EMBO/soda-nel huggingface dataset:
D-Aspartate treatment attenuates myelin damage and stimulates myelin repair (2018)
Figure 7

Figure 7. Effects of D-Aspartate treatment on corpus callosum demyelination.

A. Representative confocal double immunofluorescence images displaying MBP (red) and NF200 (green) distribution and their coexpression (white) in the middle corpus callosum of septostriatal sections of control mice (a-c), and of mice fed with cuprizone for 5 weeks in the absence (d-f) or in the presence of D-Asp (g-i). Scale bars in a-i: 20 μm. (j), Densitometric analysis of MBP immunofluorescence signal and (k) quantification of MBP-NF200 colocalized points in the middle corpus callosum of septostriatal sections from control mice, and from mice fed with cuprizone for 5 weeks in the absence or in the presence of D-Asp.

B. Western blot and densitometric analysis of MBP levels in corpus callosum lysates from control and cuprizone treated-mice for 5 weeks in the absence or in the presence of D-Asp, respectively. Data were normalized on the basis of α-tubulin and expressed as percentage of controls.

C. Representative confocal double immunofluorescence images displaying GFAP (green) and Iba1 (red) distribution in the middle corpus callosum of septostriatal sections of control mice (a-c), and of mice fed with cuprizone for 5 weeks in the absence (d-f) or in the presence of D-Asp (g-i). Scale bars in a-i: 50 μm. (j-k) Quantification of GFAP+ and Iba1+ cells in the middle corpus callosum of septostriatal sections of control mice and of mice fed with cuprizone for 5 weeks in the absence or in the presence of D-Asp. Data were normalized to the total cell number (Hoechst signal) and expressed as percentage of controls.

D. Western blot of GFAP and Iba1 protein levels in corpus callosum lysates obtained from control or cuprizone treated-mice for 5 weeks in the absence or in the presence of D-Asp, respectively.

Data information The values represent the means ± S.E.M. Level of significance was determined by using in: A, j, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=4 mice for each group); A, k, one-way ANOVA P=0.0162 followed by Newman-Keuls post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=4 mice for each group); B, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=3 mice for each group); C, j-k, one-way ANOVA P<0.0001 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=4 mice for each group); D middle panel, one-way ANOVA P=0.0146 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=3 mice for each group); D right panel, one-way ANOVA P= 0.0026 followed by Bonferroni post hoc test, *P<0.05 versus control, ^P<0.05 versus cpz (n=3 mice for each group). See the exact P‐values from comparisons tests in Appendix Table S7.

Source data are available online for this figure
D-Aspartate treatment attenuates myelin damage and stimulates myelin repair (2018)
Figure 8

Figure 8. Effects of D-Aspartate treatment on corpus callosum remyelination.

A. Confocal double immunofluorescence images depicting Olig2+ (a-e) and Olig2+-CC1+ (f-j) double labeled cells in the middle corpus callosum of control, cuprizone-treated mice at 5 weeks, in absence (cpz) or in presence of D-Asp (cpz + D-Asp), and 2.5 weeks after cuprizone withdrawal, in absence (vehicle) or in presence of D-Asp (D-Asp). (k, left panel), Quantitative analysis of Olig2+ cells (red) and Olig2+-CC1+ (blue) double labeled cells in the middle corpus callosum of control, cuprizone-treated mice, and cuprizone+D-Asp-treated mice at 5 weeks. (k, right panel), Quantitative analysis of Olig2+ cells (red) and Olig2+-CC1+ (blue) double labeled cells in the middle corpus callosum of control, vehicle, and D-Asp-treated mice 2.5 weeks after cuprizone withdrawal. Arrows point to Olig2+-CC1+ cells. Arrowheads point to Olig2+-CC1+ cells. Scale bar in a-j: 50 μm.

B. Representative electron microscopy images of middle corpus callosum sections from control mice (a), cuprizone treated-mice for 5 weeks in the absence (b) or in the presence of D-Asp (c), and from mice 2.5 weeks after cuprizone withdrawal in the absence (d, vehicle) or in presence of D-Asp (e). Scale bars in a-e: 1 μm

C. Percentage of myelinated axons in the middle corpus callosum of cuprizone (blue) and cuprizone + D-Asp-treated mice (red) for 5 weeks.

