EMBO/sd-character-level-ner huggingface dataset:
CftrF508del mice (a–d) and Scnn1b-Tg mice (e–k) treated with NAC or PBS (n = 10 per group).figf8CftrF508del mice (a-d)(a) Confocal microscopy of LC3 (top) and p62 (bottom) in lung tissues from CftrF508del mice. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm. Representative images of ten PBS-treated and ten NAC-treated mice. (b) Quantification of LC3 dots (per mm2 of tissue) in lung tissues from CftrF508del mice. Means ± s.d. Asterisk, P 0.001 versus PBS-treated mice; ANOVA. CftrF508del mice (a-d)(c) Number of macrophages per mm2 of lung tissue from CftrF508del mice (CD68-positive cells in 15-20 different randomly taken sections for each mouse lung for each condition); means ± s.d. for three separate experiments. Asterisk, P 0.05 versus PBS-treated mice; ANOVA. CftrF508del mice (a-d)(d) MPO activity in lung homogenates from CftrF508del mice (expressed as 10−3 × OD s−1). Means ± s.d. Asterisk, P 0.05 versus PBS-treated mice; ANOVA. Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (e) Confocal microscopy of LC3-positive dots in lung tissues from PBS-treated or NAC-treated mice-Tg LC3. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm. (f) Number of LC3-positive dots in Scnn1b tissues from mice-Tg PBS. Means ± s.d. Asterisk, P 0.01 versus mice-treated mice; ANOVA. Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (g) FRET analysis of TG2-Alexa 546 fluorescence after SUMO-1 Cy5 photobleaching in lung tissues from PBS-treated WT mice and PBS-treated or NAC-treated Scnn1b-Tg mice. Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (h) Immunoblot of TG2 and phospho-p42/44 in lung tissues from PBS-treated WT mice and PBS-treated or NAC-treated Scnn1b-Tg mice. αβ-Tubulin was used as loading control. Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (i) Lung histology of PBS-treated and NAC-treated Scnn1b-Tg mice. Haematoxylin staining. Scale bar, 150 μm. Black arrows indicate decrease in lung infiltration in NAC-treated in comparison with PBS-treated mice (arrowheads). Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (j) Left: confocal microscopy of COX-2 staining (left) in PBS-treated and NAC-treated Scnn1b-Tg mice. Nuclei counterstained with DAPI (blue). Scale bar, 150 μm. Right; number of COX-2-positive cells per mm2 of lung tissue in NAC-treated and Scnn1b-treated mice-Tg PBS. Means ± s.d. Asterisk, P 0.05 versus mice-treated mice; ANOVA.1 versus PBS-treated mice; ANOVA. Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (k) MPO activity in lung homogenates from PBS-treated and NAC-treated Scnn1b-Tg mice (expressed as 10−3 × OD s−1). Means ± s.d. Asterisk, P 0.05 versus PBS-treated mice; ANOVA. Densitometric analysis of the blot is shown in Supplementary Information, Fig. S9.
Representation of the mutant LAMP-2b.figf2Figure 2 Representation of the mutant LAMP-2b. 'Normal' represents the LAMP-2b amino-acid sequence. Because patient 1, who developed full DD symptoms, has a mutation in exon 9b, the LAMP-2b isoform is most probably responsible for DD. Residue 265, one of the putative disulphide-bond-creating cysteines, and two glycosylation sites are lost by exon-6 skipping in patient 3, which probably results in a significant change in the secondary structure. Black boxes represent the loops created between two cystein residues flanking each of the four loop regions; Y depicts a glycosylation site; hatched boxes show altered amino acids caused by frameshift mutations; dashes represent missing amino acids due to exon-6 skipping in patient 3. All of the other mutant proteins lack the transmembrane domain (grey bar) because of nonsense or frameshift mutations. Thus, those molecules cannot function as lysosomal 'membrane' proteins.
Fig.Figure 5A FST knockdown in MOLM-13 by shRNA effectively reduced FST expression. The morphology were measured after FST knockdown in vitro. Scale bar = 10 μm. The apoptosis (C) measured after FST knockdown in vitro. Scale bar = 10 μm. The apoptosis assays (C) were performed in triplicates. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). The clonogenicity of MOLM-13 (D and E) were measured after FST knockdown in vitro. Scale bar = 10 μm. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). The engraftment of MOLM-13 after FST knockdown was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation. The engraftment of MOLM-13 after FST knockdown was detected by flow cytometry of human CD45 and mouse CD45.1 positive cells in recipient mouse BM aspiration at week 2 post transplantation. Data information: data were presented as mean ± SEM. *P<0.05, **P<0.01 (Student's t-test). The effect of FST knockdown on the survival of NSG mice-engrafted with MOLM-13. scr: scrambled sequence control (7 mice); sh: short hairpin RNA (8 mice for sh1 and sh2 respectively). The survival curve was analyzed by Log-rank test. **P<0.01.
