EMBO/soda-nel huggingface dataset:
BCL-2-family protein tBID can act as a BAX-like effector of apoptosis (2021)
Figure 1

Figure 1. tBID induces MOMP and apoptosis in HCT AKO cells.

A) Quantification of the effect of individual BCL-2-family proteins on HCT AKO cell death, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to at least three independent experiments, n>30 cells per condition per experiment. *** p<0,001 and * p<0,05 with respect to GFP condition.

B) Representative confocal immunofluorescence and Bright Field (BF) images of an apoptotic HCT AKO cell expressing tBID-GFP (green), showing cyt c release (magenta), formation of pyknotic nuclei (cyan) and plasma membrane blebs. Scale bar 10 µm.

C) Western blot analysis of SMAC and cyt c release in GFP-BAX, tBID-GFP and GFP-BCLXL expressing cells at 12 h, 16 h and 24 h after transfection in HCT AKO. SN (supernatant, cytosolic fraction), P (pellet, membrane fraction) and PS (Ponceau Staining).

D) Quantification of caspase activity in HCT AKO cells expressing GFP-BAX, GFP-BAK, tBID-GFP, GFP-BCLXL and GFP at different time points after transfection. n=3 independent experiments. *** p<0,001, **p<0,025 and * p<0,05 with respect to GFP condition. Error bars represent S.D.

E) Representative western blot analysis of PARP1 cleavage upon tBID-GFP expression in HCT AKO cells. n=3 independent experiments.

F) Kinetics of the expression of tBID-GFP (blue) and PI intake upon tBID expression (purple) and unstransfected HCT116 AKO cells (in grey). Plots show the average fluorescence intensity (dark lines) and its standard deviation (shaded areas) from 3 technical replicates and 3 independent experiments. Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min.

G) Quantification of PI+ cells from total transfected cells with GFP-BAX, tBID-GFP, GFP-BCLXL and GFP. n=3 independent experiments with >10000 cells per condition per experiment. *** p<0,001 with respect to untransfected condition.

H) Cell death induced by tBID-GFP in HCT AKO cells in the absence and presence of ZVAD, NSA and Ferrostatin-1. Data correspond to three independent experiments, n>30 cells per condition per experiment. *** p<0,001 with respect to MOCK condition.

Data information: In A), G), and H), dots correspond to independent experiments, boxes represent 96% confidence interval, the average is represented by the line inside the box and whiskers correspond to S.D. The statistical significance was assessed by one-way ANOVA analysis of variance.
BCL-2-family protein tBID can act as a BAX-like effector of apoptosis (2021)
Figure 2

Figure 2. Endogenous BID induces apoptosis independently of BAX and BAK.

A) Representative confocal immunofluorescence and bright field (BF) images of HCT DKO cells expressing tBID-GFP (in green) treated or not with COMBO (ABT737 + S63586). Cyt c shown in magenta and nuclei in cyan. Scale bar 10 µm.

B) Effect of tBID-GFP and mCherry-BCLXL on cell death, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to at least three independent experiments, n> 30 cells per condition per experiment. *** p<0,001, ** p<0,025 and * p<0,05 with respect to tBID-GFP in HCT AKO condition.

C) Representative confocal immunofluorescence images of HCT DKO cells upon indicated treatments. Nuclei (cyan) and cyt c (magenta) are shown. Scale bar 10µm. Arrowheads represent cells showing released cytochrome c and pyknotic nuclei.

D) Representative WB showing the effect of TRAIL stimulation on endogenous BID cleavage in HCT DKO cells. n=3 independent experiments.

E) % of cell death induced by TRAIL (1µg/ml), COMBO and their combination at different treatment durations on HCT DKO measured as in panel B. Paired Student's t-test ** p<0,025 and * p<0,05 with respect to MOCK condition.

F) Effect of BID silencing on cell death upon TRAIL/COMBO treatments, measured as DRAQ7+ and normalized to siRNA control cell death. n=3 independent experiments. *** p<0,001 with respect to control condition.

G-H) Representative WB of BID levels after silencing at different time points (G) or when genetically knocked out (H). G, n=3 independent experiments.

