EMBO/SourceData huggingface dataset:
Figure 3(A) Representative images of healthy or C9orf72 iPSC-derived MNs treated with Sema3A or untreated in the distal compartment, 6 hours post treatment. Red: denotes Tubulin, Green: denotes CRMP4. Scale bar: 5 μm. (B) Quantification of CRMP4 intensity levels in healthy or C9orf72 iPSC-derived MNs with Sema3A treatment or untreated. 14 untreated healthy axons, 12 healthy axons with Sema3A treatment, 49 untreated C9orf72 axons and 31 C9orf72 axons with Sema3A treatment were monitored from 3 different chambers. One-way ANOVA, Tukey's multiple comparisons test, n = 3, Data presented as mean ±SE, *p = 0.0338; ****p<0.0001. (D) Representative images of healthy or C9orf72 human-derived MN cell somata with Sema3A treatment, Sema3A + dynein inhibitor treatment, or untreated. Gray: denotes CTX, Green: denotes CRMP4. Scale bar: 5μm. (E) Quantification of CRMP4 intensity (normalized to GAPDH+mCherry/area) levels at the somata of healthy or C9orf72 human-derived MN after Sema3A treatment, Sema3A + dynein inhibitor treatment, or untreated. Analysis performed in 3 independent chambers per condition. 19 healthy untreated cell somata, 26 healthy cell somata with Sema3A treatment, 20 healthy cell somata with Sema3A + dynein inhibitor treatment and 14 C9orf72 cell somata from each condition were monitored. One-way ANOVA, Newman-Keuls multiple comparisons test, n=3, Data presented as mean ±SE, *p=0.0207; ***p=0.0004. (F) Representative images of healthy or C9orf72 human-derived MN proximal axons with Sema3A treatment, Sema3A + dynein inhibitor treatment or untreated control. Green: denotes CRMP4, Red: denotes GAPDH. Scale bar: 5μm. (G) Quantification of CRMP4 intensity levels (normalized to GAPDH+mCherry/area) at the proximal axons in healthy or C9orf72 human-derived MN after Sema3A treatment, Sema3A + dynein inhibitor treatment, or untreated control. Analysis performed from 3 independent chambers in each condition. 21 healthy untreated proximal axons, 24 healthy proximal axons with Sema3A treatment, 16 healthy proximal axons with Sema3A + dynein inhibitor treatment, 12 C9orf72 untreated proximal axons, 8 C9orf72 proximal axons with Sema3A treatment and 13 C9orf72 proximal axons with Sema3A + dynein inhibitor treatment were monitored. One-way ANOVA, Newman-Keuls multiple comparisons test, n = 3, Data presented as mean ±SE, *p =0.0334, ****p<0.0001.
Figure 4(F) Upper panel - Immunoprecipitation assay with anti‐Flag antibody followed by Western blot analysis of dynactin (p150) in COS7 cells overexpressing Flag-CRMP4 (size of ~65 KDa). Lower panel - Total input (size of ~150 KDa). (G) Quantification of the blot in F. The dynactin intensity band was normalized to the Flag-CRMP4 intensity band in each repeat. Student's t-test, n=3, Data presented as mean ±SE, *p=0.0299. (H) Immunoprecipitation of DIC (size of ~75 KDa) followed by Western blot analysis of CRMP4 (size of ~64 KDa) in COS7 cells that were transfected with CRMP4 and AAV9-50aa or its control. IgG antibody was used as a control. (I) Quantification of the blot in H from 3 independent repeats. The CRMP4 intensity band was normalized to the DIC intensity band in each repeat. Student's t-test, n=3, Data presented as mean ±SE, *p=0.0479.