CA VII promotes experimental febrile seizures in P13–14 mice.Figure 4Representative raw traces of epidural EEG recordings during hyperthermia (HT) from a WT and a CA VII KO mouse. Seizures were reliably induced in WT (n=9) but not in CA VII KO (n=7). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces. Specimen recordings of breath rate from a WT and a CA VII KO mouse. The baseline breath rate (WT 153±11 breaths/min and CA VII KO 157±8 breaths/min) was increased during HT in both genotypes (WT 272±28 breaths/min and CA VII KO 279±12 breaths/min). The bar diagram summarizes the mean increase in breath rate in WT (177±12%) and CA VII KO mice (178±6%, P=0.98).Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces. Hyperthermia caused a comparable alkalosis in blood pH in both WT and CA VII KO mice. The blood pH did not differ between the two genotypes under control conditions (P=0.37) or during HT (P=0.44). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces. Electrographic seizures were not detected in WT mice when hyperthermia was induced in the continuous presence of 5% CO2 (n=5). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.
Figure 3Chemical structure of 14C -APR-246 and its active product MQ. 14C accumulation (cpm/mg x ml-1) in 11 cell lines after 24h 14C -APR-246 +/- MK-571 treatment (n ≥ 3). *p = 0.0006, paired t-test. 14C-accumulation and n of individual cell lines as shown in Table S3. 14C accumulation (cpm/mg x ml-1) in HCT116 (WT and R248W) cells after 24h +treatment with 14C -APR-246 +/- MK-571 or Reversan (n ≥ 3, each dots in figure). 14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W cells (n ≥ 3 and ≥ 2) transfected with MRP1 siRNAs after 24h of 14C-APR-246 treatment. Indicated values represent four individual MRP1 siRNAs and two individual negative control (Ctrl) siRNAs, where each dot is an individual siRNA. Mean of 14C-accumulation after treatment and n with individual siRNA sequences is shown in Table S4. *p = 0.04 and for R248W p = 0.06, Wilcoxon matched pairs signed rank test.
Figure 4(A) PV+ interneurons were sorted from cortices of PV-EGFP WT and Cox6a2-/- mice at P15 and processed for bulk RNA sequencing (n= 4 WT, 6 Cox6a2-/-; P15). Heatmap shows expression for top down- (red) and upregulated (blue) genes in Cox6a2-/- PV+ interneurons relative to their WT counterparts. (B) Cox6a2 expression was effectively abolished in Cox6a2-/- PV+ interneurons. In addition, Cox6a2 knockout led to a decreased expression of Gad1 and Gad2 genes (n= 4 WT, 6 Cox6a2-/-). Two-tailed unpaired Mann-Whitney tests. (C) Changes in expression of oxidative stress-related genes (green), genes coding for AMPA receptors (orange), sodium channels (red), potassium channels (magenta), and internal calcium signaling proteins (blue). Color scale bars show normalized read counts. (D) Significantly down- (red) and upregulated (blue) gene ontology (GO) terms based on top 200 down-/upregulated genes in Cox6a2-/- PV+ interneurons (Benjamini-Hochberg-adjusted p-values).
Figure 7a) Auto-inhibited HHARI structure is shown in cartoon representation (PDB 4KC9). The E2-binding site (green oval) on RING1 (pink) is separated from the active site Cys (yellow spheres) on RING2 (orange) by more than 40 Å. The IBR domain (dark blue) connects RING1 and RING2 and the Ariadne domain (grey) occludes the active site Cys. b) A model depicts the events required for Ub transfer by HHARI (with no intended order). E2~Ub binding to RING1 favors open E2~Ub conformations (middle panel) that expose the Ub hydrophobic patch (orange circle). Movement of the Ariadne domain (middle panel) exposes the active site Cys (right panel, yellow star) and may allow a disorder-to-helix transition of the IBR-RING2 linker (dotted black line in right panel, not seen in structure in A) which completes the Ub binding site on RING2 (yellow sphere, right panel). Open E2~Ub conformations make the hydrophobic patch of Ub (orange circle) available for recruitment by RING2 to ensure transfer of Ub to the active cite Cys.