I) Effect of TRAIL (1µg/ml), COMBO and their combination on HCT DKO and TKO cell death at different time points normalized to control cells. Each dot corresponds to the average of 3 technical replicates from 4 independent experiments. *** p<0,001.

J) Representative HyVolution - superresolution images of TFAM-RFP expressing U2OS BAX/BAK DKO cells in the in the absence (top) and presence of tBID-GFP (bottom). TFAM appears in red, TOM20/mitochondria in grey and tBID-GFP in green.

K) Quantification of cells with TFAM release on U2OS BAX/BAK DKO cells transfected with GFP-BAX/tBID-GFP and TFAM-RFP in the presence/ absence of COMBO. Dots correspond to individual experiments (n=3 individual experiments with more than 10 cells per condition per experiment). Unpaired Student's t-test *** p<0,001 with respect to non-treated conditions.

Data information: Unless otherwise stated, in B, E, F, G, I and K dots correspond to independent experiments, boxes represent 96% confidence interval, the average is represented by the line inside the box and whiskers correspond to S.D. and the statistical significance was assessed by one-way ANOVA analysis of variance. (T, TRAIL; C, COMBO).
BCL-2-family protein tBID can act as a BAX-like effector of apoptosis (2021)
Figure 3

Figure 3. tBID induces apoptosis independently of its BH3 domain, but requires an intact α-helix 6.

A) Structure of BID (PDB:2BID) depicting residues G94, D95, and M97-D98 localized at the BH3 domain in light orange, and K157/K158 at the α-helix 6 in blue.

B) Cell death induced by tBID-GFP and its variants on HCT AKO, measured as percentage of cells showing released cyt c or pyknotic nuclei. Data correspond to three independent experiments, with n> 30 cells per condition per experiment. *** p<0,001 and * p<0,05 with respect to tBID-GFP WT condition.

C) Structure of BID (PDB:2BID) depicting cancer-related residues localized at α-helix 6 in blue.

D) Effect of tBID wt and α6-mutants overexpression on HCT AKO cells, measured as DRAQ7+/GFP+ cells and normalized to tBID wt condition. n=3 independent experiments, ** p<0,025 and * p<0,05 with respect to tBID-GFP wt condition.

E) Effect of tBID wt and α6-mutants overexpression on MEF BID KO cells, measured as DRAQ7+/GFP+ cells and normalized to tBID wt condition. Effect of GFP overexpression on BID KO cells relative to tBID wt was assessed by pyknotic nuclei formation quantification. n=3 independent experiments *** p<0,001 with respect to tBID-GFP wt condition.

Data information: In B, D and E, dots correspond to independent experiments, boxes represent 96% confidence interval, the average is represented by the line inside the box and whiskers correspond to S.D. and t he statistical significance was assessed by one-way ANOVA analysis of variance.
BCL-2-family protein tBID can act as a BAX-like effector of apoptosis (2021)
Figure 4

Figure 4. tBID-induced mitochondrial fragmentation, MOMP and cristae remodelling does not require its oligomerization into supramolecular assemblies.

A) Representative CLEM images of mitochondria in HCT AKO cells (control) and HCT AKO cells expressing GFP-BAX or tBID-GFP. Black arrows indicate MOM disruptions.

B-D) Effect of GFP-BAX and tBID-GFP on HCT AKO mitochondrial structure. (B) Number of disruptions at the MOM per mitochondrial area, each dot represents an individual cell. (C) Mean area per mitochondrion at the condition tested, each dot represents an individual mitochondrion. (D) Analysis of cristae morphology, each dot represents an individual crista. Cristae shape calculated as length/width. Data represent n>50 mitochondria from 3-5 independent cells. In B-D), boxes represent 96% confidence interval, the average is represented by the line inside the box and whiskers correspond to S.D. The statistical significance was assessed by unpaired Student's t-test. *** p<0,001 and ** p<0,025 with respect to HCT AKO or untransfected condition.

E) Representative confocal and STED images of GFP-BAX and tBID-GFP expressed in HCT AKO. Scale bar 1 µm for BAX images, 5 µm for tBID images.

