EMBO/soda-nel huggingface dataset:
Paracaspase MALT1 regulates glioma cell survival by controlling endo-lysosome homeostasis (2019)
Figure 1

Figure 1. MALT1 Expression sustains Glioblastoma Cell Growth

A. STRING diagram representation of the network of proteins involved in NF-κB pathway.

B. The Cancer Genome Atlas (TCGA RNAseq dataset) was used on the GlioVis platform (Bowman et al, 2007) to analyze the probability of survival (log-rank p-value) of 155 GBM patients, for each gene encoding for the mediators of the NF-κB pathway.

C. Kaplan-Meier curve of the probability of survival for 155 GBM patients with low or high MALT1 RNA level, using median cut-off, based on the TCGA RNAseq dataset.

D-E. Box and whisker plot of MALT1 mRNA expression in low-grade glioma (LGG, grades II and III) or in GBM (grade IV) (TCGA GBMLGG, RNAseq dataset) (D). Horizontal line marks the median, box limits are the upper and lower quartiles, and error bars show the highest and lowest values. Alternatively, MALT1 mRNA expression was plotted in non-tumor samples versus GBM samples (TCGA RNAseq dataset) (E). Each dot represents one clinical sample.

F. Fraction of surviving cells over time in GSC#1 and GSC#9, transduced with control (shc) or bi-cistronic GFP plasmids using two different short hairpin RNA (shMALT1 sequences, seq #1 and #2). Data are plotted as the percentage of GFP-positive cells at the day of the analysis (Dx), normalized to the starting point (Day 4 post-infection, D4).

G. EdU incorporation (green, 2 hours) was visualized by confocal imagery in GSC#1 or by FACS in GSC#9 transfected with sic or siMALT1. In GSC#1 the percentage of EdU-positive cells was quantified. Nuclei (DAPI) are shown in blue. n> 240 cells per replicate. Scale bar: 10 μm. Data are presented as the mean + s.e.m. on 3 independent experiments.

H. FACS analysis of propidium iodide (PI) staining in GSC #1 and #9 transfected with non-silencing duplexes (sic) or MALT1 siRNA duplexes (siMALT1) and analyzed 72 hours later.

I. Linear regression plot of in vitro limiting dilution assay (LDA) for control (shc) or shMALT1 seq#1 and seq#2 transduced GSC#9. Data are representative of n=2. Knockdown efficiency was verified at day 3 by western-blot using anti-MALT1 antibodies. GAPDH served as a loading control.

J. Tumorspheres per field of view (fov) were manually counted in sic or siMALT1 transfected GSC#1, #4 and #9. Data are presented as the mean + s.e.m. on 3 independent experiments.

Data information: All data are representative of n=3, unless specified. Statistics were performed using pairwise comparisons (Tukey's Honest Significant Difference (HSD) with a 95% confidence interval for panels C-E), and a two-tailed t-test with a 95% confidence interval for panels (G and J), *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Source data are available online for this figure.
Paracaspase MALT1 regulates glioma cell survival by controlling endo-lysosome homeostasis (2019)
Figure 4

Figure 4. MALT1 Pharmacological Inhibition alters Endo-Lysosome Homeostasis

A. Transmission electron microscopy of GSC#9 treated with vehicle (DMSO) or MPZ (20 μM) for 16 hours. ER: endoplasmic reticulum; MVB: multivesicular bodies; lys: lysosome; mit: mitochondria; nuc: nucleus. Red stars denote lysosomes, blue stars vacuoles.

B. Confocal analysis of LAMP2 staining (red) at 0, 1, 2, 4, and 6 hours post-MPZ (20 μM) treatment. Nuclei (DAPI) are shown in blue. Scale bar: 10 μm.

C. Western-blot analysis was performed in total protein lysates from GSC#9 transfected with non-silencing duplexes (sic) or MALT1 targeting siRNA duplexes (siMALT1). Alternatively, western-blot analysis of LAMP2, CTSD, and MALT1 was done in total protein lysates from GSC#9 treated for 16 hours with MPZ (20 μM) or Z-VRPR-FMK (75 µM). DMSO was used as vehicle. GAPDH served as a loading control.

D. Confocal analysis of LAMP2 staining (red) in GSCs #1, #4, #12 treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Alternatively, GSC#9 were either treated for 16 hours with H2O or Z-VRPR-FMK (75 µM). Additionally, cells were transfected with non-silecing duplexes (sic) or MALT1 and BCL10 targeting siRNA duplexes (siMALT1 and siBCL10). Alternatively, lysotracker staining (red) was used to track for lysosomes in either GSC#9 expressing either wild-type (WT) or C464A FLAG-MALT1 (green). Scale bar: 10 μm.