GFP::MTM‐3(C459S) associates with autophagic structures A–C Fluorescent images of wild‐type embryos (1.Figure 2A-C Fluorescent images of wild‐type embryos (1.5‐fold stage) expressing GFP::MTM‐3 (A), GFP::MTM‐3(C459S) (B), or GFP::MTM‐3(C459S, del PH‐G) (C). D‐G″ Confocal fluorescent images of wild‐type (D‐D″), mtm‐3 (E‐E″), atg‐2 (F‐F″), and vps‐34 (G‐G″) embryos at the 200‐cell stage expressing GFP::MTM‐3(C459S) and stained by anti‐LGG‐1 antibody. Insets show magnified views. Arrows indicate overlapping GFP and LGG‐1 puncta. Scale bars: 5 μm. H, I Number of GFP::MTM‐3(C459S)‐positive puncta and co‐localization of GFP::MTM‐3(C459S)‐ and LGG‐1‐positive puncta in various strains [mtm‐3(tm4475), atg‐2(bp576), lgg‐1(bp500), vps‐34(h797)]. At least 10 (I) and 15 (H) embryos were scored per strain. Data are shown as mean ± SD. Data from different mutant backgrounds were compared with wild type. **P 0.0001; N.S.: not statistically different. J MTM‐3 but not MTM‐3(C459S) exhibits phosphatase activity toward PtdIns3P and PtdIns(3,5) P2 in vitro.
Figure 7A, Western blot of BT371NS showing p21 phosphorylation (pp21Thr145) in cytoplasmic lysates immunoprecipitated with p21 antibodies at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + HGF) of HGF (10 ng/ml). B, Western blot of BT463NS showing p21 phosphorylation (pp21Thr145) in cytoplasmic lysates immunoprecipitated with p21 antibodies at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of the MET inhibitor JNJ38877605 (500 nM). C, Western blot of BT308NS showing p21 localization in cytoplasmic/nuclear extracts at the indicated time points after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vinculin and lamin B were used as loading controls for cytoplasmic and nuclear fractions, respectively. D, Representative immunofluorescence staining of p21 (red) on BT308NS cells 3 h after IR (5 Gy) in the absence (vehicle) or in the presence (JNJ) of JNJ38877605. Ctrl: non-irradiated cells. Nuclei are counterstained with DAPI (blue). Scale bar, 10 µm (63× magnification). E, Quantification of the percentage of cells showing p21 cytoplasmic or nuclear localization in BT308NS represented in (D) (n = 10 HPF/group). HPF: high-power field. *: t-test (5 Gy + JNJ) vs. (ctrl or 5 Gy), p<0.0001. F, Cell cycle analysis of BT314NS, performed 24 h after IR in the absence (5 Gy) or in the presence (5 Gy + JNJ) of JNJ38877605. Vehicle: non-irradiated cells. *: one-way ANOVA, P=0.008. G, Schematic representation of the MET-driven signaling pathways that sustain DNA repair, and prevent apoptosis and cell cycle arrest. Data are represented as mean ± SEM.
Androgens divergently regulate the UPR arms.Figure 2A, B mRNA expression levels in LNCaP cells for the indicated genes were investigated using quantitative PCR (qPCR). Controls were treated with vehicle for 84 h and set to 100. Data represent the mean of three independent experiments in triplicate, and bars represent SE. P-values ranged between 1.66 × 10−5 and 0.025, and IRE1α expression in R1881 48 h was *P = 0.013 with respect to vehicle-treated cells using unpaired Student's t-test. C-E IRE1α, XBP-1S, and XBP-1U mRNA in CWR22xenografts grown in nude mice and collected at the indicated times after castration. The value at t = 0 was set to 1. Columns represent the mean of at least three independent tumors for each time point, and bars represent SE. P-values ranged between 5.7  ×  10−10 and 0.0002. IRE1α expression at 48 h post-castration was 3.17 × 10−6 with respect to t = 0 using unpaired Student's t-test. F, G Protein expression in LNCaP cells upon treatment with R1881 for the indicated times by Western blot analysis. Data presented are representative of three independent experiments.