D. Scatter plot analysis of myelin thickness, expressed as individual G-ratios against axon diameters, after 2.5 weeks of cuprizone withdrawal in the middle corpus callosum of vehicle (blue circle) or D-Asp-treated mice (red triangle). Linear regression was used to indicate the differences between the groups in myelin thickness across the range of axon diameters.

E. Histogram analysis displaying the G-ratio values of myelinated fibers across the range of axon diameters. Data represent the mean S.E.M. for at least 100 axons from each group.

F. Percentage of myelinated axons after 2.5 weeks of cuprizone withdrawal in the middle corpus callosum of vehicle (blue) or D-Asp- treated mice (red). Abbreviations: DEM, demyelination; REM, remyelination.

Data information: The values represent the means ± S.E.M. Level of significance was determined by using: in A, k left panel, one-way ANOVA P<0.0001 followed by Newman-Keuls post hoc test, *P<0.05 versus ctrl; ^P<0.05 versus cpz (n=3 mice for each group). A, k right panel, one-way ANOVA P<0.0001 followed by Newman-Keuls post hoc test, *P<0.05 versus ctrl; ^P<0.05 versus vehicle (n=3 mice for each group); C, two-tailed Student's t test, *P<0.05 versus cpz (n=4 mice for each group); E, for each axon diameter group, Student's t test, *P<0.05 versus vehicle (n=4 mice for each group); F, for each axon diameter group, Student's t test, *P<0.05 versus vehicle (n=4 mice for each group). See the exact P‐values from comparisons tests in Appendix Table S8.

Source data are available online for this figure

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Intra- and inter-tumor heterogeneity in a vemurafenib-resistant melanoma patient and derived xenografts. (2015)
Figure 2

Reactivation of MAPK pathway via amplification of a BRAF-encoding DNA fragment caused resistance to vemurafenib in three metastases

Hematoxylin–eosin (H&E) and p-ERK stainings on FFPE material of all metastases showed that all vemurafenib-resistant tumors had reactivation of the MAPK pathway. Scale bar represents 100 μm.

CNA profiles were generated from the WES data with germ line DNA as a reference. Colors represent segmented log2 ratio values with red for gain and blue for loss. Further inspection of chromosome 7 where BRAF is located revealed amplification (7q34) of this region in three vemurafenib-resistant metastases, namely M032R1, M032R2, and M032R5.

qPCR was performed on gDNA retrieved from each of the metastases, using primers for BRAF and CRAF and normalized on LINE levels. Bars represent the mean of three replicates, error bars indicate standard deviation. The results confirmed that BRAF was amplified in M032R1, M032R2, and M032R5.

Staining for BRAFV600E with a mutant epitope-specific antibody confirmed the upregulation of BRAFV600E in R1, R2, and R5. Scale bar represents 100 μm.
Intra- and inter-tumor heterogeneity in a vemurafenib-resistant melanoma patient and derived xenografts. (2015)
Figure 3

Alternative form of BRAF<sup>V600E</sup> is correlated with p-ERK and p-MEK reactivation in M032R3

Immunoblotting for MAPK pathway components confirmed reactivation of p-ERK and p-MEK in all resistant metastases.

Immunoblotting for M032 patient’s samples using an antibody directed against either the mutant BRAFV600E epitope or an N-terminal region of BRAF revealed the expression of a shorter variant of BRAFV600E in M032R3, of approximately 80 kDa. This 80-kDa band could not be detected with the N-terminus-recognizing antibody.

Source data are available online for this figure.
Intra- and inter-tumor heterogeneity in a vemurafenib-resistant melanoma patient and derived xenografts. (2015)
Figure 4

<italic>T55delinsRT</italic> mutation in <italic>MEK1</italic> is responsible for resistance to vemurafenib in M032R4

Somatic mutations present in each of these metastases, revealing that mutations were either shared by the pre-treatment tumor and all metastases, by some metastases, or only by single metastases.

