EMBO/SourceData huggingface dataset:
Figure 5Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(a) Spinning disk confocal microscopy images acquired at 160x magnification. Left panels show a maximum intensity projection imaged of a Z-stack, sampled according to the Nyquist criterion and deconvolved. Right panels shows a 3D volume rendering of the same data.Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(b) A projection of 4 planes from a structured illumination microscopy super-resolution Z-stack of the same cells.Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(c) A thin section electron microscopy image of the peri-nuclear region of a GFP-Rab34 transfected HeLa cell. * indicates lysosomes.Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(d) A cryo-immuno-EM image of a GFP-Rab34 transfected HeLa cell. GFP-Rab34 (dark black dots) is identified by immunostaining for GFP and detection with gold conjugated protein A. The lysosome is intimately associated with Rab34 positive membranes. Sites of contact are highlighted by arrows.
Figure 6(a) Confocal immunofluorescence microscopy images of HeLa cells transfected with Rab34-dsRED-Mito, FLCN-GFP and HA-FNIP2. White arrows highlight FLCN-GFP/HA-FNIP2 co-localisation distinct from Rab34-mitochondria/FLCN-GFP localisation. Scale bar = 10μm.(b) Confocal immunofluorescence microscopy images of Rab34-dsRED-Mito (WT) and FLCN-GFP DENN only or ΔDENN transfected HeLa cells showing DENN domain dependent recruitment to mitochondria. Scale bar = 10μm.(c) Western blot analysis of GST-RILP pull down showing association of endogenous FLCN, Rab34 and Rab7 with RILP. Input lane shows 1% input cell extract.(d) Western blot analysis of GST-RILP pull down from UOK257 cells showing DENN domain dependent association of FLCN with RILP N-terminal, Rab binding domain (RBD) and C-terminal fragments.(e) Western blot showing binding of His-tagged FLCN-DENN domain in E.coli extract to GST-RILP.(f) Western blot analysis of GST-RILP pull down showing direct interaction between RILP and GTP hydrolysis deficient (Q111L) Rab34 (at 200nM, in the presence of 1mM GTPγS) and FLCN-DENN domain (1μM), but not low nucleotide affinity (T66N) Rab34. FLCN-DENN enhances association of RILP with Q111L Rab34. Graph shows quantification of relative RILP / Rab34 Q111L binding. Error bars show SEM. ** = p<0.01.(g) Western blot analysis of GST-RILP / Rab34 Q111L pull down (Rab34 at 200nM) showing that enhanced association of with RILP is achieved with low concentrations of FLCN.(h) Western blot analysis of GST-RILPpull down experiments with GTPγS loaded (in the presence of 1mM GTPγS) and GDP loaded (in the presence of 1mM GDP) wildtype Rab34, showing FLCN-DENN (500nM) induced interaction with RILP.(i) Western blot analysis of a GFP-Trap immunoprecipitation experiment from HeLa cells showing that expression of HA-RILP promotes the association of GFP-Rab34 and HA-Rab7.
Figure 7(a) Western blot showing endogenous Rab7 and Rab34 expression in UOK257 and UOK257-2 cell extracts and relative levels of the active forms as measured by GST-RILP pull down. HSC70 is used as a loading control. Graphs show quantification of bound Rab7/Rab34 from 3 independent experiments, error bars show S.E.M., * = p<0.05.(b) (top) Representative confocal immunofluorescence microscopy images showing intracellular distribution of LAMP1 in UOK257 and UOK257-2 cells. Dashed line highlights cell periphery in UOK257-2 cells. Scale bar = 10μm. (bottom left) Graph showing cumulative distribution of LAMP1 intensity in UOK257 vs UOK257-2 cells. Error bars show S.E.M. from 30 cells in 3 replicates. (bottom right) Graph showing Lysotracker-Red PCC decay with time from confocal movies of UOK257 and UOK257-2 cells. Mean values from 5 cells. Error bars show S.E.M.(c) Widefield immunofluorescence images showing early endosomes (EEA1) and Golgi (golgin-97) in UOK257 and UOK257-2 cells. Scale bar = 10μm.(d) Western blot showing FLCN and FLCNΔDENN expression in stable UOK257 derived cell lines.(e) Representative widefield fluorescence microscopy images showing intracellular distribution of LAMP1 in UOK257, UOK257-FLCN and UOK257-FLCNΔDENN cells. Scale bar = 10μm. Graphs showing cumulative distribution of LAMP1 intensity in UOK257, UOK257-FLCN and UOK257-FLCNΔDENN cells. Error bars show S.E.M from 30 cells in 3 replicates.(f) (left) Western blot showing endogenous Rab7 and Rab34 expression in UOK257, UOK257-FLCN and UOK257-FLCNΔDENN cells and relative levels of activity as measured by GST-RILP pull down. HSC70 is used as a loading control. (right) Graphs show quantification of bound Rab7/Rab34 from 3 independent experiments (p<0.05, *** = p<0.001).(g) Western blot showing endogenous RILP expression in the indicated cell lines. Graph shows quantification of RILP expression from 3 independent experiments (* = p<0.05, *** = p<0.001).
