EMBO/soda-nel huggingface dataset:
Pten controls B cell responsiveness and germinal center reaction by regulating expression of IgD BCR (2019)
Figure 3

Figure 3 | IgD BCR expression requires Pten

A, B | Splenocytes from mice of the indicated genotypes, (A) Ptenf/f, Ptenf/f x mb1-cre and Ptenf/f x mb1-cre 3-83ki mice on the respective backgrounds, (B) Ptenf/f x MD4tg and Ptenf/f x mb1-cre x MD4tg mice in presence and absence of antigen (ML5tg), were analyzed by flow cytometry. B cells were pre-gated by B220 and CD19 expression and the IgM/IgD surface expression (dot plots, left) was determined. Histograms (right) show a comparison of intracellular IgD expression (Ig-δHC ic) between the splenic populations of B cells (orange, identified by B220 and CD19 expression) and non-B cells (gray). Numbers in the histograms indicate the percentages of the positive populations. Data are representative of 11 - 35 individual mice per genotype.

C | Quantification of surface IgD mean fluorescence intensity (MFI) in Ptenf/f x MD4-trangenic mice in presence (mb1-cre +/+) and absence (mb1-cre +/ki) of Pten. Single dots represent the average MFI of 4 independent measurements per mouse (n), mean ±SD. Statistical significance was calculated by using an unpaired two-tailed t-test.

D | Serum anti-HEL titers. Sera from MD4tg mice of the indicated genotypes were adjusted to an IgM concentration of 500 μg/ml and applied in triplicates to HEL-coated plates in dilution steps of 1 : 3, mean ±SEM. Statistical significance was calculated by using the Kruskal-Wallis test (see also Table S4). Statements of significance in the figure refer to the comparison between Ptenf/f x MD4tg (filled red squares) and Ptenf/f x MD4tg x MD5tg sera (filled orange triangles), ** p ≤ 0.01; *** p ≤ 0.001.

E | Immunohistochemistry of sections from spleens of Ptenf/f, Ptenf/f x mb1-cre, Ptenf/f x mb1-cre x 3-83ki and Ptenf/f x mb1-cre x MD4tg mice for CD169 (green), CD5 (red) and IgM (cyan) at 10x magnification. Pictures in the second row show enlarged areas indicated by the white squares, respectively. Double-arrows indicate the distance between T cell (red) and marginal zone (green). Shown pictures are representative of 2 - 3 mice per genotype (see also Appendix Fig. S2).
Pten controls B cell responsiveness and germinal center reaction by regulating expression of IgD BCR (2019)
Figure 4

Figure 4 | Pten activates IgD expression and development of Fo.B cells

A | Schematic overview of the transduction procedure: Purified Ptenf/f x mb1-cre x MD4tg-derived mature splenic B cells were cultured in presence of LPS 2.5 μg/ml for 1.5 days and subjected to retroviral transduction with expression vectors encoding the constitutively active FoxO1 (FoxO1-A3) or EV, respectively.

B | 3 days post transduction the surface expression of IgM/IgD (dot plots, top), IgD and CD23 (histograms, bottom) was measured by flow cytometry and compared between EV- and FoxO1-A3-transduced cells. Fluorescence minus one (FMO) stainings, lacking α-IgD or α-CD23 antibodies respectively, served as controls (ctrl). Bar diagrams below flow cytometry data display the percentages of IgD+ cells (n = 12; left) and MFI of CD23 (as relative units, RU), normalized to the MFI measured in the respective EV transduced cells; n = 12; middle). Fcer2 transcript levels were measured by qRT-PCR (right, n = 4). Mean ±SD, symbols indicate data from individual mice. Statistical significance of IgD expression was determined by applying the Wilcoxon matched-pairs signed rank test for CD23 and by applying the one sample two-tailed t-test for Fcer2 expression.