Figure 6(A-B) Epifluorecence microscopy images of HeLa cells (A) left untreated or treated with LLO (0.5 nM, 15 min). Cells were incubated with exofacial-PS probe (Alexa-568 annexin A5) (red) 30 min prior fixation, immunolabelled for NMHCIIA (green) and stained with DAPI (blue). Insets show PM damage marked by exofacial-PS sites. Arrows indicate exofacial-PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles; arrowheads indicate NMHCIIA bundles associated with exofacial-PS sites without detectable bacteria. (A-B) Epifluorecence microscopy images of HeLa cells (B) infected with GFP-expressing wt Lm for 6 h. Cells were incubated with exofacial-PS probe (Alexa-568 annexin A5) (red) 30 min prior fixation, immunolabelled for NMHCIIA (green) and stained with DAPI (blue). Insets show PM damage marked by exofacial-PS sites. Arrows indicate exofacial-PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles; arrowheads indicate NMHCIIA bundles associated with exofacial-PS sites without detectable bacteria. (C) Quantification of the % of infected cells with exofacial-PS sites in HeLa cells infected with wt Lm for 6 h. Data are the mean ±SEM (n=3) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5. (D-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa and RAW264.7 cells, infected with GFP-expressing wt or Δhly Lm for 6 h. The levels of PI incorporation by uninfected cells were subtracted from infected samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001. PM permeability was assessed in cells harbouring equivalent numbers of intracellular bacteria (Fig Appendix Fig S3).
Testing an assay for SARS-CoV-2 using synthetic RNA targets.Figure 1.Testing an assay for SARS-CoV-2 using synthetic RNA targets. We show data from both fluorescent (a) and lateral flow detection (b). Target concentrations (in cp/μl) are indicated. RPA NTC: water input into RPA; Det NTC: water input into Cas13 detection reaction. In (a), error bars indicate one standard deviation based on n=3 technical replicates.
Figure 9(A) Hypothetical model for C5 cleavage by the alternative pathway C5 convertase. The AP C5 convertase is a multimeric complex between a dimeric C3 convertase enzyme (comprised of surface-bound non-catalytic C3b in complex with protease Bb), together with additional surface-bound C3b molecules (not depicted here), which are required to strengthen the affinity for C5. Hypothetical model of C3bBb (surface representation, C3b in gray, Bb in orange) bound to substrate C5 (light green, C5d domain in dark green). C3bBb is derived from the dimeric C3bBb-SCIN complex (PDB 2WIN (Rooijakkers et al, 2009)), and C5 is modeled based on superposition of the CVF-C5 complex (PDB 3PVM (Laursen et al, 2011)) on the C3b molecule from C3bBb. The right panel shows C3bBb bound to C5b (light green, C5d in dark green). The structure of C5b is derived from the structure of the C5b6 complex (PDB 4A5W (Hadders et al, 2012)), and superimposed on C5 from the model in the left panel. (B) Superposition of C5b with the C5d domain in the pC5b6 (dark green) and C3b-like (light blue) orientation. The C3b-like conformation of C5d was generated based on superposition of the C5d structure (extracted from the pC5b6 structure, PDB 4A5W) on the C3d domain of the second C3b subunit from the dimeric C3bBb-SCIN structure (PDB 2WIN). (C) Hypothetical structural models for C5b6 assembly by convertases. (I) Model of pC5b6 bound to C3bBb, as in (A, right). (II) Model of pC5b6, with C5d-C6 superimposed on C5d in the C3b-like orientation, as in (B). Note that this orientation allows C6 to extend further toward the membrane relative to the convertase. (III) Model in which C5b6 has dissociated from C3bBb, but adopted the orientation shown in (II). All structural models and superpositions were generated using UCSF Chimera (Pettersen et al, 2004).
a, Quantification of NHS-biotin-labelled RBCs or transferred CMFDA-labelled RBCs (n = 3).figf2a, Quantification of NHS-biotin-labelled RBCs or transferred CMFDA-labelled RBCs (n = 3). b, Western blot analyses of caspases in RBCs after in vitro culture. Asterisks denote processed caspases. FL, full length. c, The relative luminescence units (RLU) of caspase activities in cultured RBCs and the suppression of annexin V staining in Nix-/- RBCs after 12-h culture in the presence of qVD-oph or solvent control (DMSO) (n = 3). d, Mitotracker versus annexin V staining of RBCs after in vitro culture. e, Mean fluorescent intensity (MFI) of ROS staining in RBCs after in vitro culture, and caspase activities in Nix-/- RBCs after 24 h culture with solvent control, ROS scavengers or qVD-oph (n = 3). BHT, butylated hydroxytoluene; CAT, catalase; TEM, tempol. f, Haematoxylin and eosin (HE) staining of spleen sections of 9-week-old wild-type and Nix-/- mice. Arrows denote iron deposits within macrophagecytoplasm. Iron deposits were also stained with Prussian blue, followed by counterstain with nuclear-fast red. Scale bar, 20 µm. g, Real-time RT-PCR of erythropoietin (Epo) (n = 12). For all relevant panels, statistical significance of the data (mean ± s.e.m.) is: *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 5A Analysis of ASCO enrichment by ChIRP using two sets of independent biotinylated probes ODD and EVEN compared to negative control with probes designed against the LacZ RNA. The fold enrichment was calculated between ODD or EVEN samples against LacZ. These samples were used for protein precipitation and Mass Spectrometry analyses (ChIRP-MS), from which PRP8a was identified as a potential ASCO partner. B Validation of PRP8a-ASCO interaction by PRP8a-RIP. U5 RNA was used as a positive control. Data information: In , the results are expressed as a percentage of the Input for PRP8a RIP followed by RT-qPCR C Immunoblot analysis was performed during PRP8a immunoprecipitation (IP). The same volume of input, unbound fraction was loaded as well as 20 % of the eluted IP fraction. α-PRP8a and α-IgG : IP performed with anti-PRP8a or control rabbit IgG antibody, respectively. : In D, the results are expressed as a percentage of the Input for PRP8a RIP followed by RT-qPCR, and IgG RIP was used as a negative control.