A 3-bp in-frame insertion in MEK1 (T55delinsRT) was identified in M032R4. This mutation was located at the base of the α-helix at the N-terminal site of MEK1.

Validation of T55delinsRT was done by PCR using insertion-specific primers. As a control, primers amplifying exon 2 of MEK1WT were used.

Transfection of pQXCIP-GFP, MEK1-WT, or MEK1-T55delinsRT into HEK293T cells showed that this mutation induces p-ERK and p-RSK.

Validation of expression of pQXCIP-GFP, MEK1WT, or MEK1T55delinsRT in A375 melanoma cells by immunoblotting.

Dose–response curves for A375 melanoma cells expressing GFP, MEK1WT, or MEK1T55delinsRT with indicated doses of BRAF inhibitor vemurafenib, ERK inhibitor SCH772984, or MEK inhibitor trametinib. Error bars indicate standard deviation.

Colony formation assays with A375 melanoma cells, infected with GFP, MEK1WT, or MEK1T55delinsRT-encoding lentivirus, and treated with indicated doses and inhibitors.

Treatment of A375 MEK1T55delinsRT melanoma cells with DMSO (−), 250 nM dabrafenib (D), 10 nM trametinib (T), or a combination (D+T).

Source data are available online for this figure.
Intra- and inter-tumor heterogeneity in a vemurafenib-resistant melanoma patient and derived xenografts. (2015)
Figure 5

<italic>T55delinsRT</italic> mutation in <italic>MEK1</italic> confers resistance to BRAF inhibition and promotes local invasion <italic>in vivo</italic>

A375 melanoma cells expressing GFP, MEK1WT, or MEK1T55delinsRT were injected into immune-deficient mice (n = 8 per group), and after the tumor size of ˜100 mm3 was reached, mice were treated with 30 mg/kg dabrafenib or vehicle. Graphs represent fold change in tumor volume normalized on the tumor volume on the day of the start of the treatment. Error bars indicate standard error of the mean.

Average tumor weight of all groups at the end of the experiment described in (A). Error bars indicate standard deviation. Unpaired t-test was used to calculate statistical significance. P-values are indicated in the graph.

Immunoblotting for MAPK pathway components performed on tumors of all groups.

Immunohistochemistry for Sirius Red (staining for collagen) and H&E on tumors from all groups, to identify grade of demarcation and local invasion. Scale bar represents 500 (upper row) and 100 (lower row) μm.

Source data are available online for this figure.
Intra- and inter-tumor heterogeneity in a vemurafenib-resistant melanoma patient and derived xenografts. (2015)
Figure 6

<italic>MEK1</italic><sup><italic>T55delinsRT</italic></sup> is a preexisting mutation

gDNA was isolated from another fragment of the tumor resection, and the PCR with the insertion-specific primers was repeated. Primers amplifying exon 2 of MEK1WT were used as an input control.

Source data are available online for this figure.
Intra- and inter-tumor heterogeneity in a vemurafenib-resistant melanoma patient and derived xenografts. (2015)
Figure 7

Patient-derived xenografts display similar resistance mechanisms as the parental metastases

Staining for hematoxylin–eosin (H&E) and p-ERK on the PDX samples M032.X1, M032R1.X1, M032R2.X1, M032R4.X1, and M032R5.X1. Stainings showed that p-ERK is higher in the PDX derived from the resistant metastases. Scale bar represents 100 μm.

Immunoblotting for components of the MAPK pathway confirmed reactivation of p-ERK in the PDX derived from the vemurafenib-resistant metastases, although the p-ERK signal is heterogeneous in the pre-treatment PDX.

Validation of the BRAF amplification was performed by qPCR on gDNA derived from the PDX, using primers for BRAF and CRAF and normalized on LINE expression. Bars represent the mean of three replicates, error bars indicate standard deviation. The results confirmed that BRAF was amplified in PDX derived from M032R2 and M032R5, but not from M032R1.