F) Zoomed-in structures of GFP-BAX supramolecular assemblies with the shape of lines (*double lines), arcs and rings in HCT AKO cells. Scale bar 100 nm.
BCL-2-family protein tBID can act as a BAX-like effector of apoptosis (2021)
Figure 5

Figure 5. BID-dependent release of SMAC during Shigella infection does not require BAX/BAK activation.

A) representative western blot of cytosolic cyt c and SMAC in HeLa cells.

B,C) Western Blot analysis of active BAX (B) and BAK (C), in HeLa cells treated with STS or infected with Shigella flexneri (MOI 30) at different time points after infection.

D) Representative Western Blot of cytosolic cyt c and SMAC in HeLa wt and HeLa BAX/BAK DKO cells upon staurosporine (STS) treatment and Shigella flexneri infection (Sf).

E) Representative Western Blot analysis of cytosolic and total (lysate) cyt c and SMAC in HCT wt, HCT DKO and HCT TKO cells infected with Shigella flexneri at the indicated time points after treatment/infection.
BCL-2-family protein tBID can act as a BAX-like effector of apoptosis (2021)
Figure 6

Figure 6. TRAIL-induced BID cleavage mediates apoptosis in Nalm6 venetoclax-resistant cells without activation of BAX and BAK.

A) Caspase-3/7 activity induced by ABT199 and etoposide on Nalm6 (venetoclax-sensitive) and Nalm6 199R (venetoclax-resistant) cells, normalized to untreated cells. Unpaired Student's t-test *** p<0,001 and ** p<0,025 (Nalm6 with respect to Nalm6 199R at different conditions). Dots correspond to technical replicates of n=3 independent experiments.

B) Caspase-3/7 activity in Nalm6 199R cells with or without 250 ng/ml TRAIL stimulation, normalized to untreated cells. Unpaired Student's t-test *** p<0,001. Dots correspond to technical replicates of n=3 independent experiments.

C) Representative western blot analysis of BID, BAX and BAK expression levels in Nalm6, Nalm6 199R, Nalm6 199R BID KO #clone1 and Nalm6 199R BID KO #clone5.

D) Representative kinetics of cell death, represented as DRAQ7+ cells in Nalm6 199R cells (grey), Nalm6 199R BID KO #1 cells (blue) and Nalm6 199R BID KO #5 cells (purple) upon 250 ng/ml TRAIL treatment. Plots show the average fluorescence intensity (dark lines) and its standard deviation (shaded areas) from 5 technical replicates. Fluorescence intensity was obtained by analysis the total fluorescence per well using incucyte system every 60 min.E) Effect of 24 h incubation with 250 ng/ml TRAIL on Nalm6 199R, Nalm6 199R BID KO #1 and Nalm6 199R BID KO #5, measured as DRAQ7+ cells and normalized to untreated Nalm6 199R cells. Each dot corresponds to the average normalized value of 3-5 technical replicates in n=3 independent experiments, *** p<0,001 with respect to untreated conditions.

F) Representative western blot of TRAIL-induced BID cleavage in Nalm6 199R cells at increasing concentrations of TRAIL (0, 25, 100 and 250 ng/ml) for 4 h.

G) Representative western blot analysis of BAK activation in Nalm6 and Nalm6 199R cells at different time points after treatment.

H) Cell death induced by GFP-BAK and GFP-BAK R127H on HCT AKO, measured as DRAQ7+/GFP+ cells. Unpaired Student's t-test. *** p<0,001 with respect to GFP BAK wt.

I) Effect of 24 h incubation with 250 ng/ml TRAIL on Nalm6 199R, Nalm6 199R BAK KO and Nalm6 199R BOK KO, measured as DRAQ7+ cells with respect of total cell surface and normalized to untreated conditions. Each dot corresponds to the average normalized value of 3-4 technical replicates in n=3 independent experiments.

Data information: Unless otherwise stated in A, B, E and H, dots correspond to independent experiments, boxes represent 96% confidence interval, the average is represented by the line inside the box and whiskers correspond to S.D and the statistical significance was assessed by one-way ANOVA analysis of variance.