C | Cstf64 (n = 8), Ell2 (n = 8) and Zfp318 (n = 11) expression levels were measured at 3 days post transduction by qRT-PCR. FoxO1-A3- and EV-transduced splenic B cells from Fig. 4B were FACS-purified according to the gating strategy shown in Fig. EV2B. Mean ±SD, symbols indicate the expression in individual mice. Statistical significance was calculated by using the one-sample two-tailed t-test.

D | Schematic overview of transduction procedure: Purified FoxO1f/f-derived mature splenic B cells were cultured in presence of 2.5 μg/ml LPS for 1.5 days and subjected to retroviral transduction with expression vectors encoding either cre or EV, respectively.

E, F | Identical to Fig. 4B-C using cells described in Fig. 4D, E: IgD (n = 6), CD23 (n = 4) and Fcer2 (n = 4). F: Cstf64 (n = 4), Ell2 (n = 4) and Zfp318 (n = 4).
Pten controls B cell responsiveness and germinal center reaction by regulating expression of IgD BCR (2019)
Figure 5

Figure 5 | Monovalent antigen controls IgD BCR activation

A | Schematic overview of reconstitution of BCR-deficient Ramos (HL-KO) cells with murine HEL-specific BCR of IgM (HH10-mu IgM) and IgD (HH10-mu IgD) isotype, respectively.

B | Representative flow cytometric analysis of Ramos cells reconstituted with HH10-mu IgM, HH10-mu IgD. Cells were stained with α-murine μHC-, δHC- and κLC-antibodies, respectively. EV-transduced HL-KO cells, expressing GFP only, were used as negative control (gray).

C | Representative flow cytometric analysis of HEL-binding in reconstituted Ramos cells after staining with fluorescently-labeled HEL. EV-transduced HL-KO cells, expressing GFP only, were used as negative control (gray).

D | Representative intracellular Ca2+ influx in HH10-mu IgM- and HH10-mu IgD-expressing cells upon stimulation with multivalent HEL (complex cHEL), monovalent (soluble sHEL) (both at a concentration of 1 μg/ml), or 10 μg/ml α-mouse κLC antibody, respectively.

E | Representative intracellular Ca2+ influx of HH10-mu IgM- (top) and HH10-mu IgD- (bottom) expressing cells upon stimulation with indicated ratios of 1 μg/ml cHEL and sHEL, and a 1 : 25 mixture of cHEL with bovine serum albumin (BSA), where 1 = 1 μg/ml.

F | Schematic overview of reconstitution of BCR-deficient Ramos (HL-KO) cells with HH10-specific human IgM (HH10-hu IgM) and IgD (HH10-hu IgD) BCR isotype.

G - J | Identical to Fig. 5B-E using HH10-hu IgM- and HH10-hu IgD-expressing cells instead of HH10-mu IgM- and HH10-mu IgD-expressing cells, respectively. Data shown in B to E and G to J are representative of minimum three independent experiments.
Pten controls B cell responsiveness and germinal center reaction by regulating expression of IgD BCR (2019)
Figure 6

Figure 6 | IgD expression modulates B cell responsiveness

A | Quantification of surface IgM (top) and IgD (bottom) MFI in B cells from mice of the indicated genotypes. Single dots represent the average MFI of 4 independent measurements per mouse (n), mean ±SD. IgD expression of Ptenf/f x MD4tg and Ptenf/f x mb1-cre x MD4tg B cells already shown in Fig. 3C. Statistical significance was calculated by using the Kruskal-Wallis test (see also Table S5), n. s. = not significant; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

B | Representative intracellular Ca2+ influx measured in Thy1.2- splenocytes, derived from mice of the indicated genotypes, upon stimulation with cHEL, monovalent sHEL (both at a concentration of 1 μg/ml) or 10 μg/ml α-mouse κLC antibody, respectively. Line diagrams below the dot plots show the overlayed MFI of the Ca2+ influx kinetics displayed in the plots above.

