4b–d were detected by the Licor system as follows. Blots were washed three times for 15 min with TBST and developed by using the LiCor system (LiCor CLx). Expression bands were analyzed by Image Studio Lite (LiCor 5.2.5) according to the manufacturer’s manual. Note: the blot in Fig. 4a was detected by the horse radish peroxidase method as follows. Membranes were washed three times for 15 min followed by probing with secondary antibody of “peroxidase-conjugated Affinipure goat anti-mouse IgG” for Sig-1R or “peroxidase-conjugated IgG fraction monoclonal mouse anti-rabbit IgG, light-chain specific” for Nup50. Blots were washed three times for 15 min with TBST and developed by using the Azure Biosystem C600. In general, the peroxidase method offers better sensitivity over the Licor method. But the Licor method is fast with less experimental steps to perform. Depending on the need of sensitivity, some experimenters prefer Licor over the peroxidase method. But some prefer the peroxidase method as a familiar routine.

RNA fluorescence in situ hybridization

The RNA FISH protocol for the detection of (G4C2)₃₁-RNA has been reported before (Rossi et al.⁵). We performed the experiment as follows. Cells were seeded on poly-l-lysine coated coverslip and fixed with 4% paraformaldehyde in PBS before incubation at 4 °C with 70% ethanol. Cells were then rehydrated with 5 mM MgCl₂ in PBS and pre-hydrated with 2X SSC buffer and 10 mM sodium phosphate PH 7.0 in 35% formamide. Commercially synthesized 250 ng/ml of Cy3-labeled (C₄G₂)₄ nucleotides (IDT company) were incubated with cells in 2X SSC buffer, 10 mM sodium phosphate PH 7.0, 10% dextran sulfate, 0.5 mg/ml tRNA, and 0.2% bovine serum albumin (BSA) in 35% formamide. After washing, cells were visualized by confocal microscopy (Perkin-Elmer Modular laser system 2.0 with Nikon Eclipse TE2000E microscope and Volocity version 6.3 software).

Protein degradation assay

Cells were cultured to 80% confluency followed by addition of cycloheximide (100–150 µg/mL, Sigma-Aldrich) to inhibit de novo protein synthesis. Cells were harvested at different time point and were lysed using the radioimmunoprecipitation assay (RIPA) lysis buffer: 10 mM Tris-HCl pH 8.0, 140 mM sodium chloride (NaCl), 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100 (Sigma-Aldrich), 1 mM ethylenediaminetetraacetic acid (EDTA), and 0.5 mM ethylene glycol tetraacetic acid (EGTA) supplemented with EDTA-free protease inhibitor cocktail (Roche). The resulting proteins were analyzed by western-blot analysis by incubating overnight at 4 °C with primary antibodies of target genes in TBST. After incubation with secondary antibodies (LiCor), blots were imaged by Odyssey infrared image system (LiCor Image Studio Lite 5.2.5). The protein turnover rate was normalized by the house-keeping gene, such as α-tubulin or actin.

RNA isolation, reverse transcription, and quantitative real-time PCR

The primer pairs used to perform real-time quantitative PCR are listed in Table S2 in the Supplementary Information. Total RNAs were isolated from HeLa cells by using TRIzol reagent (Invitrogen) and reverse transcribed by using Superscript III Reverse Transcriptase (Invitrogen) according to manufacturer’s instructions. Quantitative real-time PCR were performed by using SYBR Green PCR Master mix (Roche) and analyzed with ABI PRISM 7900HT sequence detection system (Applied Biosystems).

Drosophila Stocks

Flies were raised on standard cornmeal agar diet. The flies carrying 3 (line 370) or 30 (line 373) repeats of G4C2 hexanucleotide under the regulation of the UAS promoter were provided by Dr Peng Jin (Xu et al.⁵²). Drosophila that express wild-type human Sig-1R (line Sig-1R#2) were generated by insertion of Sig-1R coding sequence between the EcoR1 and Xho1 sites of the pUAST plasmid (Couly et al.⁴³). The cDNA encoding human wild-type Sig-1R was initially inserted in pCI-neo vector. After digestion by EcoR1 and XhoI restriction enzymes, the purified Sig-1R fragment was inserted between the EcoR1 and XhoI sites of the pUAST plasmid. The G304C mutation was generated by using Quickchange mutagenesis accordingly to the manufacturer’s instructions (Agilent Technologies, Santa Clara, California). Germ-line -mediated P-element transformation was performed by BestGene Inc. (Chino Hills, California) in a w1118 background. The GMR-GAL4 and Elav-GAL4 (line C155) strains were obtained from the Bloomington Drosophila Stock Center (BDSC, Bloomington, Indiana) and were used to target expression specifically in the whole eye or in all neurons, respectively. Female F1 progeny that carried both UAS and GAL4 were used for subsequent analyses. In alignment with the genetic background we used the w1118 (BL5905) line from BDSC as the control. Note: all Drosophila experiments in this study were done on female flies.