Presence of the MEK1T55delinsRT was analyzed by PCR using insertion-specific primers. As a control, primers amplifying exon 2 of MEK1WT were used. The insertion was found in M032R1.X1 and M032R4.X1.

Source data are available online for this figure.
Regulation of mitochondrial proteostasis by the proton gradient (2022)
Figure 1

Figure 1. Mitochondrial Ca2+ overload triggers apoptotic death of TMBIM5-/- cells

Volcano plot representation of AFG3L2E408Q-FLAG interaction partners. Mitochondria isolated from Flp-In HEK293T-REx cells expressing AFG3L2E408Q-FLAG were subjected to immunoprecipitation. Co-purifying proteins were identified by quantitative mass spectrometry (MS) (n=5 independent experiments) followed by an unpaired two-sided t-test and permutation-based FDR estimation to correct for multiple testing (FDR<0.05. Significantly enriched MitoCarta 3.0 proteins (Rath et al., 2021) are colored in red, gene names indicate previously verified AFG3L2 interactors and TMBIM5 (GHITM, MICS1). See also Dataset EV1.

Representative immunoblot of HeLa wildtype (WT) cells transfected with scrambled siRNA (control) or siRNA targeting TMBIM5 and treated with the indicated drugs for 16 h. Control (0.1% DMSO), actinomycin D (1 µM), venetoclax (1 µM), A-1155463 (1 µM), staurosporine (1 µM), thapsigargin (2 µM). n=3 independent experiments.

Representative immunoblot of HeLa WT and TMBIM5-/- cells transfected with scrambled siRNA (control) or siRNA targeting MCU for 72 h. Where indicated samples were treated with staurosporine (STS; 1 µM) for 16 h. Quantification is shown in Figure EV1G (n=3 independent experiments).

Viability of WT and TMBIM5-/- cells (depleted of MCU when indicated) after incubation with staurosporine (STS; 0.1 µM) for 16 h. Cell viability was assessed cytofluorimetrically monitoring NucView488 and RedDot2 staining and expressed as percentage of all cells. n=3 independent experiments; two-tailed t-test; mean ±SD. A p-value of <0.05 was considered statistically significant.

WT and TMBIM5-/- HeLa cells were transfected with the indicated scrambled siRNA (control) and siRNA targeting MCU for 48 h and incubated with staurosporine (0.1 µM) for 16 h in the presence of Z-VAD-FMK (50 µM) and epoxomicin (1 µM) to prevent apoptosis. Cells were subjected to immunofluorescence analysis with antibodies directed against cytochrome c (green) and TOMM20 (magenta). *Cells with cytosolic cytochrome c. Scale bar ?? Quantification is shown in Figure EV1H (n=3 independent experiments; number of cells > 150 each experiment).

Calcium retention capacity (CRC) was assessed in isolated mitochondria of HEK293T WT cells transfected with the indicated siRNA. The experiment was performed in sucrose-based medium containing respiratory substrates and the membrane-impermeable Ca2+ sensor Ca2+ Green-5N. Ca2+ Green-5N fluorescence was monitored following repeated addition of Ca2+ pulses (10 µM). Data show representative traces (2 independent experiments run in triplicate each condition).

Data information: See also Figure EV1 and Dataset EV1.
Regulation of mitochondrial proteostasis by the proton gradient (2022)
Figure 2

Figure 2. TMBIM5 acts as a Ca2+/H+ exchanger

Fluorescence (F) F/F0 ratio quantifications, where F0 is the average of the first ten seconds and F is the fluorescence peak measured after thapsigargin (2 µM) administration (left panel). The right shows mtRED-GECO ratio traces of HeLa cells transfected with scrambled siRNA (control) and siRNA targeting TMBIM5. Each measurement was performed in at least 10 cells from 6 different preparations. Mean ±SD; two-tailed t-test. A p-value of <0.05 was considered statistically significant.

Schematic representation of experiments shown in (A).

Matrix pH was measured with Sypher3smito in wildtye (WT) HeLa cells transfected with scrambled siRNA (control) or siRNA targeting TMBIM5 for 72 h. The probe was calibrated in KRB buffer by the stepwise addition/removal of permeant weak acid. Each measurement was performed in at least 80 cells from 7 different preparations. Mean ±SD; two-tailed t-test. A p-value of <0.05 was considered statistically significant.

