EMBO/soda-nel huggingface dataset:
Direct interaction with DRP1 activates BAX and induces apoptosis (2021)
Figure 6

Figure 6. Induced dimerization of BAX and DRP1 induces MOMP.

A) Hierarchy of BAX and DRP1 recruitment to mitochondria in apoptotic cells. Confocal microscopy image of U2OS BAX/BAK DKO cells transfected with mCherry-DRP1 (red), mEGFP-BAX (green) and 4xmts-mTurquoise (cyan) to label mitochondria. After apoptosis induction, translocation of DRP1 to mitochondria and foci formation (set to 0 min) was observed before BAX foci formation (5 min later). Zoomed images correspond to crop regions as indicated. Arrowheads highlight co-localizing foci of DRP1 and BAX. Scale bar 20 µm, zoomed images 2 µm.

B) Representative confocal microscopy image of BAX foci formation in apoptotic U2OS BAX/BAK DKO cells transfected with mEGFP-BAX (green) and 4xmts-mTurquoise (cyan) after siRNA-mediated DRP1 depletion. Scale bar 20 µm.

C) Forced dimerization of BAX and DRP1 in healthy cells induces their translocation to mitochondria and foci formation. Confocal microscopy images of U2OS BAX/BAK DKO cells transfected with FKBP-mCherry-DRP1 (red) and FRB-EGFP-BAX (green) and stained with MitoTracker Deep Red FM (cyan) were acquired before (0 min) and after induction of BAX/DRP1 dimerization (5-60 min). From top to down, the individual rows correspond to fluorescence signal of BAX and DRP1 and the merge of both together with the MitoTracker signal, respectively. Scale bar 20 µm.

D) Quantification of mitochondrial morphology normalized to the number of transfected cells before (Ctrl) and after induced dimerization of BAX and DRP1 (Dim). Significance was tested using paired two-tailed Student´s t-test (** p < 0.01) compared to untreated control.

E) Induced dimerization of BAX and DRP1 causes mitochondrial depolarization. U2OS BAX/BAK DKO cells were transfected as described in C) and stained with the mitochondrial membrane potential-sensitive dye MitoSpy NIR (cyan). Confocal microscopy images were acquired before (0 min) and after induced dimerization of BAX and DRP1 (10 min). Scale Bar 20 µm.

F) Quantification of mitochondrial depolarization normalized to the number of transfected cells before (Ctrl) and after induced dimerization of BAX and DRP1 (Dim). Significance was tested using paired two-tailed Student´s t-test (* p < 0.05) compared to non-induced control.

G-K) Induced dimerization of DRP1 with itself (G), BAX with itself (H), BAX with TOM20 (I) in U2OS BAX/BAK DKO cells, or BAX with DRP1 in HCT OctaKO cells (J,K), transected with FKBP- and FRB-mCherry DRP1 (G, red), FKBP- and FRB-EGFP-BAX (H, green), TOM20-mCherry-FKBP (red) and FRB-EGFP-BAX (green, I), respectively. Mitochondria were stained using MitoTracker Deep Red FM or MitoSpy NIR (cyan) as indicated. Scale bar 20 µm (G-I) and 10 µm (J,K ). All images are representative of n=3 independent experiments.

Data information: Values in D and F are presented as mean (bar, line) ± SD of n=3 independent biological experiments (with n=100 cells each). All images are representative of n=3 independent experiments.

Extended View Figure Captions
The E3 ubiquitin ligase Peli1 regulates the metabolic actions of mTORC1 to suppress antitumor T cell responses (2020)
Figure 1

Figure 1. Peli1 deficiency promotes antitumor immunity.

A,B Tumor growth curve (left) and summary of end-point tumor masses (right) of 6-8 week-old wildtype (WT) and Peli1-KO (KO) mice inoculated s.c. with B16F10 (A; WT, n=7; KO, n=6) or E.G7 (B; WT, n=5; KO, n=5) tumor cells.