Expanded View Figure Legends
O-GlcNAcylation of TDP-43 suppresses proteinopathies and promotes TDP-43's mRNA splicing activity (2021)
Figure 1

Figure 1. TDP-43 is O-GlcNAcylated by OGT in mammalian cells.

O-GlcNAcylation signals of TDP-43 were examined by immunoblotting with immunoprecipitated TDP-43 from SH-SY5Y cells. IgG immunoprecipitation served as a negative control.

The O-GlcNAcylated TDP-43 was immunoprecipitated by the RL2 antibody and examined by immunoblotting against a TDP-43 antibody.

HEK 293T cells were treated with the indicated compounds for 36 h, and the O-GlcNAc levels of TDP-43 were examined.

SH-SY5Y cells expressing Flag-TDP-43 constructs were treated with TMG, and the O-GlcNAc levels of TDP-43 were examined. Endogenous (endo.) and exogenous (exo.) TDP-43 were indicated.

Cells overexpressing Flag-TDP-43 were treated with increasing doses of UDP-GlcNAc, and the O-GlcNAcylation levels of TDP-43 were detected.

Quantification of (E). Mean ± SD, unpaired two-tailed t-test, n = 4 biological replicates.

Overexpression of WT OGT, but not catalytic-inactive OGT mutant, increased the O-GlcNAc levels of TDP-43. OGT protein levels were examined by immunoblotting.

Quantification of (G). Mean ± SD, unpaired two-tailed t-test, n = 3 biological replicates.

Overexpression of WT OGA, but not catalytic-inactive OGA mutant, abated the O-GlcNAc levels of TDP-43. OGA protein levels were examined by immunoblotting.

Quantification of (G). Mean ± SD, unpaired two-tailed t-test, n = 3 biological replicates.

Data information: The black arrows in different panels indicate O-GlcNAcylated TDP-43 bands; the asterisks in different panels indicate heavy-chain bands. ** P < 0.01, **** P < 10-4.
O-GlcNAcylation of TDP-43 suppresses proteinopathies and promotes TDP-43's mRNA splicing activity (2021)
Figure 2

Figure 2. OGT suppresses TDP-43 induced cellular toxicity and protein aggregation in budding yeast.

Yeast cells that express galactose-inducible GFP-tagged hTDP-43 only or co-expressing mCherry-tagged WT or catalytic-inactive mutant of OGT, were spotted onto plates containing glucose or galactose. Cell growth was assessed after 2 days.

Yeast cells expressing the indicated constructs were induced in galactose-containing medium, and representative images were shown using fluorescence microscopy to visualize TDP-43 aggregates. DIC: Differential interference contrast. Scale bar: 2 μm.

Expression levels of TDP-43 in strains of (B) were examined by immunoblotting.

TDP-43 aggregate foci numbers from individual cell of (B) were quantified from over 50 cells each of the indicated yeast strains. The foci numbers in individual cell > 3 were labelled with yellow, and the foci numbers in individual cell < 3 are labelled with blue. Mean ± SD, unpaired two-tailed t-test, n = 3 for each samples, biological replicates. ** P < 0.01.
O-GlcNAcylation of TDP-43 suppresses proteinopathies and promotes TDP-43's mRNA splicing activity (2021)
Figure 3

Figure 3. OGT suppresses TDP-43 associated proteinopathies in mammalian cells.

SH-SY5Y cells overexpressing Flag-TDP-43 were treated with GlcNAc, and the O-GlcNAcylated and phosphorylated (pTDP-43) levels of TDP-43 were examined by immunoblotting.

Relative O-GlcNAcylation levels (top) and phosphorylation levels (bottom) of TDP-43 shown in (A) were quantified. Mean ± SD, unpaired two-tailed t-test, n = 3 biological replicates.

SH-SY5Y cells overexpressing Flag-TDP-43 were treated with either 20 μM EA or 2 mM GlcNAc or a combination. The O-GlcNAcylated or phosphorylated levels of TDP-43 were examined by immunoblotting. The asterisks indicate heavy-chain bands.