C | Quantification of intracellular Ca2+ influx from Fig. 6B. The median area under the curve of the Ca2+ influx kinetics was calculated, single dots represent data from individual mice, mean ±SD. Statistical significance was calculated by applying the Mann-Whitney-U test (see also Table S6), n. s. = not significant, ** p ≤ 0.01.
Pten controls B cell responsiveness and germinal center reaction by regulating expression of IgD BCR (2019)
Figure 7

Figure 7 | Abnormal germinal center reaction in IgD-deficient mice

A | Schematic overview over the procedure of mouse immunization with 2,4,6-trinitrophenyl hapten conjugated to ovalbumin (TNP-Ova) and analysis of germinal center (GC) reactions at different time intervals. Control animals were injected intraperitoneally (i. p.) with PBS and quantified as day 0 of immunization.

B | Analysis of GC B cells in splenic B220+ B cells from wild type (WT, top) and IgD-/- (bottom) animals after 4, 7 and 10 days (left to right) post immunization. Percentages of follicular B cells (Fo.B; CD38+/CD95-) and GC B cells (CD38-/CD95+) are depicted in the plots. GC B cells (CD38-/CD95+) were further analyzed for GL-7 expression, compared to Fo.B cells and quantified.

C | Quantification of Fo.B cells (top, percentages) and CD38-/CD95+/GL-7+ GC cells (percentages and absolute numbers, bottom panels) in WT (black) and IgD-/- (green) animals after 4, 7, 10 and 14 days post immunization, mean ±SD (numbers of animals indicated in the top plot). Statistical significance was analyzed by a two-tailed unpaired t-test.

D | Analysis of GC in spleen sections from WT and IgD-/- animals after 4, 7 and 10 days post immunization. Representative images of 2 x 2 mm sections stained with DAPI (nuclei) and peanut agglutinin (PNA) are shown for the indicated time points. Scale bar represents 500 μm. Dashed white or yellow lines mark distinct GC foci in the merged images.

E | Representative 800 x 800 μm regions of areas marked by yellow dashed lines within the images of D are shown as enlarged single GCs. As no distinct GC foci were observed in IgD-/- samples from days 4 and 7, an identical 800 x 800 μm region is shown in each case.

F | Quantification of number of GCs per mm2 area of spleen sections from WT (black) and IgD-/- (green) animals after 4, 7 and 10 days post immunization. Data represent mean ±SD of two sections (from 3 animals per group and time point) and were analyzed by two-tailed unpaired t-test.

G | Total serum α-TNP IgG. Sera from PBS- or TNP-Ova-immunized WT (left) and IgD-/- (middle) mice, collected at day 7 following immunization, were adjusted to an IgG concentration of 1 μg/ml and applied in duplicates and dilution steps of 1 : 3 to TNP-coated plates, respectively, mean ±SEM. Right: Overlay of α-TNP IgG titers from TNP-Ova-immunized WT and IgD-/- mice. Statistical significance was calculated by using the Mann-Whitney-U test (see also Table S7), n. s. = not significant, * p ≤ 0.05, ** p ≤ 0.01.

Figure Legends - Expanded View Figures
Tamoxifen mechanically reprograms the tumor microenvironment via HIF-1A and reduces cancer cell survival (2018)
Figure 1

Figure 1: Tamoxifen decreases hypoxia and increases vascularisation: (A) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control of tamoxifen, scale bar 100 μm. (B,D) Quantification of GLUT1 (hypoxia marker) and CD31 (endothelial cell marker). Control (n=5), 2mg (n=5), and 5mg (n=4). In all cases, bars represent mean ± s.e.m. (C) Relative values of protein levels for Glut1 in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates). (E,F) Expression levels of DEGs relevant to response to hypoxia (left) and blood vessel morphogenesis (right). The values were normalized by tubulin family genes. (G) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control and 5mg of tamoxifen, scale bar 100 μm. (H) Quantification of HIF-1A in PDAC tissues Control (n=5), and 5mg (n=4). In the box-and-whisker plot, the central box represents values from the lower to upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum value. (I) qPCR levels of HIF-1A in PSCs, normalized to RPLP0 and relative to control. (J) Western blot bands for protein expression in PSCs (p-Tmod is post-translational modification). The plot shows the quantification of the sum of band intensities corresponding to isoform 1, isoform 2, isoform 3, and post-transcriptionally modified HIF-1A (n=8 control and n=8 tam). All histogram bars represent mean±s.e.m.; *P<0.05,**P<0.01,***P<0.001 (t-test for H; Anova and Tukey's post hoc test for B, C, I). For A and G n≥ 5 sections per animal. Results collected during 3 or more separate experiments.
Tamoxifen mechanically reprograms the tumor microenvironment via HIF-1A and reduces cancer cell survival (2018)
Figure 2