C,D Flow cytometry analysis of CD4+ and CD8+ T cells in CD45.2+ TILs and dLN cells from B16F10 tumor-bearing wildtype and Peli1-KO mice, presented as a representative plot (C) and summary graph based on multiple mice (D; WT, n=6; KO, n=6).

E,F Flow cytometry analysis of IFNγ-producing CD4+ and CD8+ T cells in TILs, and dLN cells of B16F10 tumor-bearing (day 19) wildtype and Peli1-KO mice, presented as a representative plot (E) and summary graph (F). (WT, n=6; KO, n=6)

G,H Flow cytometry analysis of IFNγ+CD8+ T cells in TILs of E.G7 tumor-bearing (day 24) wildtype and Peli1-KO mice, presented as a representative plot (G) and summary graph (H). (WT, n=3; KO, n=3)

Data information: Data are representative of 3 independent experiments, and bar graphs are presented as mean±SEM with P values being determined by a two-way ANOVA with Bonferroni correction (left panel of A and B) and two-tailed unpaired Student's t-test (right panel of A and B; D,F,H). *P<0.05; **P<0.01; ***P<0.001.
The E3 ubiquitin ligase Peli1 regulates the metabolic actions of mTORC1 to suppress antitumor T cell responses (2020)
Figure 2

Figure 2. T cell-specific Peli1 deficiency promotes antitumor immunity.

A,B Tumor growth curve (left) and summary graph of end-point tumor masses (right) of wildtype (WT) and Peli1-TKO (TKO) mice inoculated s.c. with B16-OVA (A; WT, n=5; KO, n=5) or E.G7 (B; WT, n=6; KO, n=4) tumor cells.

C Flow cytometry analysis of IFNγ-producing CD4+ and CD8+ T cells in TILs and dLN cells from B16OVA tumor-bearing wildtype and Peli1-TKO mice, presented as a representative plot (upper) and summary graph (lower). (WT, n=5; KO, n=5)

D Flow cytometry analysis of GZMB+CD8+ T cells in TILs from B16-OVA tumor-bearing wildtype and Peli1-TKO mice, presented as a representative plot (upper) and summary graph (lower). (WT, n=5; KO, n=5)

E Tumor growth curve (left) and summary graph of end-point tumor masses (right) of B16-OVA tumor-bearing wildtype and the indicated Peli1 conditional KO mice. (Treg KO model: WT, n=5; Treg KO, n=6; MKO model: WT, n=3; MKO, n=5; BKO model: WT, n=9; BKO, n=9)

Data information: Data are representative of 2 (E) or 3 (A-D) independent experiments, and bar graphs are presented as mean±SEM. P values are determined by a two-way ANOVA analysis with Bonferroni correction (left panel of A,B,E) and two-tailed unpaired Student's t-test (C, D and right panel of A,B,E). *P<0.05; ***P<0.001.
The E3 ubiquitin ligase Peli1 regulates the metabolic actions of mTORC1 to suppress antitumor T cell responses (2020)
Figure 3

Figure 3. Peli1 regulates metabolic reprograming of CD8 T cells.

A,B Seahorse analysis of basal (glucose injection) and maximal (oligomycin injection) ECAR or basal (no treatment) and maximal (FCCP injection) OCR in WT and Peli1-KO OT-I CD8 T cells that were either untreated (naïve) or activated with plate-bound anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) for 16 h. Data are presented as a representative plot (A) or summary graph based on 6 wildtype and 6 KO mice (B).

C,D qRT-PCR analysis of the indicated genes using RNAs isolated from wildtype or Peli1-KO OT-I CD8 T cells stimulated with anti-CD3 and anti-CD28 for 6 h.

E Immunoblot analysis of HK2 and Glut1 in wildtype or Peli1-KO OT-I CD8 T cells that were stimulated with plate-bound anti-CD3 and anti-CD28 for 24 h.