Figure 5(E) Representative images from the proximity ligation assay (For explanation of PLA technique; please refer to method section) for CRMP4 and dynein in SODG93A and WT primary MNs axons that were exposed to either control or Sema3A 8h post treatment. Scale bar: 5µm. (F) Quantification of the CRMP4-DIC puncta number per primary motor neuron axon in each condition. We analyzed ~20 axons per condition from 3 independent chambers per group (One-way ANOVA, Tukey's multiple comparisons test, n=3, Data presented as mean ±SE, **p=0.01 *p=0.04) (G) Representative images of proximity ligation assay for CRMP4 and dynein in healthy and C9orf72 human-derived proximal axons post peptides treatment, Sema3A treatment, Sema3A + peptides treatment or untreated controls. Scale bar: 5µm. (H) Quantification of the CRMP4-DIC puncta number per axon in healthy or C9orf72 human-derived MN proximal axons after Sema3A treatment, Sema3A + peptides treatment or untreated controls. Data collected from 3 independent chambers in each condition. Total of 37 healthy untreated proximal axons, 61 healthy proximal axons with peptides treatment, 59 healthy proximal axons with Sema3A treatment and 52 healthy proximal axons with Sema3A + peptides treatment. 67 C9orf72 untreated proximal axons, 63 C9orf72 proximal axons with peptides treatment, 41 C9orf72 proximal axons with Sema3A treatment and 50 C9orf72 proximal axons with Sema3A + peptides treatment monitored. Data presented as mean ±SE. One-way ANOVA, Tukey's multiple comparisons test, n=3, ****p<0.0001.
Figure 6(A) Representative images of CTX signal in healthy and C9orf72 human IPSC-derived MNs before and after Sema3A application. Green: denotes CTX-positive cells. Yellow circles are numbered CTX positive cells. Purple circles are cells that are missing post Sema3A treatment. Scale bar: 40 μm. (B-C) Quantification of CTX signal in healthy and C9orf72 IPSC-derived MNs before and 3 days after applying Sema3A to distal compartment, compared with untreated control. 3 independent chambers in each condition were analyzed. Average of ~150 neurons per condition monitored. Student's t-test, n=3, Data presented as mean ±SE, *p<0.05. (D) Representative images of CTX signal in WT or SOD1G93A primary MNs before and 2 days after Sema3A application to the distal compartment in the presence of either Dynein inhibitor+Sema3A, Dynasore+Sema3A or untreated. Green: denotes CTX-positive cells. Yellow circles are numbered CTX positive cells. Purple circles are cells that are missing post Sema3A treatment. Scale bar: 30 μm. (E) Quantification of CTX signal in a SOD1G93A explant before and 2 days after Sema3A application to the distal compartment in the presence of either Dynein inhibitor+Sema3A, Dynasore+Sema3A. 3 independent chambers in each condition were analyzed. ~200 neurons were monitored per each condition. One-way ANOVA, Tukey's multiple comparisons test, n = 3, Data presented as mean ±SE, *p<0.05, **p<0.01. Dynein inhibitor and Dynasore treatments were used as a negative control.
Figure 7(A) Representative images and insets of P90 WT and SOD1G93A SC cross sections at P90. Blue: denotes DAPI, Red: denoted NeuN, and White: denotes activated caspase 3. Scale bar: 20 µm.(C) Representative images of P90 SOD1G93A mice SC cross sections that were injected with AAV9-GFP/AAV9-50aa-GFP. Blue: denotes DAPI, Red: denoted NeuN, and White: denotes activated caspase 3. Scale bar: 10 µm.
Figure 1Kaplan Meier plots representing the survival difference between SKCM patients stratified according to the 40% highest or lowest BNIP3 expression.Representative images of BNIP3 immunostaining in a benign nevus (n=5), primary melanoma and their paired metastasis (n=7). In the representative pictures, nevi or tumor tissue is circled in yellow and highlighted with a yellow asterisk (*). Histopathological scoring of BNIP3 protein levels in nevi and melanoma patients with paired primary and metastases harboring weak and in gradient (+/-), weak and homogeneous (+) or strong and homogeneous (++) BNIP3 protein levels based on histopathological staining intensity and distribution scoring. Scale bars represent 150µm.Representative images of BNIP3 immunohistochemistry in TMAs containing (n=158) melanoma metastases and related Kaplan Meier plot of survival difference comparing melanoma patients with different BNIP3 histopathological scores. Scale bars represent 50µm.