Figure 2: Tamoxifen reduces LOX-L2 levels in PSCs and PDAC tissues. (A) qPCR levels of LOX-L2 in PSCs, normalized to RPLP0 and relative to control. (B) Western blot levels of LOX-L2 in PSCs (n=3 experimental replicates, **P<0.01). (C) Expression of LOX family genes obtained from RNA-seq data in control and tamoxifen treated PSCs (n = 3 experimental replicates). Expression value was normalized by tubulin family genes. (D) qPCR levels of LOX family in PSCs, normalized to RPLP0 and relative to control. (E) Relative values of protein levels for LOX members in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates) (F) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxifen, scale bar 50 μm. (G) Quantification of LOX-L2 for images in F. n ≥ 4 mice per condition, and n> 10 sections per animal. (H) qPCR levels of LOX-L2 and HIF-1A in PSCs, normalized to RPLP0 and relative to 1 kPa. (I) qPCR levels of LOX-L2 and HIF-1A in PSCs, normalized to RPLP0 and relative to control. (J) Quantification of average forces applied by PSCs on pillars. BBI = blebbistatin. In the box and whisker plot, the central box represents values from the lower and upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum. ***P<0.001 Three experimental repeats. (K) qPCR levels of LOX-L2 in PSCs, normalized to RPLP0 (60S acidic ribosomal protein P0) and relative to control. In all cases, histogram bars represent mean±s.e.m., ***P<0.001 (t-test for B, D, I; Anova and Tukey's post hoc test for A, H, and J).
Tamoxifen mechanically reprograms the tumor microenvironment via HIF-1A and reduces cancer cell survival (2018)
Figure 4

Figure 4. Tamoxifen decreases synthesis and secretion of collagen and MMP-2 in PSCs and PDAC tissues. (A) qPCR levels of collagen in PSCs, normalized to RPLP0 and relative to control. (B) Western blot analysis of collagen normalised to total protein and relative to control. (C) Quantification of time-lapse collagen synthesis and deposition by PSCs. (D) Representative immunofluorescent images used for the quantification in C, scale bar 20 μm, collagen-I was assessed with a specific primary antibody staining. (E) Relative values of protein levels for collagen in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates). (F) qPCR levels of MMP-2 in PSCs, normalized to RPLP0 and relative to control (G) MMP-2 activity on control and tamoxifen treated PSCs assayed by gelatin zymography; above signal intensity of the representative bands used for the quantification presented in the plot below. (H-I) Immunohistochemistry images and quantification of MMP-2 levels in PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxifen, scale bar 100 μm (n=4 animals per condition and n≥ 5 sections per animal). (J) Relative values of protein levels for MMP-2 in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates). (K) qPCR levels of MMP-2 in PSCs, normalized to RPLP0 and relative to 1 kPa. (L) qPCR levels of MMP-2 in PSCs, normalized to RPLP0 and relative to control. Histogram bars represent mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001 (t-test for group of two data sets and Anova and Tukey post hoc test for the rest).
Tamoxifen mechanically reprograms the tumor microenvironment via HIF-1A and reduces cancer cell survival (2018)
Figure 5