Figure 1(A) Immunoblotting for Dnmt1, Dmnt3a and Dmnt3b in the spinal cords of AR-97Q, AR-24Q, and wild-type mice. (B) Quantitative densitometry analysis indicated that Dnmt1 spinal cord level was up-regulated in AR-97Q mice (n = 3). Dnmt3a and Dnmt3b spinal cord level was not significantly different among wild-type, AR-24Q, and AR-97Q mice (n = 3).(C) Quantification of the Dnmts mRNA levels of the spinal cord in wild-type, AR-24Q, and AR-97Q mice using RT-qPCR (n = 3).(D and E) Representative immunohistochemical images depicting the expression of Dnmt1, Dmnt3a and Dmnt3b in the spinal cords of male AR-97Q, AR-24Q, and wild-type mice and quantification of the immunoreactivity for Dnmts (n = 25 neurons from three mice of each group).(F) Double-immunofluorescence staining for Dnmt1 and 1C2 in the spinal motor neurons of an AR-97Q mouse.(G) Immunoblotting for Dnmt1 of the skeletal muscle of wild-type and AR-97Q mice. (H) Quantification of the signal intensity of Dnmt1 bands (n = 3).
Figure 2(A) Immunoblotting of NSC34 cells expressing AR-24Q or AR-97Q (NSC24Q and NSC97Q, respectively) that were treated with DHT for Dnmt1, Dnmt3a and Dnmt3b, and quantification of the signal intensities of bands corresponding to Dnmt1, Dnmt3a and Dnmt3b (n = 3).(B) Relative mRNA levels of Dnmt1 in NSC24Q and NSC97Q cells measured with RT-qPCR (n = 3).(C) Protein levels of Dnmt1, Dnmt3a and Dnmt3b in DHT-treated and -untreated NSC97Q cells by Western blotting analysis.(D) RT-qPCR analysis of Dnmt1 in DHT-treated and -untreated NSC97Q cells (n = 3).(E, F) Quantitative assessment of cell viability changes due to the siRNA-mediated knock down of Dnmt1, Dnmt3a or Dnmt3b in NSC97Q cells with DHT (E) or without DHT (F) using the WST-8 assay (n = 3).(G) Quantitative assessment of the cell viability of NSC97Q cells treated with DHT and RG108 using the WST-8 assay (n = 3 per group).(H) Immunoblotting of NSC97Q cells treated with DHT and RG108 for Dnmt1, Dnmt3a and Dnmt3b.(I, J) Cell viability analysis with siRNA-mediated knockdown of Dnmts in DHT-untreated NSC97Q (I) and DHT-treated NSC24Q (J).
Figure 3(A-D) Grip power (A), body weight (B), rotarod performance (C) (Two-way ANOVA with Tukey's test) and survival rate (D) (Kaplan-Meier analysis and log-rank test) of AR-97Q mice treated with or without RG108 (DMSO, n =20; 0.5 mg/dL, n = 20; 2.0 mg/dL, n = 20). All parameters were significantly improved in AR-97Q mice treated with 2.0 mg/dL RG108 compared with those treated with DMSO: *P < 0.05 (grip); *P < 0.05 (body weight); *P < 0.05 (rotarod); and *P < 0.05 (survival).(E) Representative photograph of a 13-week-old AR-97Q mouse treated with DMSO (left) and an age-matched AR-97Q mouse treated with 2.0 mg/dL RG108 (right). AR-97Q mouse medicated with RG108 maintained muscle volume and body size compared with DMSO-treated AR-97Q mouse of same age. The white compact masses (arrow) of mice head are the cement covering the surgical site.(F,G) Immunoblots (F) and relative intensities (G) of the Dnmt1-immunoreactive bands of spinal cords from AR-97Q mice treated with or without RG108 (n = 3 per group).(H,I) Dnmt1 immunohistochemistry in spinal cord sections from AR-97Q mice treated with or without RG108 (n = 3 per group).