SH-SY5Y cells treated with the indicated compounds were immunostained with TDP-43 or pTDP-43 and observed by immunofluorescence. Scale bar: 10 μm.

Quantification of signal intensities of pTDP-43 staining (left) or foci numbers per cell (right). Cell number counted: left panel, "Mock" n = 58, "GlcNAc" n = 35, "EA" n = 45, "GlcNAc+EA" n = 52; right panel, "Mock" n = 1167, "GlcNAc" n = 1051, "EA" n = 542, "GlcNAc+EA" n = 815. Mean ± SD, unpaired two-tailed t-test.

SDS-soluble and insoluble fractions were isolated from SH-SY5Y cells expressing WT or catalytic-inactive mutant OGT treated with 20 μM EA, and the fractions were analyzed by immunoblotting.

Quantification of (F). Mean ± SD, unpaired two-tailed t-test, n = 3 biological replicates.

SDS-soluble and insoluble fractions were isolated from SH-SY5Y cells expressing WT OGT and/or Flag-TDP-43 in the presence or absence of EA, and the truncated TDP-43 fragments were examined by immunoblotting. L.E.: long exposure; S.E.: short exposure.

Quantification of (H). Mean ± SD, unpaired two-tailed t-test, n = 3 biological replicates.

Data information: * P <0.05, *** P < 0.001., **** P < 10-4.
O-GlcNAcylation of TDP-43 suppresses proteinopathies and promotes TDP-43's mRNA splicing activity (2021)
Figure 4

Figure 4. Identification of O-GlcNAcylation sites of TDP-43.

TDP-43 interacts with OGT examined by immunoblotting.

In vitro glycosylation assay was performed using immunoprecipitated OGT incubated with recombinant His-tagged TDP-43 in the presence of UDP-GlcNAc. The O-GlcNAcylated TDP-43 protein was detected by immunoblotting.

Sequence alignment of TDP-43 segments containing the identified O-GlcNAcylation sites in different species. The amino acids with red color display the conserved O-GlcNAcylated sites.

Structural modeling of the RRM2 domain bound to a single strand DNA illustrates the O-GlcNAcylation sites T199 (pink) and T233 (red) of TDP-43 with surrounded DNA (light orange).

In vitro glycosylation assays were performed using immunoprecipitated Flag-OGT incubated with recombinant GST-tagged TDP-43 fragments (a.a. 102-269) in the presence of UDP-GlcNAc. The O-GlcNAcylated TDP-43 proteins were detected by immunoblotting.

Quantification of (E). Mean ± SD, unpaired two-tailed t-test, n = 4 biological replicates. **** P < 10-4.

Cells expressing the indicated forms of GFP-TDP-43 were subjected to immunoprecipitation, and the O-GlcNAcylated levels of TDP-43 were examined.

Quantification of (G). Mean ± SD, unpaired two-tailed t-test, n = 3 biological replicates. * P < 0.05, **** P < 10-4, n.s. not significant.
O-GlcNAcylation of TDP-43 suppresses proteinopathies and promotes TDP-43's mRNA splicing activity (2021)
Figure 6

Figure 6. TDP-43 O-GlcNAcylation regulates mRNA splicing.

CFTR splicing assay was examined in cells expressing various TDP-43 constructs by PCR analysis. The numbers represented the relative ratios of exon 9 exclusion (-) vs. inclusion (+) (top panel). Both endogenous (endo.) and exogenous (exo.) TDP-43 protein levels in the indicated samples were examined by immunoblotting (bottom panel).

CFTR splicing assay was performed in cells with knockdown of either OGT, TDP-43, or combination. ACTB levels served as loading controls (top panel). The indicated protein levels were examined by immunoblotting (bottom panel).

CFTR splicing assay was examined in siTDP-43 cells overexpressing the WT or mutant OGT by PCR analysis. ACTB levels served as loading controls (top panel). Various protein levels were examined by immunoblotting (bottom panel).

Representative gel images for alternative exons in Dnajc5 and Sort1 genes in Neuro 2a cells expressing the indicated Flag-TDP-43 constructs were shown (top panel). TDP-43 protein levels were examined by immunoblotting (bottom panel).