Figure 5: Tamoxifen decreases fibronectin levels in PSCs and PDAC tissues. (A) qPCR levels of fibronectin (FN), fibronectin extracellular domain A (FN-EDA), and fibronectin extracellular domain B (FN-EDB) in PSCs, normalised to RPLP0 and relative to control. (B) Western blot analysis of FN normalised to total protein and relative to control. (C) Quantification of fibronectin intensity density of images presented in D. (D) FN fluorescence and SHG collagen images for matrigel collagen gels remodelled by PSCs. (E) Immunohistochemistry images of PDAC tissues from KPC mice treated with vehicle control, and 2mg, and 5mg of tamoxifen. (n=4 animals per condition, and n ≥ 5 sections per animal). (F) FN fiber thickness colour-code map in E represented through the BoneJ plugin. (G) Relative values of protein levels for FN in PDAC tumors assessed by proteomic analysis. (H-J) Quantification of fibronectin immunohistochemistry staining, fiber thickness and alignment scored of images presented in e. In all cases, histogram bars represent mean ± s.e.m.; *P<0.05,**P<0.01,***P<0.001 (t-test for A, B, C; Anova and Tukey's post hoc test for H, I, J). Scale bar is 100 μm.
Tamoxifen mechanically reprograms the tumor microenvironment via HIF-1A and reduces cancer cell survival (2018)
Figure 6

Figure 6: Tamoxifen treatment decreases proliferation and increases apoptosis in epithelial cells of PDAC tissues. (A) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control, and 2mg of tamoxifen, scale bar is 100 μm. Upper panels: Ki67 staining is used as a surrogate of proliferation. White arrows indicate Ki67 positive nuclei in epithelial cells. Lower panels: Cc3 staining shows the cells undergoing caspase-mediated apoptosis. Tamoxifen panels show higher levels of yellow staining, which indicates higher percentage of apoptotic epithelial cells. (B) Quantification of staining in panel A (n=4 animals per condition, and n≥ 5 sections per animal, two experimental repetitions). Histogram bars represent mean ± s.e.m.; ***P<0.001, t-test.
Tamoxifen mechanically reprograms the tumor microenvironment via HIF-1A and reduces cancer cell survival (2018)
Figure 7

Figure 7: Tamoxifen treatment decreases HIF-1A levels and proliferation and increases apoptosis in Suit-2 pancreatic cancer cells. (A, E, F) Immunofluorescence images of control and tamoxifen treated suit-2 cells, scale bar is 20 μm. Panel A represents HIF-1A staining in hypoxia and non-hypoxia conditions, panels E and F show Ki67 and Cc3 staining as markers of proliferation and caspase-mediated apoptosis, respectively. Panel E: Red - F-actin, green - Ki67, blue - nuclei. Tamoxifen negatively regulates HIF-1A in hypoxia and non-hypoxia conditions. (B,C,D) Quantification of immunofluorescence staining in panels A, E, F. For quantification, eight fields of view (n>50 cells) per condition. Histogram bars represent mean ± s.e.m.; ***P<0.001, t-test. All panels include data collected during 3 independent experiments.
SIRT5 impairs aggregation and activation of  MAVS through catalyzing lysine desuccinylation (2020)
Figure 1

Figure 1. SIRT5 suppresses the MAVS-mediated type-I IFN signaling.

A-B IFN-β promoter activity (A) and ISRE reporter activity (B) in Myc empty vector (200 ng) or Myc-SIRT5 (200 ng)-transfected HEK293T cells with or without SeV infection (SeV or UI) for 18~24 h.

C-D IFN-β promoter activity (C) and ISRE reporter activity (D) in Myc empty vector (200 ng) or Myc-SIRT5 (200 ng)-transfected H1299 cells with or without SeV infection (SeV or UI) for 18~24 h.

E-F IFN-β promoter activity (E) and ISRE reporter activity (F) in the indicated siRNA-transfected H1299 cells (si-NC, si-SIRT5#1 and si-SIRT5#2) with or without SeV infection (SeV or UI) for 18~24 h. NC, negative control.