Figure 2Growth curves of B16-F10 tumors generated with cells transduced with either empty vector (CntlKD), shRNA against BNIP3 (BNIP3KD) or ATG5 (ATG5KD) (n=7 per condition) and analyzed with a two-way ANOVA with Tukey's multiple comparisons test.Haematoxylin and eosin staining of sections representative of CntlKD, BNIP3KD or ATG5KD B16-F10 tumors. Necrotic areas are highlighted with an asterisk (*). Quantification of TUNEL+ tumor area (representing the fraction of dying/dead cell population) of CntlKD, BNIP3KD or ATG5KD B16-F10 tumors (n= 5 per condition). Quantification of the Ki67+ cells within the tumor area (representing the fraction of proliferative cell populations in non-necrotic areas) of CntlKD (n=7), BNIP3KD (n=6) and ATG5KD (n=8) B16-F10 tumors.Immunofluorescent staining for Ki67 (red) representative of CntlKD, BNIP3KD or ATG5KD B16-F10 tumor sections. Immunofluorescent staining for LC3B (orange granularity) representative of CntlKD, BNIP3KD or ATG5KD B16-F10 tumor sections. A zoomed-in area is shown in the lower-left corner.
Figure 3Confocal microscopy images of co-localization analysis of the BNIP3-proficient (shCntl, n=27 cells) or deficient (shBNIP3, n=31 cells) B16-F10 cells transiently transfected with GFP-LC3 and immunostained for TOMM20 (mitochondrial marker). Colocalization events of GFP-LC3 puncta (green) with TOMM20 (red) are highlighted with white arrows. The percentage of GFP-LC3 puncta colocalizing with TOMM20 was calculated in n cells from 3 biologically independent experiments and analyzed with Mann-Whitney's non-parametric test. Scale bars represent 10µm.Glut1, Hk2, Pkm1, Pkm2, Pdk1, Mct1, Ldha, Ldhb and Mct4 transcript levels measured by qPCR from the corresponding B16-F10 cell lysates. Transcript expression is represented as the fold-change relative to their corresponding shCntl value. Heatmap shows the average values in each condition (n=4) and they were analyzed with a one-sample T-test only against shCntl, except for Glut1 shATG5 that was analyzed with a Wilcoxon rank test.
Figure 4Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA and ACTIN protein levels from lysates of B16-F10 cells (n=4) or B16-F10 tumors [CntlKD (n=5), BNIP3KD (n=5) and ATG5KD (n=7)]). The HIF-1α-OH /HIF-1α ratio is shown in a bar graph below. Densitometric quantifications relative to ACTIN levels are shown below each corresponding band. Analysis was performed as indicated in the Data Information section, unless for HIF-1α/Actin ratio (in vivo), which was analyzed with Kruskal-Wallis non-parametric test with Dunn's multiple comparisons test, and LDHA/Actin ratio for shATG5 condition (in vitro), which was analyzed with Wilcoxon's rank test.Immunohistochemical staining for HIF-1α (brown) representative from CntlKD, BNIP3KD and ATG5KD, B16-F10 tumor tissues. Higher magnification of the highlighted (with a white square) stained sections is shown below.
Figure 5Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA, PHD2 and ACTIN protein levels from normoxic lysates of B16-F10 cells transfected in presence of non-targeting siRNA sequences (Scr) or siRNA against PHD2.
Figure 6Relative intracellular Fe2+ levels in normoxic B16-F10 cells (n=4). Representative pictures of FerroOrange intensity are shown on the left and analyzed using a RM one-way ANOVA (Geisser-Greenhouse correction) with Holm-Sidak's multiple comparisons test. Scale bars represent 50µm.Immunoblot detection of Hydroxylated HIF-1α (HIF-1α-OH), total HIF-1α, LDHA, NCOA4 and ACTIN levels from normoxic lysates of B16-F10 cells transfected in presence of non-targeting siRNA sequences (Scr) or siRNA against NCOA4 (n=4).
Figure 7Growth curves of B16-F10 tumors generated by shCntl or shBNIP3 cells expressing Luciferase (Luc) or an undegradable HIF-1α (HIF-1α-AA) construct (n=7 for CntlKD Luc/BNIP3KD HIF-1α-AA, n=8 CntlKD HIF-1α-AA and n=5 for BNIP3KD Luc) and analyzed with a two-way ANOVA with Tukey's multiple comparisons test. Representative double immunostaining for CD31 and αSMA in B16-F10 tumors (n=5 per condition). Scale bars represent 50µm.