E. Quantification of LAMP2 staining pixel intensity on GSC#9 treated as described in panel (D). Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>30.

F. Cryosections from GSC#9-xenografted tumors in vehicle and MPZ-challenged animals (as described in Fig 2J) and assessed for LAMP2 staining (green). Nuclei (DAPI) are shown in blue. Scale bar: 10 μm. Western-blot analysis of LAMP2 was performed in tumor lysates. GAPDH served as a loading control.

G. Confocal analysis of lysotracker staining (red) in GSC#9 treated for 16 hours with vehicle (DMSO) or MPZ (20 μM). Alternatively, GSC#9 were either treated for 16 hours with H2O or Z-VRPR-FMK (75 µM) (upper panel) or transfected with sic and siMALT1 (bottom panel). As indicated, number of lysotracker-positive puncta and lysotracker pixel intensity (arbitrary unit, AU) were quantified per cell. Data are presented as the mean + s.e.m. on 3 independent experiments. Each dot represents one cell. n>30. Nuclei (DAPI) are shown in blue. Scale bars: 10 μm.

Data information: All data are representative of n=3, unless specified. Statistics were performed using a two-tailed t-test with a 95% confidence interval. ***p<0.001.

Source data are available online for this figure.
Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1 (2021)
Figure 1

Figure 1: OFD1 interacts with both myosin VI isoforms.

(A) List of myosin VI interactors commonly found in the four cell lines tested. See Dataset EV1 for the full list of interactors.

(B) Total lysates from HEK293T transfected with Flag-OFD1 were IP with anti-myosin VI antibodies (1295 and 1296) and an unrelated rabbit antibody (anti-GST, as control). IB was performed with anti-Flag and anti-myosin VI antibodies.

(C) Total lysates from HEK293T transfected with Flag-OFD1 or Flag (as control) were IP with anti-Flag antibody-conjugated beads. IB was performed with anti-Flag and anti-myosin VI antibodies.

(D) Selected sections deriving from TEM analysis of A549 wild-type versus myosin VI KO cells. The cells were immunogold-labelled with anti-myosin VI antibody. The centrioles in the images are indicated with white dashed circles. Scale bar, 200 nm. Top: representation of the estimated localisation of myosin VI at the centrioles.

(E) GST pulldown assay using the indicated myosin VI constructs or GST alone (as control) and lysates from HEK293T cells transfected with Flag-OFD1 construct. IB was performed with anti-Flag antibody. Ponceau staining as indicated.

(F) GST pulldown assay using myosin VI tail (835-1295) construct (or GST alone as control) and lysates from HEK293T cells transfected with the indicated Flag-OFD1 constructs (or Flag alone, as control). IB was performed with anti-Flag antibody. Ponceau staining as indicated.
Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1 (2021)
Figure 2

Figure 2: Myosin VI depletion leads to increased OFD1 and Cep164 recruitment at the centrioles.

(A) Total lysates from hTERT-RPE1 cells were IP with anti-myosin VI antibody and an anti-rabbit IgG antibody (as control). IB was performed with anti-OFD1 and anti-myosin VI antibodies.

(B) A scheme of the IF analysis performed to calculate the total intensity of OFD1 staining at the mother and daughter centrioles.

(C,D) IF analysis of centriole-associated OFD1 signal. hTERT-RPE1 cells were transfected with siRNA against myosin VI. Four days after transfection, cells were treated with nocodazole (1 hour, 6 μg/ml) and immunostained with anti-OFD1, anti-centrin1 and anti-Cep164 antibodies. Mother centrioles were identified by the coincident staining of centrin1 and Cep164, while daughter centrioles were centrin1-only stained. A scheme of the position of the markers used is depicted above. (C) Representative images, scale bar, 1 μm. (D) Quantification of OFD1 intensity at the mother or daughter centrioles. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ± SD. Mother centrioles: Mock, n=148 cells; MyoVI KD, n=199 cells, from four independent experiments. Daughter centrioles: Mock, n=101 cells; MyoVI KD, n=148 cells, from three independent experiments. **** P<0,0001 by Mann-Whitney test.

(E) IB analysis of hTERT-RPE cells treated with the indicated siRNAs with anti-myosin VI and anti-OFD1 antibodies. Anti-GAPDH was used as loading control.