Real-time quantitative PCR: Drosophila

Total mRNA was purified from 10 heads of flies (female, 4 days old, reared at 25 °C) in Trizol Reagent (Ambion) then submitted to trituration using plastic pestles. Chloroform (Carlo Erba) was added, and after centrifugation the upper aqueous phase was collected. RNA was then precipitated using isopropanol (VWR) and was washed in 70% ethanol and dissolved in RNase-free water. RNA concentration and purity were measured using a spectrophotometer (NanoDrop One^c, Thermo Scientific). RNA samples were treated with DNase from the DNA-free kit (Invitrogen) accordingly to the manufacturer’s protocol. Reverse transcription was performed using M-MLV Reverse Transcriptase (Promega) following the manufacturer’s instructions. Reaction plates were prepared with diluted cDNAs and Sybr No-Rox Mix (Sensifast, Bioline) by an Echo 525 acoustic liquid handler (Labcyte) and RT-qPCR experiments were performed by using a LightCycler 480 (Roche). The following primers were used for: (G4C2)30 forward 5′-GGGATCTAGCCACCATGGAG-3′ and reverse 5′- TACCGTCGACTGCAGAGATTC-3′; actin (house-keeping control gene) forward 5′- GCGCGGTTACTCTTTCACCA-3′ and reverse 5′- ATGTCACGGACGATTTCACG-3′. The primers for (G4C2)30 were designed to amplify a 3′ region immediately after the G4C2 repeats as previously published (Zhang et al.¹²). RT-qPCRs were conducted for 45 cycles (10 s at 95 °C, 10 s at 60 °C, and 10 s at 72 °C). Fold changes of gene expression were analyzed using the 2-ΔΔCp method. Data collected from at least four independent experiments were averaged and presented as mean ± SEM. Statistical analysis was performed using Student’s t test.

Western blot: Drosophila

Heads of flies (n = 4; female; four days old, reared at 25 °C) were homogenized in 50 μl RIPA lysis buffer (50 mm Tris-HCl, pH 8, 150 mm NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 1% Igepal CA-630) supplemented with cOmpleteTM protease inhibitor cocktail (Merck, Darmstadt, Germany). Following a 1-min centrifugation, 1/4 (v/v) sample Laemmli buffer was added to supernatants. Total proteins were separated through a 10% polyacrylamide resolving gel, transferred to a nitrocellulose membrane (AmershamTM, Merck). Membrane was blocked for 1 h in the blocking solution (1X PBS, 0.1% Tween 20, and 5% dry milk) and incubated overnight with primary antibodies at 4 °C. Sig-1R protein was detected using a rabbit polyclonal antibody (1:100) generated by Abliance (Compiègne, France) and raised against residues 142-161 (KSEVFYPGETVVHGPGEATAV) of human Sig-1R. Rat anti-Elav antibody (1/700, 7E8A10, Developmental Studies Hybridoma Bank, Iowa City, Iowa) was used as a loading control. Secondary peroxidase-conjugated antibodies (1/5000, Jackson ImmunoResearch, Cambridge, UK) were incubated for 2 h in blocking solution. Chemiluminescence was revealed by using the ClarityTM Western ECL Blotting substrates (Bio-Rad) and the ChemiDoc2 Touch Imaging System (Bio-Rad).

External eye morphology: Drosophila

For examination of external eye phenotype, flies were reared at 29 °C. At least 48 flies from four independent groups were examined. Data are shown as the percent of flies with necrotic spots on their eyes at 15–20 days of age. Statistical analysis was performed by using the Student’s t test.

Negative geotaxis test: Drosophila

Startle-induced climbing response was assessed by using the negative geotaxis test. Flies were reared during 4 days posteclosion at 25 °C. Then they were anesthetized with CO2 and eight flies were placed in a plastic column (1.3 cm diameter × 30 cm). After 20 min recovery, columns were disposed vertically and flies were tapped to the bottom of the column. Flies that remained at the bottom or climbed above the 22 cm mark were counted after 1 min. The test was repeated three times for each batch of flies at 1 min intervals. The data are the mean of at least four trials and are presented as percentages of flies to the top or at the bottom. Statistical significance was assessed by ANOVA followed by Tukey’s multiple-comparison test.