Mitochondrial membrane potential was monitored with TMRM in WT and TMBIM5-/- HeLa cells. Fluorescent intensity was calculated as ratio to the value upon CCCP addition (15 µM). Values are expressed as mean ±SD relative to control. Each measurement was performed in triplicate from 4 different preparations.

Matrix pH was measured with Sypher3smito in WT HeLa cells transiently expressing TMBIM5 or TMBIM5D294K D325K for 24 h. The ratio of absorbance at 488 nm and 405 nm is shown in arbitrary units (a.u.). Each measurement was performed in at least 20 cells from 3 different preparations. Representative immunoblot of WT HeLa cells transfected with TMBIM5 or TMBIM5D294K D325K for 24 h (right panel). Mean ±SD; two-tailed t-test. A p-value of <0.05 was considered statistically significant.

Liposome assay. Recombinant TMBIM5 was reconstituted in liposomes containing 100 µM Fura-2. CaCl2 was added later at the concentration of 100 µM. Rmax was obtained at the end of the experiment after the addition of ionomycin (10 µM). Data are expressed as mean of R/Rmax, where R is the peak after CaCl2 addition. Where indicated GdCl3 (100 µM) was added 10 min before the experiment. n=3 independent experiments, shown as black, white and grey dots. Mean ±SD; two-tailed t-test. A p-value of <0.05 was considered statistically significant.

Liposome assay (as in E). Slope was calculated in the 100 s after 100 µM CaCl2 addition as expressed as mean ±SD. n=3 independent experiments, shown as black, white and grey dots. Two-tailed t-test. A p-value of <0.05 was considered statistically significant.

Data information: See also Figure EV2.
Regulation of mitochondrial proteostasis by the proton gradient (2022)
Figure 5

Figure 5. Loss of TMBIM5 promotes proteolysis by AFG3L2

Pie chart showing the localization of proteins accumulating at decreased levels in TMBIM5-/- HeLa cells. Localization according to MitoCarta 3.0. OM, outer membrane; IMS, intermembrane space; IM, inner membrane.

Alterations in mitochondrial proteome in WT and TMBIM5-/- HeLa cells upon depletion of AFG3L2. WT and TMBIM5-/- HeLa cells were transfected with scrambled siRNA (control) or siRNA targeting AFG3L2 for 48 h. Proteins, which were downregulated in the absence of TMBIM5 and whose steady state levels was altered upon depletion of AFG3L2, were identified using a two-way ANOVA. An interaction p-value of 0.01 was used as a cutoff. Log2-transformed LFQ intensities of MitoCarta 3.0 proteins were Z-Score normalized and visualized in a hierarchical cluster analysis (Euclidean distance, complete method). The dendrogram is omitted. Proteins accumulating upon depletion of AFG3L2 (cluster 1, C1) and proteins whose steady state levels are decreased upon depletion of AFG3L2 (cluster 2, C2) are shown. See also Dataset EV6.

Representative immunoblot of TMBIM5-/- HeLa cells transfected with scrambled siRNA (control) or siRNA targeting AFG3L2 for 48 h. Samples were treated with the protein synthesis inhibitor cycloheximide (CHX; 10 μg/ml) and collected at the indicated time points (n=3 independent experiments).

Data information: See also Table EV 6.
Regulation of mitochondrial proteostasis by the proton gradient (2022)
Figure 6

Figure 6. Hyperpolarization induces degradation of TMBIM5 allowing AFG3L2-mediated remodeling of the mitochondrial proteome.

Venn diagram of significantly downregulated proteins (FDR ≤0.05) in oligomycin-treated wildtype (WT) HeLa cells (0.01% DMSO vs oligomycin 10 μM for 16 h; blue) and in TMBIM5-/- HeLa cells (WT vs TMBIM5-/- HeLa cells; red). See also Dataset EV7.