Data information: Data are representative of 3 independent experiments, and bar graphs are presented as mean±SEM with P values being determined by two-tailed unpaired Student's t-test. *P<0.05; **P<0.01; ***P<0.001.
The E3 ubiquitin ligase Peli1 regulates the metabolic actions of mTORC1 to suppress antitumor T cell responses (2020)
Figure 4

Figure 4. Peli1 deficiency promotes mTORC1 signaling in CD8 T cells.

A Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of freshly isolated CD8 T cells from wildtype (WT) or Peli1 KO (KO) mice (6 weeks old). Data are presented as a representative blot (left) and summary graphs of densitometric quantifications of the indicated proteins (presented as phosphorylated/total protein ratio) based on three independent experiments.

B,C Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype or Peli1-KO OT-I CD8 T cells that were first rested on ice for 15 min and then stimulated in low-serum (1% FBS) medium with anti-CD3 and anti-CD28 for the indicated time periods.

D,E Seahorse analysis of basal and maximal ECAR (D) or OCR (E), as described in Fig 3, using wildtype or Peli1-KO OT-I CD8 T cells that were activated for 16 h with plate-bound anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) in the presence of solvent control (DMSO), rapamycin (Rapa, 10 nM), or Torin 1 (100 nM). Data are presented as a representative plot (left) and summary graphs (right).

F,G Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype or Peli1-KO OT-I CD8 T cells that were either not treated (-) or stimulated (+) for 1 h (F) or 24 h (G) with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) in the presence (+) or absence (-) of rapamycin (10 nM) and Torin 1 (100 nM).

H qRT-PCR analysis of Ifng gene expression in naïve wildtype or Peli1-KO OT-I CD8 T cells that were either not treated (-) or stimulated for 6 h (+) with anti-CD3 plus anti-CD28 in the presence (+) or absence (-) or rapamycin.

I-K Schematic of experimental design (I), tumor growth curve (J), and summary of day 15 tumor weight (K) of B6.SJL mice inoculated with B16-OVA melanoma cells (2 x 105) and adoptively transferred on day 7 with in vitro activated and rapamycin- or DMSO-treated wildtype or Peli1-KO OT-I CD8 T cells (4 x 105).

Data information; Data are representative of 2 (J,K) or 3 (A-H) independent experiments and bar graphs are presented as mean±SEM with P values being determined by two-tailed unpaired Student's t-test (A,D,E,H,K) or two-way ANOVA with Bonferroni correction (J). *P<0.05; **P<0.01; ***P<0.001.
The E3 ubiquitin ligase Peli1 regulates the metabolic actions of mTORC1 to suppress antitumor T cell responses (2020)
Figure 5

Figure 5. Peli1 regulates mTORC1 activation by growth factors in T cells and MEF cells.

A,B Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) OT-I CD8 T cells that were activated for 16 h with plate-coated anti-CD3 (1 μg/ml) plus anti-CD28, starved for 6 h in serum-free RPMI medium, and then treated for indicated time periods with FBS (10%) (A) or insulin (B).

C-E Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) MEFs that were starved for 16 h in serum-free DMEM medium and then treated for the indicated time periods with FBS (C), insulin (D) and EGF (E).
The E3 ubiquitin ligase Peli1 regulates the metabolic actions of mTORC1 to suppress antitumor T cell responses (2020)
Figure 6

Figure 6. Peli1 regulates the ubiquitination and stability of TSC1-TSC2 complex.

A Immunoblot analysis of the indicated proteins in whole-cell lysates of wildtype (WT) and Peli1-KO (KO) CD8 T cells that were either not treated (NT) or activated for 16 h with plate-bound anti-CD3 and anti-CD28. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC1 (middle panel) and TSC2 (right panel) protein bands relative to the level of α-Tubulin.

B,C Immunoblot analysis of the indicated proteins in whole-cell lysates of wildtype and Peli1-KO CD8 T cells that were activated for 1 h with anti-CD3 and anti-CD28 and then treated with cycloheximide (CHX) for the indicated time periods. Data are presented as a representative blot (B) and a summary graph of quantified TSC1 and TSC2 protein bands presented as percentage of the level in untreated (0 min) cells (C).