Figure 4(A) Immunohistochemistry of the spinal cords of AR-97Q mice treated with DMSO or RG108 for polyglutamine using a 1C2 antibody. (B) Quantification of 1C2-positive motor neurons in the spinal cords (n = 5 per group).(C) Immunoblotting of the spinal cords of AR-97Q mice treated with or without RG108 for AR. (D) Densitometric analyses to quantify AR accumulation in the spinal motor neurons of AR-97Q mice treated with DMSO or RG108 (n = 3).(E) Relative mRNA expression levels of human AR in AR-97Q mice spinal cord with or without RG108 treatment (n = 3).Immunohistochemistry for ChAT in the spinal cord anterior horns of AR-97Q mice (n = 5 per group).quantitative analysis of the neuron sizes in the spinal cord anterior horns of AR-97Q mice (n = 5 per group).(H,I) Western blot analysis (H) and relative signal intensities (I) of the ChAT-immunoreactive bands of spinal cords from AR-97Q mice treated with or without RG108 (n = 3).
Figure 5(A) Immunoblotting of SH-SY5Y cells stably expressing AR-24Q (SH24Q) or AR-97Q (SH97Q) for Dnmt1, Dnmt3a and Dnmt3b. (B) Densitometry quantification showing that Dnmt1 level was up-regulated in SH97Q cells compared with SH24Q cells (n = 3).(C, D) Immunoblots for Dnmts of SH97Q with or without DHT treatment (C) and quantification data (D) (n = 3).(E) Seven genes with DNA hyper-methylation in their CpG islands were identified by DNA methylation array analysis.(F) The relative mRNA levels of CDC25B, GFRA3, NPY, HES5, SCTR, LEF1-AS1 and CABS1, normalized to beta-2 microglobulin, in DHT-treated SH24Q and SH97Q cells were measured using RT-qPCR (n = 3).(G) RT-qPCR analysis indicated the decrease of Hes5 mRNA level in DHT-treated SH97Q cells compared with DHT-untreated SH97Q cells (n = 3).(H) Methylation-specific PCR of SH24Q and SH97Q cells that were treated with DHT.(I) Hes5 immunoreactivity of spinal motor neurons in wild-type, AR-24Q, and AR-97Q mice (n = 3).(J) Hes5 mRNA levels in the spinal cords of wild-type, AR-24Q, and AR-97Q mice (n = 3).
Figure 6DNA methylation levels of the HES5 promoter region of SH97Q cells treated with DHT and RG108.HES5 mRNA levels of SH97Q cells treated with DHT and RG108.DNA methylation levels of the Hes5 promoter region in DHT-treated NSC97Q cells treated with or without RG108.mRNA levels of Hes5 (n = 3) in DHT-treated NSC97Q cells treated with or without RG108.(E) Hes5 mRNA levels in the spinal cords of AR-97Q mice treated with or without RG108 (n = 3).(F, G) Hes5 immunoreactivity of spinal motor neurons in AR-97Q mice treated with or without RG108 (n = 3).
Figure 7(A) Viability of DHT-treated NSC97Q cells treated with Hes5 and control siRNA measured by the WST-8 assay (n = 3).(B) Immunoblots of DHT-administrated NSC97Q cells treated with Hes5 siRNA (n = 3).(C) Quantitative cell viability analysis revealed that siRNA-mediated Hes5 knock down negated the therapeutic effect of RG108 in NSC97Q cells that were treated with DHT (n = 3), as determined by the WST-8 assay.Cell viability measured with the WST-8 assay of DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 3).Cell viability measured with immunoblotting of DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 3).(F) Quantification of LDH release from DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 4).(G) Immunoblots of DHT-administrated NSC97Q cells transfected with a mock plasmid or the Hes5 vector for AR. (H) AR aggregation as quantified by densitometry (n = 3).
Figure 8(A) Immunoblots of NSC34 cells treated with Hes5 siRNA. (B) Quantification of protein levels of pSmad2, Smad2, phospho-IkBα and heat shock factor-1 (Hsf1) (n = 3).(C) siRNA-mediated knockdown of Hes5 down-regulated Hes5 protein level (n = 3).(D) Western blots of Smad2 and pSmad2 in NSC97Q cells with or without DHT treatment (n = 3).(E) Protein levels of Smad2, pSmad2, and Hes5 in NSC97Q cells transfected with Hes5 vector (n = 3).(F) Immunoblots of Smad2, pSmad2, and Hes5 in primary cortical neurons treated with lentiviral vector containing AR24Q and AR97Q (n = 3).(G) Relative mRNA levels of Hes5 in primary cortical neurons expressing AR24Q and AR97Q (n = 3).(H) Relative mRNA levels of Hes5 in primary motor neurons expressing AR24Q and AR97Q (n = 6).(I) Western blots of Smad2, pSmad2, and Hes5 in primary cortical neurons expressing AR97Q treated with mock plasmid or Hes5-containing lentiviral vector.