Volcano plot of mitochondrial protein (MitoCarta 3.0) changes in WT HeLa cells treated with oligomycin (10 μM for 16 h) when compared to untreated cells. Before MitoCarta 3.0-filtering, significantly altered proteins were identified using a two-sided t-test followed by permutation-based FDR estimation to 0.05 and are colored in blue (n=5 independent experiments). See also Dataset EV7.

Representative immunoblot of WT HeLa cells treated with the indicated drugs for 16 h. Antimycin A (AA; 10 μM), piericidin A (10 μM) and oligomycin (10 μM). Quantification is shown in Figure EV5B (n=7 independent experiments).

Representative immunoblot of WT HeLa cells transfected with scrambled siRNA (control) and siRNA targeting AFG3L2 for 48 h, treated when indicated with oligomycin (10 μM) for 16 h. Quantification of TMBIM5 levels is shown on the right panel. TMBIM5 levels in untreated WT cells was set to 1 (n=3 independent experiments; mean ±SD).

TMBIM5 abundance determined by mass spectrometry in WT HeLa cells treated with oligomycin (10 µM; 16 h) and depleted of AFG3L2 when indicated. n=5 independent experiments. P-value was calculated using a two-sided unpaired t-test. The central band of each box is the median value, and the box defines the 25th (lower) and 75th (higher) quantile. The whiskers represent the minimum and maximum value in the data excluding outliers (greater than 1.5 * inter quantile range distance to median - no outliers found here).

Zoom in to selected clusters of hierarchical clustering of Z-Score normalized log2 LFQ intensities. Proteins that were significantly (FDR<0.05) accumulated between HeLa WT and TMBIM5-/- cells after oligomycin treatment (10 µM) for 16 h were clustered using Euclidean distance and the complete method on protein features. The figure shows identified cluster C4 and C5 with gene names and MitoCarta 3.0 localization annotations. The row dendrogram is omitted. See also Dataset EV6.

Data information: See also Table EV 6, EV 7.
Reduced symmetric dimethylation stabilizes vimentin and promotes metastasis in MTAP deficient lung cancer (2022)
Figure 1

Figure 1. MTAP loss promotes invasion/metastasis of lung adenocarcinoma

A. The Venn diagram showing 207 differentially expressed genes (DEGs) between CL1-0 and CL1-5 cells (fold change greater than 5-fold in Log2 scale) and 66 genes with copy number variation (CNV, Frequency ≧5%) in lung adenocarcinoma (LUAD) from TCGA database (PanCancer Atlas) and the 4 intersected genes.

B. Comparisons of MTAP mRNA and protein expression between CL1-0 and CL1-5 cells detected by RT-qPCR (top, mean ± SD, Student t test, n=3, biological replicates, *p < 0.05) and Western blot assays using anti-MTAP antibody (bottom, representative of three independent experiments).

C. Cell invasion abilities of MTAP-overexpressing CL1-5 or MTAP-knockout CL1-0 and H1650 were determined by Boyden chamber invasion assays (mean ± SD, Student t test, n=3, biological replicates, *p < 0.05). sgCTRL indicates cells transduced with a non-targeting control sgRNA, and sgMTAP indicates cells transduced with a sgRNA targeting MTAP. Bottom: the protein expression levels of V5-MTAP and endogenous MTAP were detected by Western blots.

D. Migration assays using a single-cell tracking, time-lapse video microscopy system. Representative trajectories and quantification of averaged velocity of cells (mean ± SD, Student t test, n=20, technical replicates, *p < 0.05). Data shown are representative of three independent experiments.

E. Effect of MTAP overexpression on lung cancer metastasis in vivo was demonstrated by orthotopic implantation assays. Top: representative photographs of lungs and H&E staining of the lung sections. The primary tumors are indicated by arrowheads and the metastatic nodules are indicated by arrows. Bottom: quantification of averaged primary tumor sizes, metastatic incidence and nodule number.

F. Kaplan-Meier analyses of overall survival (top) and progression-free survival (bottom) for 101 patients with lung adenocarcinoma grouped into high- or low-MTAP mRNA expression measured by RT-qPCR. p values were obtained by log-rank test.