Figure 3(A) Gene expression analysis of IL12b in whole brain (n=3 per gender, Il12b undetected), Cd11b-negative non-microglial cells (n=3 per gender, Il12b undetected) and CD11b-positive microglia (n=5 per gender, p=0.3540) in male and female APP23 mice. Gapdh expression was used as an internal reference gene. Mean ± s.e.m., statistical analysis: two-tailed unpaired t-test for each fraction. (B) ELISA measurements of the IL12p40 concentration in the TBS-soluble protein fraction derived from wild-type (WT) (n=5), APP23p40+/+(n=17) and APP23p40‑/- (n=16) mice. Male mice are depicted by blue squares while female mice are shown as red squares. Mean ± s.e.m., statistical analysis: one-way ANOVA, Tukey post-hoc test, *p=0.0276, ***p=<0.0001.
Figure 6A. Plasma urea levels were elevated to a greater extent in tunicamycin-treated Nox4 KO mice than WT. Co-treatment with guanabenz (Gbz) reduced urea levels in both groups. Numbers of animals are indicated within bars. B. 48 hours after systemic tunicamycin treatment, kidneys of Nox4 KO mice showed a marked surface pallor (bottom right). C. TUNEL staining revealed a significantly higher number of apoptotic cells in tunicamycin-treated Nox4 KO mice. n=4/group. D. Immunoblotting of kidney homogenates showed significantly elevated cleaved caspase-12 and cleaved PARP levels in tunicamycin-treated Nox4 KO mice compared to WT. E. Survival curves showed that a very high proportion of Nox4 KO mice died after AKI. Guanabenz (Gbz) treatment dramatically improved survival in tunicamycin-treated KO mice. Number of animals as indicated. Levels of significance by Kaplan Meier analysis are reported to the right. F. Schematic depicting the effect of Nox4-generated ROS on PP1 activity and the balance between eIF2α phosphorylation and dephosphorylation. Nox4 is upregulated by ATF4 and binds to GADD34. It inhibits GADD34-bound PP1 through the local generation of H2O2 and oxidation of the metal (M) center of the serine-threonine phosphatase. The consequent prolongation of eIF2α phosphorylation promotes cell survival in the face of acute protein unfolding stress. M = iron or manganese, which are oxidized from the M (II) to the M (III) species.
Figure 6A. HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 were incubated with 250 μM LLOMe, fixed at different time points as indicated and stained for ubiquitin. As presented, CHMP4B is recruited before ubiquitin and Galectin3 upon lysosomal membrane damage Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments B. HeLa cells stably expressing CHMP4B-eGFP were treated as in (A) and stained for p62. CHMP4B is recruited before p62 on the damaged lysosomes. The number of LAMP1-positive foci appears stable independently of LLOMe treatment Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments C. Experimental setup as in (A and B) where HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 were stained for LC3. As shown, CHMP4B is recruited prior to LC3 Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments
Figure 4(A) Left panel, immunoblotting against the HA tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with a construct expressing 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA in the GA frame and treated with 10 µM of the indicated compound for 15 hours. Right panel, quantification of polyGA expression relative to the GAPDH. (B) Left panel, immunoblotting against the HA tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with a construct expressing 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA tag in the PG frame and treated with 10 µM of the indicated compound for 15 hours. Right panel, quantification of polyPG expression relative to the GAPDH. (C) Cell viability (TO-PRO-3 FACS staining) of GT1-7 neuronal cells treated with 1, 3 or 10 µM of promethazine and co-transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA and a construct expressing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA or PG frame.