D Immunoblot analysis of TSC1 and TSC2 in wildtype or Peli1-KO CD8 T cells stimulated with anti-CD3 plus anti-CD28 for 1 h and then incubated with (+) or without (-) cycloheximide (CHX) and/or MG132 for 2 h. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC1 (middle panel) and TSC2 (right panel) protein bands relative to the level of α-Tubulin.

E,F Ubiquitination analysis of immunoprecipitated TSC2 (E) and TSC1 (F) from activated (anti-CD3 plus anti-CD28 for 2 h) wildtype or Peli1-KO CD8 T cells by immunobloting using anti-ubiquitin Abs detecting total (Ub), K48 (Ub-K48), or K63 (Ub-K63) polyubiquitin chains.

G-J, Analysis of ubiquitin conjugation to TSC1 (G, H) or TSC2 (I, J) in 293T cells transiently transfected (for 48 h) with the indicated expression vectors.

K Co-immunoprecipitaton analysis of TSC1-TSC2 interaction in 293T cells transiently transfected (for 48 h) with the indicated expression vectors.

L Co-immunoprecipitation analysis of endogenous TSC1-TSC2 interaction in wildtype and Peli1-KO CD8 T cells that were either not treated (NT) or stimulated for 2 h with anti-CD3 plus anti-CD28. The right panel is a summary graph of TSC2/TSC1 ratio based on quantification of the blot from anti-TSC1 IP (the right four lanes of the top two panels).

Data information: Summary graphs in A, C, D, and L are densitometric quantification of the indicated protein bands based on three independent experiments, and bar graphs are presented as mean±SEM. P values are determined by two-tailed unpaired Student's t-test. *P<0.05, **P<0.01.
The E3 ubiquitin ligase Peli1 regulates the metabolic actions of mTORC1 to suppress antitumor T cell responses (2020)
Figure 7

Figure 7. Peli1 regulates the stability of TSC complex through ubiquitination of TSC1.

A Analysis of ubiquitin conjugation to wildtype (WT) TSC1 and the indicated TSC1 mutants in 293T cells transiently transfected Flag-tagged TSC1 and its mutants along with (+) or without (-) the indicated expression vectors.

B Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in lysates of TSC1-KO MEFs transfected with (+) or without (-) TSC1 wildtype or K30A expression vectors. The cells were serum starved and then left either untreated (NT) or stimulated with 10% FBS for 30 mins. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC2 (right panel) protein bands relative to the level of β-Actin.

C Analysis of TSC2 ubiquitination in 293 cells transfected (for 48 h) with (+) or without (-) the indicated expression vectors.

D Co-immunoprecipitation analysis of the interaction between endogenous TSC2 and exogenous TSC1 wildtype and K30A in TSC1-KO MEFs transfected (for 48 h) with (+) or without (-) the indicated expression vectors. KO MEF cells were transfected with indicated expression plasmids. The right panel is a summary graph of TSC2/TSC1 ratio based on quantification of the last four lanes of the anti-Flag IP blot in left panel.

E Analysis of ubiquitin conjugation to endogenous TSC2 in TSC1-KO MEFs transfected (for 48 h) with (+) or without (-) TSC1 WT or K30A and treated with MG132 for 2 h before lysis.

F Immunoblot analysis of endogenous TSC2 in TSC1-KO MEFs transfected (for 48 h) with Myc-TSC1 wildtype or K30A and subsequently treated with cycloheximide (10 μg/ml) for the indicated time periods before lysis. The right panel is a summary graph of quantified TSC2 WT and K30A protein bands presented as percentages of the level in the untreated (0 min) lane.

Data information: Summary graphs in B, D, and F are densitometric quantification of the indicated protein bands based on three independent experiments, and bar graphs are presented as mean±SEM. P values are determined by a two-tailed unpaired Student's t-test. *P<0.05.