(F-G) IF analysis of Cep164 signal. hTERT-RPE1 cells were transfected with siRNA against myosin VI and/or OFD1. Four days after transfection, cells were treated with nocodazole (1 hour, 6 μg/ml) and immunostained with anti-OFD1, anti-centrin1 and anti-Cep164 antibodies. (F) Representative images. Scale bar, 1 μm. (G) Quantification of Cep164 intensity at the mother centrioles. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=147 cells; OFD1 KD, n=146 cells; MyoVI KD, n=150 cells; MyoVI + OFD1 KD, n=151 cells, from four independent experiments. ** P<0,005; **** P<0,0001 by Kruskal-Wallis test.
Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1 (2021)
Figure 3

Figure 3: Myosin VI depletion leads to p53 activation and cell cycle arrest.

(A) Growth curve of hTERT-RPE1 cells transfected with myosin VI siRNA. A representative plot of three independent experiments is shown.

(B) Growth curve of BJ-hTERT cells transfected with myosin VI siRNA. A representative plot of two independent experiments is shown.

(C) Analysis of DNA content in hTERT-RPE1 cells stably expressing a myosin VI shRNA. After 10 days of doxycycline induction, the cells were stained with Propidium Iodide (PI) and analysed by FACS. Ctrl = control cells, not induced.

(D) Senescence-associated β-gal assay (SA-β-gal) of cells treated as in C. Scale bar, 100 μm.

(E) IB analysis of hTERT-RPE1 cells transfected with myosin VI siRNA, with anti-myosin VI, anti-p53 and anti-p21 antibodies. Anti-GAPDH was used as loading control.

(F) Growth curve of hTERT-RPE1 cells transfected with the indicated p53 and myosin VI siRNAs. A representative plot of three independent experiments is shown.

(G) Representative bright field images of cells treated with the indicated p53 and myosin VI siRNAs. Scale bar, 200 μm.

(H) IB analysis with anti-myosin VI, anti-p53 and anti-p21 antibodies of hTERT-RPE cells treated with Nutlin-3, or not treated as control. Anti-GAPDH was used as loading control.

(I) Quantification of OFD1 intensity at the mother or daughter centrioles. hTERT-RPE1 cells treated or not with Nutlin-3, were incubated with nocodazole for 1 hour (6 μg/ml) and immunostained with anti-OFD1, anti-centrin1 and anti-Cep164 antibodies. Mother centrioles were identified by the coincident staining of centrin1 and Cep164, while daughter centrioles were centrin1-only stained. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mother centrioles: Mock, n=148 cells; Nutlin-3, n=169 cells, from four independent experiments. Daughter centrioles: Mock, n=101 cells; Nutlin-3, n=122 cells, from three independent experiments. ns, not significant by Mann-Whitney test.

(J) Quantification of Cep164 intensity at the mother centrioles in hTERT-RPE1 cells treated as in (I). Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=195 cells; Nutlin-3, n=196 cells, from four independent experiments. ns, not significant by Mann-Whitney test.
Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1 (2021)
Figure 4

Figure 4: Lack of myosin VI alters the mobility and localisation of OFD1 at the centrioles.

(A) Super-resolution analysis of OFD1 localisation at the centrioles. hTERT-RPE1 cells were transfected with siRNA against myosin VI, immunostained with anti-OFD1 and anti-acetylated tubulin antibodies and visualised using structured illumination microscopy (SIM). Representative images are shown, scale bar, 500 nm. A scheme of the estimated localisation of OFD1 in the two conditions is depicted on the right side.

(B) Two-colour SIM images in control and myosin VI-depleted cells illustrating the localisation and distribution of the distal appendage markers Cep164 and ODF2 at the centrioles, stained with anti-acetylated tubulin antibody. Representative images are shown; scale bar, 500 nm.

(C) dSTORM super-resolution analysis of the distal appendage markers FBF1 and ODF2 in control and myosin VI-depleted cells. To emphasise the symmetry of the structures, the signals from all nine appendages were averaged (bottom). Representative images are shown; scale bar, 200 nm.

(D-F) FRAP analysis of centriole-associated GFP-OFD1. hTERT-RPE cells stably expressing GFP-OFD1 and centrin1-dTomato were transfected with siRNA against myosin VI. After four days, cells were treated with nocodazole (1 hour, 6 μg/ml) and subjected to live-cell imaging. (D) Left: a scheme of the localisation of GFP-OFD1 and the centriole marker centrin1-dTomato. Right: a scheme of photobleaching and recovery of GFP-OFD1 at the centrioles. (E) A representative graph of one out of three experiments. For each time point, the fraction of recovery of GFP-OFD1 is shown. Results are expressed as means with 95% confidence interval. n=12 cells (Mock), n=13 cells (MyoVI KD). (F) Quantification of the half-time of fluorescence recovery (t1/2,) and of the mobile fraction of GFP-OFD1. Results are expressed as mean ±SD. n=41 cells (Mock), n=31 cells (MyoVI KD), from three independent experiments. ns, not significant; *** P<0,0005 by Unpaired T-test.
Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1 (2021)
Figure 5

Figure 5: Myosin VI depletion affects the centriolar satellites via p53.

