EMBO/soda-nel huggingface dataset:
Exercise-induced α-ketoglutaric acid stimulates muscle hypertrophy and fat loss
through OXGR1-dependent adrenal activation (2020)
Figure 5

Figure 5. OXGR1 is required for the stimulatory effects of AKG on thermogenesis and lipolysis

(A). The mRNA expression of AKG-sensing genes in the adrenal gland tissue of 12-weeks male C57BL/6 mice (n = 4 per group).

(B). The mRNA expression of OXGR1 in different tissues of 12-weeks male C57BL/6 mice (n = 3 per group).

(C). OXGR1 localization in adrenal gland medulla indicated by fluorescent staining of OXGR1 (green).

(D). The mRNA expression of OXGR1 in the adrenal gland of male mice 3 hrs after i.p. injection of saline or 10 mg/kg AKG, or immediately after 40-mins resistance exercise (n=8 per group).

(E). Immunoblots and quantification of OXGR1 protein expression in adrenal chromaffin cells treated with negative control (NC) siRNA or siOXGR1 (n = 3 per group).

(F). E level in the medium from adrenal chromaffin cell cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 30 mins (n = 8 per group).

(G). Intracellular calcium ion [Ca2+] changes in adrenal medulla cell cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 (n = 30 per group).

(H-I). Body weight gain (H) and cumulative food intake (I) of male WT control (littermates) or OXGR1 global knock out (OXGR1KO) mice. At 12 weeks of age, both control and KO mice were switched to HFD and further divided into two groups, receiving tap water or water supplemented with 2% AKG for 13 weeks (n = 8 per group).

(J-K). Representative images of body composition (J) and fat and lean mass index (K) of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).

(L-M). Weight index of iWAT (L) and gWAT (M) in male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).

(N-O). Immunoblots (N) and quantification (O) of p-HSL and ATGL protein in gWAT of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 4 per group).

(P-S). Oxygen consumption (P-Q) and RER (R-S) of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).

Data information: Results are presented as mean ± SEM. In (A-B), (F), (K-M), (O), (Q) and (S), different letters between bars indicate p≤0.05 by one-way ANOVA followed by post hoc Tukey's tests. In (D), **p≤0.01 by one-way ANOVA followed by post hoc Dunnett's tests. In (E), **p≤0.01 by non-paired Student's t test. In (G), (H-I), *p≤0.05 by two-way ANOVA followed by post hoc Bonferroni tests.
Exercise-induced α-ketoglutaric acid stimulates muscle hypertrophy and fat loss
through OXGR1-dependent adrenal activation (2020)
Figure 6

Figure 6. OXGR1 expressed by adrenal gland mediates the stimulatory effects of AKG on thermogenesis and lipolysis

(A-B). Body weight gain (A) and cumulative food intake (B) of male OXGR1 adrenal-specific reexpression mice (OXGR1REAG). Male OXGR1KO mice (8 weeks) were adrenal-specifically injected with control HBAAV2/9-GFP (OXGR1KO control) or HBAAV2/9-OXGR1 (OXGR1REAG). Two weeks after injections, mice were switched to HFD and further divided into two groups, receiving tap water or water supplemented with 2% AKG for 12 weeks (n = 8 per group).

(C-D). Representative image of body composition (C) and fat and lean mass index (D) of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).

(E-F). Weight index of gWAT (E) and iWAT (F) in male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).

(G-H). Immunoblots (G) and quantification (H) of p-HSL and ATGL protein in gWAT of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).

(I-L). Oxygen consumption (I-J) and RER (K-L) of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).