Figure 1B Observed dsDNA processing patterns of Sgs1, including an overview over consecutive bursts and pauses (upper panel) as well as detailed views into individual bursts containing multiple unwinding events (lower panels). A typical unwinding event of Sgs1 starts with slow, gradual unwinding of the dsDNA followed by DNA rehybridization, that can be almost instant (76% of events, see blue sections in lower right panel) or contain slow rewinding sections (24% of events, see orange sections in lower right panel).C Histograms of the observed unwinding and rewinding velocities for Sgs1. The mean unwinding rate was 65 ± 2 bp/s (N = 899). The mean rewinding rate was 115 ± 6 bp/s (N = 287). We attribute the large rewinding rate in the absence of RPA to an increased error in inferring the rewinding rate from the short rewinding sections.
Figure 2B Observed dsDNA processing pattern of Sgs1 in the presence of RPA including an overview over successive bursts and pauses (upper panel) and detailed views of single bursts (lower panels). Unwinding events are always followed by slow rewinding events (orange sections).C Histograms of the observed unwinding and rewinding velocities for Sgs1 in the presence of RPA (brown bars). The mean unwinding rate was 51 ± 3 bp/s (N = 2139). The mean rewinding rate was 66 ± 3 bp/s (N = 2091). White bars show for comparison the distributions of unwinding and rewinding velocities for Sgs1 aloneD Force-dependence of the Sgs1 unwinding (black squares) (N = 2139) and rewinding rates (black diamonds) (N = 2091) in the presence of RPA (errors given as standard deviations). Red circles represent the force-dependent DNA opening and closure rates measured for RPA alone
Figure 3B Typical dsDNA processing event. The dashed red line connects the minima at the end of the rewinding sections The inset shows a DNA unwinding event in the presence of nuclease-dead Dna2 E675A and RPA.C Histograms of the observed unwinding and rewinding rates (green bars) with mean values of 69 ± 3 bp/s (N = 365) and 74 ± 5 bp/s (N = 282), respectively. White bars show for comparison the unwinding and rewinding velocities of Sgs1 in the presence of RPA
Figure 4B dsDNA processing events observed for Sgs1 in the presence of Top3-Rmi1. Typical events consist of periods with slow gradual unwinding typically followed by instant DNA rezipping as seen also for Sgs1 alone.C Histogram of the unwinding rate of Sgs1 in the presence of Top3-Rmi1 (violet bars) with a mean rate of 86 ± 3 bp/s (N = 1101). For comparison, the distribution of unwinding rates for Sgs1 alone is depicted with white barsE dsDNA processing by Sgs1 in the presence of Top3-Rmi1 and RPA. In contrast to the absence of RPA, DNA closure is seen as a slow rewinding.F Histograms of unwinding and rewinding rates of Sgs1 in the presence of Top3-Rmi1 and RPA (purple bars). The mean rates for unwinding and rewinding are 66 ± 2 bp/s (N = 502) and 70 ± 4 bp/s (N = 369), respectively. For comparison, the velocity distributions for Sgs1 in the presence of RPA are shown as white bars
Figure 5A Mean duration of bursts (violet bars) (N > 40) and pauses (green bars) (N > 40) for the different enzyme combinations. Error bars represent standard error of the mean.B Mean duration of a unidirectional unwinding event (N > 350). Error bars represent standard error of the mean.C Mean processivity of unidirectional DNA unwinding (N > 350). Error bars represent standard error of the mean.