G-H IFN-β promoter activity (G) and ISRE reporter activity (H) in SIRT5-deficient H1299 cells (SIRT5 -/-) or the wildtype (WT) H1299 cells (SIRT5 +/+) with or without SeV infection (SeV or UI) for 18~24 h.

I ISRE reporter activity in Myc-SIRT5 (0, 100 ng or 200 ng)-transfected HEK293T cells with or without poly(I:C) transfection for 18~24 h.

J ISRE reporter activity in co-transfection of Myc-RIG-I (200 ng) together with Myc-SIRT5 (0, 100 ng or 200ng)-transfected HEK293T cells for 24 h.

K ISRE reporter activity in co-transfection of Myc-MDA5 (200 ng) together with Myc-SIRT5 (0, 100 ng or 200 ng) in HEK293T cells for 24 h.

L ISRE reporter activity in co-transfection of HA-MAVS (200 ng) together with Myc-SIRT5 (0, 100 ng or 200 ng) in HEK293T cells for 24 h.

M ISRE reporter activity in co-transfection of Myc-TBK1 (200 ng) together with Myc-SIRT5 (0, 100 ng or 200 ng) in HEK293T cells for 24 h.

N ISRE reporter activity in co-transfection of HA-IRF3 (200 ng) together with Myc-SIRT5 (0, 100 ng or 200 ng) in HEK293T cells for 24 h.

O ISRE reporter activity in co-transfection of HA-MAVS (200 ng) together with Myc-SIRT5 (200ng) or Myc-SIRT5-H158Y(200ng) respectively in HEK293T cells for 24 h.

Data information: Graphs represent fold-induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).
SIRT5 impairs aggregation and activation of  MAVS through catalyzing lysine desuccinylation (2020)
Figure 2

Figure 2. SIRT5 interacts with MAVS.

A Confocal microscopy image of endogenous SIRT5 co-localized with endogenous MAVS in H1299 cells detected by immunofluorescence staining using anti-SIRT5 and anti-MAVS antibodies. Mito, Mitotracker; Scale bar = 8 µm.

B-C Co-immunoprecipitation of Myc-SIRT5 with HA-MAVS and vice versa. HEK293T cells were co-transfected with indicated plasmids for 24 h. Anti-Myc (B) or anti-HA antibody conjugated agarose beads (C) were used for immunoprecipitation and the interaction was detected by immunoblotting with the indicated antibodies.

D Endogenous interaction between MAVS and SIRT5. Anti-MAVS antibody was used for immunoprecipitation, and normal mouse IgG was used as a control.
E Endogenous interaction between MAVS and SIRT5 in the wildtype (WT) (MAVS +/+) or SIRT5-deficient (MAVS -/-) H1299 cells. Anti-MAVS antibody was used for immunoprecipitation, and the interaction was detected by immunoblotting with anti-SIRT5 antibody.
F GST pull-down assay for GST-tagged MAVS and His-tagged SIRT5. GST-tagged MAVS and His-tagged SIRT5 were expressed in Escherichia coli (BL21), respectively. The association of GST-MAVS with His-SIRT5 was detected by immunoblotting with anti-SIRT5 antibody. GST and GST-MAVS proteins were stained with Coomassie blue.

G Schematic of MAVS domains interacted with SIRT5. The interaction is indicated by plus (+) sign.

H-I Co-immunoprecipitation analysis of HA-SIRT5 with Flag-MAVS-truncated mutants. HEK293T cells were co-transfected with the indicated plasmids. Anti-Flag antibody conjugated agarose beads were used for immunoprecipitation and the interaction was analyzed by immunoblotting with the indicated antibodies. Flag-MAVS fragments (WT: full length; ΔC1, 1-173 aa; ΔC2, 1-513 aa; ΔN1, 91-540 aa; ΔN2, 173-540 aa; ΔCARD, Δ10-77 aa; ΔPR, Δ103-173 aa; ΔTM, Δ514-535 aa).

Data information: IP, immunoprecipitation; TCL, total cell lysates.