(A) A scheme of the IF analysis performed to calculate the total intensity of satellite staining that surrounds the centrioles. The centriole marker Cep135 or Cep164 are used to define the centre of a 3 μm circle, in which the intensity of the satellite marker PCM1 was calculated.

(B) IF analysis of PCM1 signal. hTERT-RPE1 cells were transfected with siRNA against myosin VI and immunostained with anti-PCM1 and anti-Cep135 antibodies. Upper panel, representative images, scale bar, 2 μm. Lower panel, quantification of PCM1 intensity. Results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock, n=96 cell; MyoVI KD, n=98cells, from two independent experiments. **** P<0,0001 by Mann-Whitney test.

(C) IF analysis of PCM1 signal. hTERT-RPE1 cells were treated with Nutlin-3 for 24 hours and immunostained with anti-PCM1 and anti-Cep135 antibodies. Panels as in B. Mock, n=96 cells; Nutlin-3, n=100 cells, from two independent experiments. **** P<0,0001 by Mann-Whitney test.

(D) IF analysis of PCM1 signal. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI and immunostained with anti-PCM1 and anti-Cep164 antibodies. Panels as in B. Quantification of PCM1 intensity refers to a 3 μm circle around the mother centriole, identified with anti-Cep164 staining. Mock, n=128 cells; MyoVI KD, n=114 cells from three independent experiments. ns, not significant by Mann-Whitney test.
Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1 (2021)
Figure 6

Figure 6: Myosin VI contributes to OFD1 removal from the centriolar satellites required for ciliogenesis.

(A) A scheme of the IF analysis performed to calculate the total intensity of OFD1 staining at the centriolar satellites.

(B) IF analysis of OFD1 signal at the centriolar satellites upon serum starvation. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI. After four days, cells were fixed (growing) or serum starved for 24 hours (SS). Cells were immunostained with anti-OFD1 and anti-PCM1 antibodies. The intensity of OFD1 signal in the area covered by PCM1 was quantified and normalised against the intensity of PCM1 staining in the same area. Left, representative images, scale bar, 2 μm. Right, results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. Mock_growing, n=150 cells; Mock_SS, n=149 cells; MyoVI KD_growing, n=149 cells; MyoVI KD_SS, n=150 cells, from three independent experiments. ns, not significant; * P<0,05; **** P<0,0001 by Kruskal-Wallis test.

(C) IB analysis of OFD1 after serum starvation in control and myosin VI-depleted cells. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI. After four days, cells were serum starved for 24 hours (SS). Lysates were analysed by IB with anti-OFD1, anti-myosin VI, anti-p62, and anti-LC3 antibodies. Anti-GAPDH was used as loading control. The amount of OFD1 protein was normalised against GAPDH signal and is expressed as percentage of OFD1 levels in cells grown in serum-starved conditions compared to cells grown in media containing serum. Bars represent mean ±SD. n=5 independent experiments. * P<0,05 by Kruskal-Wallis test.

(D) IF analysis of primary cilium upon serum starvation. hTERT-RPE1 p53 KO cells were transfected with siRNA against myosin VI. After four days, cells were fixed (growing) or serum starved for 24 hours (SS). Cells were immunostained with anti-acetylated tubulin (to identify the cilia), anti-Cep135 (to identify the centrioles) and DAPI. Left, representative images, scale bar, 2 μm. Right, results are expressed as fold change with respect to mock average intensity. Bars represent mean ±SD. n=3 independent experiments. 100-200 cells/condition were counted for each experiment. ***P < 0.001 by two-way ANOVA test.

EXPANDED VIEW FIGURE LEGENDS
Recruitment of the major vault protein by InlK: a Listeria monocytogenes strategy to avoid autophagy. (2011)
Figure 1

InlK is a virulence factor of <italic>L.</italic> monocytogenes.