Figure 1The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Spinning disc confocal live cell imaging of Alexa 555-EGF (555-EGF) shown as vesicle tracking with time represented as a colour spectrum. Tracking started 5 min after addition of 20 ng/mL 555-EGF for the indicated durations. Scale bar: 10 μm. The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). B: Immunofluorescence staining of EGFR following 5 or 15 min stimulation with 20 ng/mL EGF. Scale bar: 20 μm.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). C: Quantification of EGFR vesicles in a perinuclear region (within 30 μm diameter of the centre of the nucleus) Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). D: Cells were stimulated with 20 ng/mL 555-EGF for 15 or 30 min before immunofluorescence staining against EGFR. Scale bar: 10 μm.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). E: Quantification of percentage of total EGFR vesicles that colocalise with 555-EGF Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). F: Cells stably expressing mCherry-Rab5 were stimulated with 20 ng/mL EGF for indicated times before immunofluorescence staining against EGFR. Scale bar: 10 μm.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). G: Pearson's colocalisation coefficient between mCherry-Rab5 and EGFR Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01.
Figure 2The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. A: Control, sgAtg7, or sgAtg16l1 cells were treated with 2 ng/mL EGF for 15 min. Cells were then processed for staining using anti-EEA1 antibodies and PI(3)P probe (Alexa 488-labelled 2XFYVE domains). To ensure the specificity of the probe, control cells were pre-treated with 5 mM 3'MA for 30 min. Scale bar: 10 μm. B: Quantification of PI(3)P+ label intensity per cell (in A). C: Pearson's colocalisation coefficient between PI(3)P and EEA1 (in A). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. D: Control and sgAtg7 cells were stimulated 20 ng/mL EGF for 5 or 15 min before immunofluorescence staining against EEA1 and EGFR. Scale bar: 10 μm. E: Pearson's colocalisation coefficient between EEA1 and EGFR (in D). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. F: Endogenous VPS34 was immunoprecipitated from control and sgAtg7 cells that were treated with 2 ng/mL EGF for 15 min then lysed in CHAPS-containing detergent buffer and binding partners detected by western blotting. G: Densitometry analyses of proteins coimmunoprecipitated with endogenous VPS34 (in F). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, * p<0.05, ** p<0.01, *** p<0.001.
Figure 3The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Cells transiently expressing YFP-Galectins (YFP-Gal3, -Gal8, or -Gal9) were stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1. Scale bar: 10 μm. B: Quantification of percentage of total EEA1 vesicles that colocalise with YFP-Galectins (in A).Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, ** p<0.01, *** p<0.001.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). C: Cells transiently expressing YFP-Gal8 were stimulated with 20 ng/mL Alexa 555-EGF (555-EGF) for 15 min. Scale bar: 10 μm. D: Quantification of the percentage of YFP-Gal8-labelled vesicles that colocalise with 555-EGF in control or ATG7-deficient cells (in C). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, ** p<0.01, *** p<0.001.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). E: Cells transiently expressing YFP-Gal8 were treated 100 μM monensin for 1 hr and stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1. Scale bar: 10 μm. F: Quantification of percentage of total EEA1 vesicles that colocalise with YFP-Gal8 upon monensin treatment (in E). Data information: White arrows indicate colocalisation. Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: NS p>0.05, ** p<0.01, *** p<0.001.
Figure 5The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). A: Cells were treated for 15 min with 2 ng/mL EGF before fixation and immunofluorescence staining against EEA1 and p-TBK1. White arrows indicate colocalisation. Scale bar: 10 μm. B: Quantification of percentage of total EEA1 vesicles that colocalise with p-TBK1 (in A). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). C: Cells stably expressing Flag-S-ATG16L1 were pre-treated for 1 hr with TBK1 inhibitors (100 μM MRT68601 or 5 μM momelotinib). Control cells were also treated 100 μM monensin for 1 hr as indicated. All cells were stimulated for 15 min with 2 ng/mL EGF followed by fixation and immunofluorescence staining against Flag tag and EEA1. Scale bar: 10 μm.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). D: Quantification of total EEA1 vesicles that colocalise with Flag-S-ATG16L1 Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). E: Western blotting of shNf-1/shTp53 glial cells expressing gRNA sequences targeting Gal8.The following experiments were performed in glial shNf-1/shTp53 glial cells serum starved 4 hr before assaying. Cells were expressing either Cas9 alone (control) or Cas9 and sgRNA targeting Atg7 (sgAtg7 #1 or #2). F: Control and sgGal8 cells stably expressing Flag-S-ATG16L1 were treated 100 μM monensin for 1 hr and stimulated with 2 ng/mL EGF for 15 min before fixation and immunofluorescence staining against EEA1 and Flag tag. White arrows indicate colocalisation. Scale bar: 10 μm. G: Quantification of percentage of total EEA1 vesicles that colocalise with Flag-S-ATG16L1 in sgGal8 cells (in F). Data information: Statistical analyses were performed on at least 3 independent experiments, where error bars represent SEM and p values represent a two-tailed Student's t test: * p<0.05, ** p<0.01, *** p<0.001.