Electrophysiological recordings after electroconvulsive stimulation: Drosophila

In previous studies, the bang-sensitive phenotype was associated to long firing discharge at the neuromuscular junction after a high-frequency electroconvulsive stimulation of the giant fiber pathway (Kuebler and Tanouye⁵⁶; Lee and Wu⁵⁷). Flies were reared 1–2 days posteclosion at 25 °C. Briefly, head and thorax of each fly were glued on a needle under CO2 anesthesia. Bipolar tungsten electrodes were introduced into the head to stimulate the giant fiber circuit. As a reference, an Ag/AgCl electrode was placed into the abdomen. To record evoked responses, a borosilicate glass micropipette filled with 3 M KCl was inserted into a muscular fiber of the dorsal longitudinal indirect flight muscles. Stimulation was induced by a Grass S88 Stimulator (GRASS Instruments). Recordings were made with an Intracellular Electrometer IE-210 amplifier (Warner Instruments) connected to a PowerLab 4/35. Recordings were digitized at a frequency of 20 KHz. High-frequency stimulation at 200 Hz was delivered to the brain neurons during 2 s at 30 V. Quantitative analysis was performed only on flies showing electroconvulsion by using LabChart 8 software. Data from 20 flies per condition were averaged and presented as mean ± SEM. Statistical analysis was performed using the Student’s t test.

Statistics and reproducibility

For all experiments subjected to statistical analyses, data were collected from at least three independent experiments and were compared for statistical significance by using Prism (version 8.2 at the NIDA USA lab, or version 5.01 at the INSERM France lab; GraphPad, San Diego, CA, USA). No samples were pre-allocated to specific groups to maintain randomization. Data were collected from experiments performed in replicates and were expressed as means ± standard error of means (SEM). Comparisons among multiple groups were performed for most of the experiments in this study by using a two-way ANOVA with appropriate post hoc tests. A p-value of p ≤ 0.05 was considered statistically significant. For comparisons between non-linear regression curves (i.e., Fig. 3d–f), the second order polynomial (quadratic) model was first used for the curve fittings. Next, the ‘extra sum-of-squares F-test’ was used to test if the best-fit curve of a group is the same as the global (shared) fitting curve. A p-value ≤ 0.05 was considered statistically significant. Comparisons between two experimental conditions were performed by using the unpaired Student t test (i.e., Fig. 8f, h, i). A p-value ≤ 0.05 was considered statistically significant. For comparisons among multiple groups with Drosophila (i.e., Fig. 8c–e, k), statistical significance was assessed by ANOVA followed by Tukey’s multiple-comparison test. A p-value ≤ 0.05 was considered statistically significant.