Figure 1C: Time course of PIKfyve downregulation in brains of prion-infected mice (3 mice/time point). PIKfyve (but not FIG4, calnexin, NeuN and synaptophysin) was suppressed between 60 and 120 dpi. T: terminal disease stage.E: Prion-infected Gt1 cells (75 dpi) stained with Alexa-Fluor647-succinimidyl ester which stains vacuoles negatively (arrows).G: tga20 COCS were treated with pooled IgG or POM1 (72 hours). POM1 suppressed PIKfyve but not FIG4 and VAC14. Right: Quantification of immunoblots. Each dot represents an independent experiment. **: p<0.01; ***: p<0.001; unpaired t-test Error bars represent s.e.m.H: Immunoblots of human CJD brains (red: sampled region). Gt1Sc: prion-infected Gt1 cells. Loading controls: actin and calnexin (Cnx)
Figure 2C: Treatment of COCS with POM1 reduces the levels of PIKfyve starting 48h post treatment. Each dot represents an individual experiment. Statistics: Unpaired t-test. **: p<0.01.G: Left: partially restored PIKfyve levels in prion-infected tga20 mice treated with LIN5044 (LCP) or vehicle. Right: quantification of the western blot. Each dot represents an individual biological replicate. *: p0.05; unpaired t-test Error bars represent s.e.m.H: Left, tga20 COCS were inoculated with RML or NBH, optionally treated with GSK2606414 starting at 21 dpi, and lysed at 35 dpi. Depletion of PIKfyve in prion-infected samples was largely rescued by GSK2606414 (GSK). Right, quantification of western blot. Each dot represents an individual biological replicate. Statistics: Unpaired t-test. **: p<0.01; unpaired t-test; Error bars represent s.e.m.
Figure 3E, F: Acyl-rac of Gt1 cells subjected to siRNA against zDHHC9, zDHHC21 or both (72 hours post transfection), showing synergistic decrease of PIKfyve acylation by suppression of zDHHC9 and 21 (quantified in F). Each dot represents a separate experiment (unpaired t-test). **: p<0.01. Error bars represent s.e.m.H-I: Flag-tagged zDHHC9 or zDHHC21 were expressed in Gt1 cells were transfected with plasmids encoding. After 72 hours, zDHHC9/21 (magenta) were localized to the Golgi apparatus (GM130+, cyan) in NBH-exposed cells but not in prion-infected cells. n=3 individual biological replicates.
Figure 4A: Upper row: prion-infected Gt1 cells (75 dpi) immunostained for LAMP1 showing prominent vacuoles. Control: NBH-inoculated cells. Middle row: Gt1 cells transfected with shRNA targeting PIKfyve or luciferase (control) for 5 days and immunostained for LAMP1. Depletion of PIKfyve resulted in LAMP1+ vacuoles (yellow). DAPI: nuclear stain. Lower row: prion-infected and NBH treated cells (75 dpi) contained LAMP2- vacuoles. n=3 individual biological replicates.D: tga20 COCS treated with POM1 or control IgG (10 days) and immunostained with NeuN and LAMP1. Nuclei: DAPI. POM1-treated COCS showed increased LAMP1 expression (quantification: Appendix Fig. S4B). NeuN+ cells exhibited vacuoles (arrowheads).G: Lysosomal enzymes, but not β-actin, were elevated in brains of terminally scrapie-sick mice. Panels show independent triplicates. Statistics: ANOVA.. **: p<0.01. Error bars represent s.e.m.
Figure 5A: Time course of TFEB phosphorylation in prion-infected C57BL/6 mice. Total TFEB was unchanged but reduced TFEB phosphorylation was evident at ≥90 days. Quantification: Each dot represents an individual biological replicate. Statistics: Unpaired t-test. *: p0.05; **: p<0.01. Error bars represent s.e.m.B: TFEB-controlled transcripts in brains of terminally scrapie-sick C57BL/6 mice were measured by qPCR. Prion infection led to upregulation of these genes, but not of STAT3. Control: NBH-inoculated mice. ANOVA on independent triplicates. ***: p<0.001. Error bars represent s.e.m.E: Gt1 cells (as in D) were fixed and stained with anti-TFEB antibody. Prion-infected cells showed nuclear translocation of TFEB. DAPI: Blue. Quantification: Each dot represents an individual biological replicate (30 images were quantified per replicate). Statistics: Unpaired t-test. **: p<0.01. Error bars represent s.e.m.F: TFEB responsive genes were upregulated in Gt1 cells chronically infected with RML prions (75 dpi). Panels depict independent triplicates. Statistics: ANOVA. *: p0.05. Error bars represent s.e.m. G: TFEB responsive genes were upregulated in cells transfected with shPIKfyve for 5 days compared to shCtrl (shRNA against luciferase). Panels depict independent triplicates. Statistics: ANOVA. *: p0.05. Error bars represent s.e.m.