A. The inlK gene locus in L. monocytogenes compared with the same genomic region in the related non-pathogenic species L. innocua. The stem and circle represent transcription terminators. B. Kaplan-Meier curve represents the survival of BALB/c mice over time. Four BALB/c mice in each experimental group were infected i.v with 104 L. monocytogenes wild-type (EGD-e) or ΔinlK mutant. C. The L. monocytogenes EGD-e wild-type strain (WT), the ΔinlK mutant (ΔinlK) and the complemented strain (ΔinlK+pPL2 inlK) 104 CFU were inoculated i.v into BALB/c mice. Animals were euthanized 24 h, 48 h, 72 h or 96 h after infection and organs were recovered, homogenized, and homogenates serially plated on BHI. The number of bacteria able to colonize liver (left panel) and spleen (right panel) is expressed as log10 CFU. Four animals per bacterial strain, per time points and per experiment were used. Statistical analyses were performed on the results of 3 independent experiments using the Student t test. P values of <0.05 were considered statistically different and are labeled here as *.
Recruitment of the major vault protein by InlK: a Listeria monocytogenes strategy to avoid autophagy. (2011)
Figure 2

InlK is expressed <italic>in vivo</italic>.

A. InlK amino acid sequence. The signal sequence is underlined and the different regions of leucine rich repeats (LRRs) are outlined. The consensus pentapeptide LPXTG at the C-terminal end is boxed. B. Detection by immunofluorescence microscopy of InlK over-expressing in L. monocytogenes EGD-e (WT), ΔinlK, WT+pADc-inlK, ΔinlK+pPRT-inlK and the ΔsrtA mutant over-expressing inlK (ΔsrtA+pPRT-inlK) grown in BHI medium using the rabbit polyclonal anti-InlK antibody. InlK was detected at the surface of InlK over-expressing bacteria (WT+pADc-inlK and ΔinlK+pPRT-inlK), whereas it was undetectable at the surface WT bacteria or at the surface of the ΔstrA mutant over-expressing inlK. C. Detection of InlK by Western blot on total lysates of L. monocytogenes EGD-e (WT), ΔinlK and ΔinlK+pPRT-inlK grown in BHI using the rabbit polyclonal anti-InlK antibody. Decreased concentrations of recombinant purified InlK were used as a positive control. D. Detection of secreted InlK in the supernatant of ΔsrtA mutants over-expressing InlK. Western blotting was carried out on trichloroacetic acid precipitates of ΔsrtA and ΔsrtA+pPRT-inlK culture (OD600 = 1) supernatants using the rabbit polyclonal anti-InlK antibody. E. Detection of purified recombinant InlK protein with rabbit polyclonal anti-live Listeria antibody, rabbit polyclonal anti-killed Listeria antibody, rabbit polyclonal anti-InlK and a rabbit pre-immune serum. InlK was detected only with the rabbit polyclonal anti-live Listeria antibody indicating that it is expressed during the in vivo infectious process. BSA was used as control protein. Two different amounts of proteins were tested (500 ng and 200 ng) to access signal specificity.
Recruitment of the major vault protein by InlK: a Listeria monocytogenes strategy to avoid autophagy. (2011)
Figure 3

InlK interacts with the Major Vault Protein.

A. Bacterial pull-down of purified GST-MVP with the L. monocytogenes strains WT+pPRT-empty, ΔinlK+pPRT-empty, ΔinlK+pPRT-inlK and WT+pPRT-inlJ. GST-MVP bound to InlK over-expressing bacteria (ΔinlK+pPRT-inlK) but not to other bacteria. GST-ScarA was used as control of the specificity of the MVP precipitation by InlK over-expressing bacteria. B. Bacterial pull-down of MVP-GFP from transfected HeLa cell lysates with the L. monocytogenes strains WT+pPRT-empty, ΔinlK+pPRT-empty, ΔinlK+pPRT-inlK and WT+pPRT-inlJ. MVP-GFP bound to InlK over-expressing bacteria (ΔinlK+pPRT-inlK) but not to other bacteria. C. Co-immunoprecipitation (Co-IP) of purified InlK (20 µg) with endogenous MVP of HT29 cells. The right panel shows anti-MVP co-IP performed on HT29 cell lysates. The left panel shows the control anti-MVP co-IP performed on lysis buffer. D. Detection of MVP recruitment at the surface of InlK over-expressing bacteria. HeLa cells were transfected with MVP-GFP (green), infected with L. monocytogenes EGD-e wild-type (WT), ΔinlK or ΔinlK+pPRT-inlK for 4 h, fixed for fluorescence light microscopy, and stained with anti-Listeria antibodies (red). Inset regions are magnified. The scale bar represents 1 µm.