Extended View Figure Legends
Path-seq identifies an essential mycolate remodeling program for mycobacterial host adaptation (2019)
Figure 2

Fig. 2. The expression levels of desA1 and desA2 from Path-seq of MTB infected BMDMs and over a hypoxia time course. (A) Path-seq profiles of desA1, desA2 in MTB infected BMDMs. Error bars show the standard deviation from three biological samples. Representative results from two experiments are presented. (B) Upper panel: dissolved oxygen curve over 120 h time course showing controlled depletion (1), sustained hypoxia (2) and controlled reaeration (3). Points represent the average of three biological replicates and were measured via fiber optic technology that non-invasively probes oxygen levels in the culture (PreSens Precision Sensing GmbH). Lower panel: expression profiles (RNA-seq) of desA1 and desA2 over the time course and oxygen levels. Error bars show the standard deviation from three biological samples.
Path-seq identifies an essential mycolate remodeling program for mycobacterial host adaptation (2019)
Figure 3

Fig. 3. Systems approach to identify active intracellular regulatory networks. (A) Schematic of network analysis to identify TFs with activity (increased or decreased) in controlling the transcriptional state of MTB during infection of host cells. Abbreviations: dec; decreased, inc; increased. (B) Heatmap of TFs with decreased (purple) or increased (green) activity at specific time points during in vitro or in vivo infection. (C) Rv0681 regulon genes differentially expressed in vitro and the evidence for predicted high Rv0681 activity (induced expression of target genes). (D) Zur regulon genes differentially expressed in vitro and the evidence for decreased Zur activity (de-represses target genes). (E) Rv0691c regulon genes differentially expressed in vitro and in vivo; evidence for increased Rv0691c activity (increased up- and down-regulation of target genes) at 24 h from both in vitro and in vivo infection.
Path-seq identifies an essential mycolate remodeling program for mycobacterial host adaptation (2019)
Figure 4

Fig. 4. EGRIN model predicts module 276 relevant to in vivo infection and regulation of desA1 and desA2 by Rv0472c (MSMEG_0916). (A) EGRIN model of MTB predicts a module enriched with lipid metabolism genes from Path-seq data of in vivo infection. Overexpression data confirms the regulation of desA1 and desA2 by Rv0472c in both MTB and MSM. Graphic representation of linkages between module 276 genes, regulatory influences, functional associations, cell wall modifications, and homology to MSM. (B) Plot of read pile-ups from MSM with inducible overexpression of MSMEG_0916 shows ChIP-binding in the promoters of desA1 and desA2. (C) Boxplots representing RPKM values from RNA-seq of MSM with inducible overexpression of MSMEG_0916. Significant log2 fold change (FC) between uninduced (-ATc) and induced (+ATc) samples for MSMEG_0916 (log2 FC = 4.99), desA1 (log2 FC = -1.32) and desA2 (log2 FC = -1.8) with multiple hypothesis adjusted P-values < 0.0001. Data is from three biological samples (induced) and nine biological samples (uninduced).
Path-seq identifies an essential mycolate remodeling program for mycobacterial host adaptation (2019)
Figure 5

Fig. 5. Cell viability and mycolic acid characterization. (A) Serial ten-fold dilutions of MTB H37Rv wild type (wt) and MTB with inducible overexpression of Rv0472c were spotted on 7H10 agar plates with or without ATc. (B) Argentation TLC of 14C-labeled methyl esters of mycolic acids (MAMEs) obtained from apolar lipids and delipidated cell wall fractions of MSM wt and MSM with inducible overexpression of MSMEG_0916. The α, αʹ, epoxy (e) and cyclopropanated α- (X1) MAMES species are labeled. Faster-migrating species that co-migrated with α-MAMES and accumulate with induced MSMEG_0916 overexpression are indicated as X2 and X3. (C) BCG wt and BCG with inducible overexpression of Rv0472c cultures, labeled with 14C-acetate, were induced (+ATc) or uninduced (-ATc) for 4 h or 8 h. The total FAMEs and MAMEs were extracted and analyzed by autoradiography-TLC using equal counts (15,000 cpm) for each lane.
Inheritance of OCT4 predetermines fate choice in human embryonic stem cells (2018)
Figure 1