Figure 6C: Prion-infected Gt1 cells were lentivirally transduced with GADD34 at 75 dpi. Control: empty lentiviral vector. At 79 dpi, GADD34 overexpression had normalized eIF2α phosphorylation and restored PIKfyve levels. Right: Quantification of western blots (n=3; unpaired t-test). Each dot represents an individual experiment. **: p<0.01. Error bars represent s.e.m.E: tga20 COCS were infected with RML and optionally treated with bPIP (5µg/ml). At 45 dpi, NeuN morphometry revealed ablation of cerebellar granule layer (CGN) in prion-infected slices and its rescue by bPIP. Control: NBH treated COCS. Each dot represents an individual slice (Statistics: ANOVA). *: p<0.05. **** p<0.0001.F: tga20 COCS were treated with POM1 (optionally pre-blocked with recPrP) and treated with bPIP (5µg/ml). At 14 dpi NeuN morphometry revealed POM1-induced ablation of cerebellar granule layer (CGL) and rescue by bPIP.
Figure 1(A) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (right panel) were transfected with either non‐targeting siRNA (control siRNA) or a pool of LC3 siRNAs (A, B, C), using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS medium in the absence or presence of 0.1 μM Baf A and subjected to western blot analysis after lysis with RIPA extraction buffer.(B) Quantification of relative p62 and GFP‐GATE‐16 level (upper panel) and degradation (lower panel) was performed as described in 'Materials and methods'. Mean±s.d. of three independent experiments is presented.(C) HeLa cells stably expressing GFP‐GATE‐16 were transfected with non‐targeting siRNA or a pool of LC3 siRNAs and treated as in (A). The cells were subjected to immunostaining with anti‐p62 after fixation, and analysed by confocal microscopy. The GFP intensity of three independent experiments was measured as described in 'Materials and methods' (right panel). Scale bar: 20 μm.
Figure 2(A) HeLa cells stably expressing GFP‐GATE‐16 (left panel) or GFP‐LC3B (right panel) were transfected with either non‐targeting siRNA (control siRNA) or a pool of GABARAP siRNAs (GABARAP, GABARAPL1, and GATE‐16) using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS medium in the absence or presence of 0.1 μM Baf A and subjected to western blot analysis after lysis with RIPA extraction buffer.(B) Quantification of relative p62 and GFP‐LC3B level (upper panel) and degradation (lower panel) were performed as described in 'Materials and methods'. Mean±s.d. of three independent experiments is presented.(C) HeLa cells stably expressing GFP‐LC3B were transfected with non‐targeting siRNA or a pool of GABARAP siRNAs and treated as in (A). The cells were subjected to immunostaining with anti‐p62 after fixation, and analysed by confocal microscopy. The GFP intensity of three independent experiments was measured as described in 'Materials and methods' (right panel). Scale bar: 20 μm.
Figure 3(A) Bulk protein degradation was tested in HeLa cells that were transfected with non‐targeting siRNA (control siRNA), a pool of GABARAP/GATE‐16 siRNAs, or a pool of LC3 siRNAs. The rate of long‐lived protein degradation was measured in cells incubated in either αMEM (control) or EBSS (starvation) medium, in the presence or absence of 10 mM of 3‐MA, 72 h after siRNA transfection. Values expressing the protein degradation percentage are represented as the mean±s.d. of three determinations.(B) HeLa cells stably expressing GFP‐LC3B (left panel) or GFP‐GATE‐16 (right panel) were transfected with a pool of GABARAP/GATE‐16 siRNAs or a pool of LC3, respectively, and with non‐targeting siRNA. Seventy‐two hours after transfection, the cells were collected after 5 h of starvation and the relative level of GFP‐LC3B or GFP‐GATE‐16 was measured using flow cytometry. Values represent the mean±s.d. of three experiments.