Data information: Results are presented as mean ± SEM. In (A-B), *p≤0.05 by two-way ANOVA followed by post hoc Bonferroni tests. In (D-F), (H), (J) and (L), different letters between bars indicate p≤0.05 by one-way ANOVA followed by post hoc Tukey's tests.
Exercise-induced α-ketoglutaric acid stimulates muscle hypertrophy and fat loss
through OXGR1-dependent adrenal activation (2020)
Figure 7

Figure 7. OXGR1 is required for metabolic beneficial effects of resistance exercise

(A). Body weight gain in male WT littermates and OXGR1KO mice. At 8 weeks of age, male C57BL/6 WT control or OXGR1KO mice were switched to HFD. After 12 weeks of HFD feeding, mice were further divided into two groups, receiving non-exercise or resistance exercise for 14 days. (n = 8 per group).

(B). Exercise-induced body weight loss in male WT littermates and OXGR1KO mice. Body weights from exercise mice were subtracted by the average body weight of non-exercise control group for each genotype (n = 8 per group).

(C). Exercise-induced fat mass loss in male WT littermates and OXGR1KO mice. Fat mass from exercise mice were subtracted by the average fat mass of non-exercise control group for each genotype (n = 8 per group).

(D). Cumulative food intake of male WT littermates and OXGR1KO mice after 14-day resistance exercise (n = 8 per group).

(E-F). Weight index of gWAT (E) and iWAT (F) of male OXGR1KO mice after 14-days resistance exercise (n = 8 per group).

(G). Body composition of male OXGR1KO mice after 14-days resistance exercise (n = 8 per group).

(H). Serum AKG levels of male WT and OXGR1KO mice after resistance exercise. Male WT and OXGR1KO mice (10 weeks) fed with normal chow were receiving resistance exercise for 40 min (n = 8 per group). The serum AKG levels were tested before and immediately after exercise.

(I). Serum E level in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).

(J-L). The mRNA expression (J) and protein expression of UCP1 (K) in the BAT or the mRNA expression of HSL and ATGL (L) in the gWAT of male OXGR1KO mice after 14-day resistance exercise (n = 4 per group).

(M-P). Oxygen consumption (M-N) and RER (O-P) in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).

Data information: Results are presented as mean ± SEM. In (A-D) *p≤0.05, ** p≤0.01 by two-way ANOVA followed by post hoc Bonferroni tests. In (E-L), (N) and (P), different letters between bars indicate p≤0.05 by one-way ANOVA followed by post hoc Tukey's tests.
Exercise-induced α-ketoglutaric acid stimulates muscle hypertrophy and fat loss
through OXGR1-dependent adrenal activation (2020)
Figure 8

Figure 8. p65/NF-κB inflammatory pathway is required for the stimulatory effects of AKG on E release of adrenal chromaffin cell in vitro.

(A). Ingenuity Pathway Analysis (IPA) of AKG-induced transcriptome signature in adrenal chromaffin cell treated with AKG. The mRAN was extracted from adrenal chromaffin cells after 3-hrs incubation of vehicle or AKG (100 μM). (n = 3 per group).

(B-C). Immunoblots (B) and quantification (C) of p-IKK/IKK, p65, p-IκB/IκB and OXGR1 protein in adrenal chromaffin cells cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 3 hrs (n = 3 per group).

(D). p65 translocation in adrenal chromaffin cells cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 3 hrs (n = 3 per group). Scale bars, 100 μm.

(E-F). Immunoblots (E) and quantification (F) of p-IKK/IKK, p65 and p-IκB/IκB protein in adrenal chromaffin cells cultured with vehicle, AKG (100 μM), IKK inhibitor IKK16 or AKG + IKK16 for 3 hrs.

(G). E level in the medium from adrenal chromaffin cell cultured with vehicle, AKG (100 μM), IKK16 or AKG + IKK16 for 3 hrs (n = 10 per group).

Data information: Results are presented as mean ± SEM. In (C), (F) and (G), different letters between bars indicate p≤0.05 by one-way ANOVA followed by post hoc Tukey's tests.
Host succinate inhibits influenza virus infection through succinylation and nuclear retention of the viral nucleoprotein (2022)
Figure 1

Figure 1. Influenza virus infection increases succinate levels in airways.