The relative ratios of exclusion to inclusion (-/+) were quantified. Mean ± SD, unpaired two-tailed t-test, n = 3 biological replicates.

Neuro-2a cells expressing doxycycline-inducible shRNA knockdown of TDP-43 were transfected with the indicated TDP-43 constructs, and the alternative exons in Dnajc5 and Sort1genes were examined by PCR. Representative gel images were shown (top panel). TDP-43 protein levels were examined by immunoblotting (bottom panel).

The relative ratios of exclusion to inclusion (-/+) were quantified. Mean ± SD, unpaired two-tailed t-test, n = 3 biological replicates.

Representative PCR analysis to examine expression levels of the spliced mRNA isoform containing exon 2a of STMN2 in SH-SY5Y cells with the indicated constructs (top panel). TDP-43 protein levels were examined by immunoblotting (bottom panel).

Quantification of the relative levels of truncated STMN2. Mean ± SD, unpaired two-tailed t-test, n = 3 biological replicates.

Quantification of the relative mRNA levels of STMN2. Results were normalized to expression of the gene encoding ACTB and were quantified by the change-in-threshold method (ΔΔCT). Mean ± SD, unpaired two-tailed t-test, n = 6 biological replicates.

Data information: * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant.

Expanded View Figure legends:
MET Mutation Causes Muscular Dysplasia and Arthrogryposis (2019)
Figure 1

Figure 1. MET p.Y1234C mutation caused arthrogryposis in a four-generation Chinese family. (A) The MET p.Y1234C mutation segregated with disease phenotype in the arthrogryposis family. Filled symbols denote affected individuals, open symbols indicate unaffected individuals, and symbols with slashes represent decreased individuals. Asterisks indicate a mutation is present, # means wild type. (B) Phenotypes of affected individuals. Camptodactyly, absent flexion crease, and limited forearm supination were observed. (C-E) T1 weighted MRI scan on upper limbs of subject IV:7. (C) The pronator quadratus absence of affected side was indicated by a red arrow. (D) No difference was found in palmar muscles. (E) Loss of lumbrcalis and interosseus muscles of fifth finger of affected side was indicated by a red arrow. (F-H) T1 weighted MRI scan on upper limbs of subject IV:8. (F) Increased epimysial fat was indicated by red arrow. (G) Completely loss of thenar eminences of both hands were indicated by red arrows. (H) Loss of radial lumbrcalis and interosseus muscles of both hands were indicated by red arrows. (I) The MET variants by Sanger sequencing was indicated by red arrow. (J) 293T cells were transfected with FLAG-tagged MET/METMut/Vector plasmids, and 48 h post transfection, cells were treated with 10 ng/mL recombinant human HGF for 1 h. Then MET/METMut protein purification and tyrosine kinase assay were conducted. Western Blot pictures representative of n = 3 experiments. METMut means p.Y1234C mutant MET. EGFR means epidermal growth factor receptor and serves as a positive control. NC means negative control.
MET Mutation Causes Muscular Dysplasia and Arthrogryposis (2019)
Figure 2

Figure 2. Met mutation caused the reduction of both appendicular and paraspinal muscles of P0 heterozygotes. (A) Gross appearance of wild-type and heterozygous newborns. (B) Graph showing weight of P0 mice. Sample size: WT (n = 4) and Hetero (n = 5). *P < 0.05, by one-way ANOVA and follow by Dunnett's post hoc test. (C-G) Sections of spine, forelimbs, hindlimbs, hand, and foot from wild types and heterozygotes at P0, were conducted with HE staining and immunofluorescence staining using anti-myosin heavy chain antibody. Scale bars, 100 μm. (H) The mean number of myofibers in the muscles of the spine, forelimb, hindlimb, hand and foot was qualified. NS means no statistic significance. *P < 0.05, **P < 0.01, by two-tailed independent student's t test. In (C-H), n = 3. (I) Transmission electron microscope analysis of gastrocnemius from wild-type and heterozygous newborns. Black arrows denote mitochodria. Scale bars, 2 μm. WT means wild types, Hetero means heterozygotes.