Figure 1. Single-cell dynamics of OCT4 in human embryonic stem cells. A. Individual hESCs have the potential to generate another stem cell through self-renewal or to differentiate into a more lineage-specific cell type. B. Before differentiation, hESCs show uniformly high expression of OCT4. Treatment with BMP4 produces a mixture of OCT4+/CDX2- self-renewing hESCs and OCT4-/CDX2+ mesodermal cells. C. Quantification of OCT4 and CDX2 expression by immunofluorescence after 24 h of BMP4 treatment reveals two populations of hESCs. Cells were assigned to one of two distinct populations based on a 2-component mixed Gaussian distribution (see Appendix Figure S6). D. A fluorescent mCherry coding sequence was introduced into the endogenous OCT4 locus of H9 hESCs using CRISPR-mediated homologous recombination. E. Filmstrip of OCT4 dynamics in an undifferentiated hESC throughout its cell cycle duration. Yellow outlines indicate the region used to quantify mean nuclear fluorescence intensity. F. Distribution of OCT4 levels in individual hESCs. A single OCT4 level was quantified for each cell by averaging the mean nuclear mCherry intensity over the lifetime of the cell. G. Single-cell traces of OCT4 signaling. The length of each cell's trace indicates its cell cycle duration. H. Distribution of cell cycle durations for 120 hESCs.
Inheritance of OCT4 predetermines fate choice in human embryonic stem cells (2018)
Figure 2

Figure 2. Differences in OCT4 dynamics before differentiation stimulus predict eventual fate decisions. A. Single-cell traces of hESCs before and after treatment with 100 ng/mL BMP4. Cells were imaged for 42 h prior to BMP4 treatment. Mean nuclear OCT4 levels were quantified every 5 minutes and individual cells were tracked from the cell division event that created the cell until its own division. B. 24 h after BMP4 treatment, cells were fixed, stained for expression of CDX2, and returned to the microscope for registration with the final time-lapse image. Mean nuclear OCT4-mCherry and CDX2 were quantified for each cell, and the resulting distribution was fit to a 2-component mixed Gaussian distribution representing self-renewing (OCT4+/CDX2-) and differentiated (OCT4-/CDX2+) cells (Appendix Figure S6). hESCs that could be assigned to either distribution with >99% confidence (red and green dots, Pdiff < 0.01 or Pdiff > 0.99) were considered for pro-fate analysis. Cells that did not reach this threshold (gray dots) were not used to determine pro-fate. Because cells in the final frame were assigned to only self-renewing or differentiated categories, pro-mixed cells (yellow traces in panel A do not persist to the final frame). C. Distributions of OCT4 levels in pro-self-renewing, pro-mixed, and pro-differentiated cell populations. D. Distributions of OCT4 pulse frequencies in pro-pluripotent, pro-mixed, and pro-differentiated cell populations. E. Distributions of cell cycle durations in pro-pluripotent, pro-mixed, and pro-differentiated cell populations. To gain an unbiased look at preexisting determinants of cell fate in panels D-E, only cells who completed their entire cell cycle duration before BMP4 addition (t = 0) were included in the analysis. F. Decision surfaces for logistic regression classifier. Cells with self-renewing (red) or differentiated (green) pro-fates are plotted above the predicted class surfaces. (left) Slice of decision surface for pulse frequency vs. OCT4 levels at a cell cycle duration of 14 h. (right) Slice of decision surface for cell cycle duration vs. OCT4 level at a pulse frequency of 0.25 h-1. * P < 0.05, ** P < 0.0005; ns, not significant.