(a) C57Bl/6 mice were infected (n=9) or not (n=8) intranasally with 150 pfu of influenza A virus (IAV; A/Scotland/20/74 (H3N2)). Bronchoalveolar lavage fluids (BALs) were collected 4 days post-infection. Samples were further processed for metabolomic analysis by NMR. Left panel shows a volcano-plot representing the comparison of "IAV-infected" versus "control, non-infected" animals. The graph plots the -log (p-value) against the fold change (log2 (ratio)) for individual metabolites. Vertical dashed blue lines mark the two-fold change and horizontal dashed blue line marks the cut-off p-value of 0.0001. Metabolites in the upper right square in red are significantly more abundant in "IAV-infected" compared to "control, non-infected" samples. Succinate is one of these compounds with a FC=2.2 and a FDR-adjusted p-value=8.10-7. The boxplots whiskers correspond to the minimum and maximum values, while the central band is the mean. The first and third quartiles (boxes) are merely the average values between the median and the extrema.

(b) shows succinate quantification by NMR from tracheal aspirates collected in mechanically-ventilated patients with (pink bar, n=9) or without (orange bar, n=7) diagnostic of IAV pneumonia. *P < 0.05.

(c) IL-6 and IL-8 measurements by ELISA from these same tracheal aspirates.

(d) Main baseline characteristics of the patients included in the study.

Data information: Data are mean ± SEM and statistical analysis was performed using the Mann-Whitney U-test.
Host succinate inhibits influenza virus infection through succinylation and nuclear retention of the viral nucleoprotein (2022)
Figure 6

Figure 6. The anti-influenza virus effect of succinate involves the nuclear retention of NP and NS1 proteins.

(a-c) Human bronchial epithelial BEAS-2B cells were infected with influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, then washed and treated or not with succinate (Suc; 4 mg/mL) for 20 h. (a, c) Expression of IAV proteins (i.e. PA, PB2, M2, M1, NP, NS1) was analyzed by confocal microscopy using specific antibodies. Viral proteins are stained in green, DNA in blue and actin in red. For a given antibody, pictures were taken on the exact same day with the same laser power settings, Pictures are representative of five independent experiments. Scale bar 10 µM. (b) Relative nuclear intensity of NS1 or NP was determined by using the Intensity Ratio Nuclei Cytoplasm Tool. Seven (NS1) or nine (NP) random images were collected per treatment with at least 5 cells per field. Data are represented as the mean ± SEM. Statistical analysis was performed using the Mann-Whitney test, (*P < 0.05).
Host succinate inhibits influenza virus infection through succinylation and nuclear retention of the viral nucleoprotein (2022)
Figure 8

Figure 8. Succinate reduces the assembly of influenza virus vRNPs by inducing succinylation of NP at K87, a highly conserved amino acid probably involved in vRNA binding.

Human bronchial epithelial BEAS-2B cells were infected with the influenza A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1 for 4 h, and subsequently treated or not with 4 mg/mL of succinate (Suc) for 20 h.

(a, b) Cells were stained using a monoclonal antibody that specifically recognizes "NP-vRNA" complexes, but not RNA-free NP, and confocal microscopy (a) was performed (green staining). The value raw integrated density (RawIntDen, which is the sum of all pixel values in the ROI (region of interest)) was further measured (b).

(c) Cells were lysed and proteins were digested to peptides with Lys-C and trypsin. Then, succinylated peptides were isolated directly from protease-digested protein extracts by immunoaffinity purification (IAP) using an antibody specific for the succinyl-Lysine motif. Next, the modified peptides were further analyzed by LC-MS/MS. Box plots show the distribution of intensities of the succinylated NP "YLEEHPSAGK87succDPK" peptide, computed as the integrated mass spectrometry peak area of the modified K87 succinyl peptide (n=3). The boxplots whiskers correspond to the minimum and maximum values, while the central band is the median. The first and third quartiles (boxes) are merely the average values between the median and the extrema.

(d) Interaction of recombinant of wild-type NP (NP K87) or a K87E mutant protein (NP K87E) with the indicated synthetic polyUC RNAs as measured by fluorescence anisotropy.