Note: because of the limit of words in a figure legend, the statistical details of Fig. 8c–k are given as follows for the sake of clarity. (Fig. 8c) The quantification of flies presenting necrotic spots in the eyes were from four independent studies for each group with the total number of 91 in control, 48 in (G4C2)₃₀, and 68 in (G4C2)₃₀ + Sig-1R group. Data are presented as means ± SEM; n = 4 independent studies for each group; one-way ANOVA followed by Tukey’s multiple comparisons test, 95% CI of difference for control vs. (G4C2)₃₀, control vs. (G4C2)₃₀ + Sig-1R and (G4C2)₃₀ vs. (G4C2)₃₀ + Sig-1R are −54.46 to −22.74, −18.55 to 13.18, and 20.05 to 51.78, respectively; ***p < 0.001. (Fig. 8d) Climbing performances of 4-day-old flies expressing no transgene (control), 3 ((G4C2)₃) or 30 G4C2 repeats ((G4C2)₃₀). Transgenes were expressed in neurons. In each experiment, the proportions of flies that climbed to the top of the column or that remained at the bottom were determined after 1 min. n = 8 flies per group; number of trials: control, 4; (G4C2)₃, 5; (G4C2)₃₀, 5. ***p < 0.001 versus control. Data are presented as means ± SEM; n = 4–5 trials for each group; one-way ANOVA followed by Tukey’s multiple comparisons test. In the flies climbing to the top groups, 95% CI of difference for control vs. (G4C2)₃, control vs. (G4C2)₃₀ and (G4C2)₃ vs. (G4C2)₃₀ are −0.9948 to 18.08, 83.17–102.2, and 75.18–93.16, respectively. In the flies remaining at the bottom groups, 95% CI of difference for control vs. (G4C2)₃, control vs. (G4C2)₃₀ and (G4C2)₃ vs. (G4C2)₃₀ are −11.41 to 9.328, −98.91 to −78.17, and −97.28 to −77.72, respectively; ***p < 0.001 vs. control. (Fig. 8e) Climbing performances of 4-day-old flies expressing (G4C2)₃₀ alone or with Sig-1R or the green fluorescent protein GFP (GFP65T) in neurons. n = 8 flies per group; number of trials: (G4C2)₃₀, 7; (G4C2)₃₀ + Sig-1R, 7; (G4C2)₃₀ + GFP65T, 5. Data are presented as means ± SEM; one-way ANOVA followed by Tukey’s multiple comparisons test. In the flies climbing to the top groups, 95% CI of difference for (G4C2)₃₀ vs. (G4C2)₃₀ + Sig-1R, (G4C2)₃₀ vs. (G4C2)₃₀ + GFP65T and (G4C2)₃₀ + Sig-1R vs. (G4C2)₃₀ + GFP65T are −30.64 to −8.642, −14.55 to 9.550, and 5.093 to 29.19, respectively. In the flies remaining at the bottom groups, 95% CI of difference for (G4C2)₃₀ vs. (G4C2)₃₀ + Sig-1R, (G4C2)₃₀ vs. (G4C2)₃₀ + GFP65T, and (G4C2)₃₀ + Sig-1R vs. (G4C2)₃₀ + GFP65T are 25.67 to 64.80, −3.458 to 39.41, and −48.70 to −5.828, respectively; ***p < 0.001 vs. (G4C2)₃₀. (Fig. 8f) Sig-1R does not modify G4C2₃₀ mRNA expression. Total RNAs were extracted from heads of flies expressing G4C2₃₀ alone or together with human Sig-1R. Transgenes were expressed in neurons. The quantitative real-time PCR was performed to measure mRNA levels of G4C2₃₀ by using specific primers for amplifications. The mRNA expression levels of G4C2₃₀ (n = 5) or (G4C2)₃₀ + Sig-1R (n = 4) were normalized to house-keeping gene actin. Data are presented as means ± S.E.M. Statistical analysis was performed using unpaired two-tailed t-test (p = 0.2950). (Fig. 8g) Representative traces of evoked responses after an electroconvulsive stimulation (30 V, 200 Hz) in flies expressing no transgene (control) or (G4C2)₃₀. (Fig. 8h) Number of spikes in firing discharges induced by an electroconvulsive stimulation of flies expressing (G4C2)₃₀ alone or together with Sig-1R. Data from 20 flies were averaged and are presented as means ± S.E.M. Statistical analysis was performed using unpaired two-tailed t-test (*p = 0.0385). (Fig. 8i) Duration of firing discharges for flies expressing (G4C2)₃₀ alone or together with Sig-1R. Data from 20 flies were averaged and are presented as means ± S.E.M. Statistical analysis was performed using unpaired two-tailed t-test (*p = 0.0375). (Fig. 8j) Expression of Sig-1R-E102Q in retina leads to rough eye phenotype. Representative external eye morphology of 1-day aged flies expressing G4C2₃₀ or human Sig-1R-E102Q alone or together. Note: transgenes were expressed in eyes only. (Fig. 8k) Sig-1R-E102Q failed to ameliorate climbing performances of flies expressing expanded G4C2 repeats. Climbing performances of 4-day-old flies expressing no transgene (control), G4C2₃₀ alone or together with Sig-1R-E102Q or Sig-1R-E102Q alone. Note: transgenes were expressed in neurons. n = 8 flies per group; number of trials: Control, 6; (G4C2)₃₀, 7; (G4C2)₃₀ + Sig-1R- E102Q, 8; Sig-1R- E102Q, 6. Data are presented as means ± S.E.M. Statistical analysis was performed using ANOVA followed by Tukey’s multiple-comparison test (***p < 0.001 versus control, ns: not significant). In the flies climbing to the top groups, 95% CI of difference for control vs (G4C2)₃₀, control vs (G4C2)₃₀ + Sig-1R-E102Q, control vs Sig-1R-E102Q, (G4C2)₃₀ vs (G4C2)₃₀ + Sig-1R-E102Q, (G4C2)₃₀ vs Sig-1R-E102Q, and (G4C2)₃₀ + Sig-1R-E102Q vs Sig-1R-E102Q are 88.03 to 101.6, 86.45 to 99.66, −4.981 to 9.148, −8.119 to 4.547, −99.57 to −85.95 and −97.58 to −84.36, respectively. In the flies remaining at the bottom groups, 95% CI of difference for control vs (G4C2)₃₀, control vs (G4C2)₃₀ + Sig-1R-E102Q, control vs Sig-1R-E102Q, (G4C2)₃₀ vs (G4C2)₃₀ + Sig-1R-E102Q, (G4C2)₃₀ vs Sig-1R-E102Q, and (G4C2)₃₀ + Sig-1R-E102Q vs Sig-1R-E102Q are −78.81 to −33.89, −67.29 to −23.68, −26.09 to 20.53, −10.03 to 31.76, 31.11 to 76.03, and 20.91 to 64.51, respectively.

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