(e) Human bronchial epithelial BEAS-2B cells were infected with a A/Scotland/20/74 (H3N2) virus (IAV) carrying a wild-type NP (NP WT) or with the corresponding mutated virus bearing a NP with a K87R substitution (NP K87R). Cells were infected by either virus at MOI=1 for 4 h, then washed and treated or not with 4 mg/mL of succinate for 20 h. Localization of NP proteins was analyzed by confocal immunofluorescence microscopy. Scale bar: 10 µM. Pictures are representative of three independent experiments.

(f) Relative nuclear intensity of NP was determined by using the Intensity Ratio Nuclei Cytoplasm Tool.

(g) Plaque-Forming Unit assays determined the production of infectious viral particles in cell supernatants.

Data information: Data are represented as the mean ± SEM of 3 biological replicates with (g) or without (a, c-e) 2 technical replicates each. 8 (b) or at least 10 (f) random images were analysed per treatment with a minimum of 5 cells per field. Statistical analysis was performed using the Mann-Whitney test (b, c, d, f) or paired t-test (g), (*P < 0.05).
Sensitivity towards HDAC inhibition is associated with RTK/MAPK pathway activation in gastric cancer (2022)
Figure 5

Fig5: Targeting the EGFR/MAPK pathway at different levels of the signaling cascade.

(A) Scheme of EGFR/MAPK signaling pathway and targeting inhibitors.

(B-E) Drug response curves upon treatment with inhibitors for (B) EGFR (afatinib, gefitinib), (C) B‑RAF (LY3009120, TAK‑632, vemurafenib, dabrafenib), (D) MEK1/2 (selumetinib, binimetinib) and (E) ERK1/2 (ravoxertinib, ulixertinib) (biological replicates n=3, data are shown as mean ±SD).
Sensitivity towards HDAC inhibition is associated with RTK/MAPK pathway activation in gastric cancer (2022)
Figure 6

Fig6: Treatment of human patient derived organoids with MEK1/2 and HDAC inhibitors.

(A) Brigthfield images of human patient derived organoids (PDOs) from gastric cancer with and without RTK/MAPK alterations (scale bar 100µm, zoom in 40µm).

(B) Drug response curves of PDOs upon treatment with the MEK1/2 inhibitor trametinib (biological replicates n=3, data are presented as mean, SD values are shown in Table EV2).

(C) Drug response curves upon HDAC inhibition with panobinostat (PAN) and entinostat (ENT) (biological replicates n=3, data are presented as mean, SD values are shown in Table EV2).

(D) Comparison of the relative area under the curve (AUCrel) upon trametinib treatment in RTK/MAPK altered (mean AUCrel: 0.5308; biological replicate n=7) vs. non‑altered (mean AUCrel: 0.7101; biological replicates n=6) human PDOs (two‑tailed Student's t‑Test; *p= 0.0356).

(E) Z scores from PDOs and normal organoids treated with HDAC inhibitors (green: PDOs with RTK/MAPK alterations; black: PDOs without RTK/MAPK alterations; orange: normal PDOs).

(F) Correlation coefficients for the MEK1/2 inhibitor trametinib and the HDAC inhibitors PAN, ENT, tacedinaline (TAC) and vorinostat (VOR) by Pearson correlation.

(G) Comparison of the mean AUCrel of all RTK/MAPK altered vs. unaltered human PDOs upon HDAC inhibition with PAN, ENT, TAC and VOR (biological replicates n=4; two-tailed paired Student's t‑Test; **p= 0.0013).

Expanded View Figure Legends
Bclaf1 regulates c-FLIP and protects cells from TNF induced apoptosis and tissue injury (2021)
Figure 1

Figure 1. Bclaf1 deficiency sensitizes cells to apoptosis in response to TNF+CHX.

A. Bclaf1 wild type (WT) and two Bclaf1 knockout HeLa cell lines (KO-1 and -2) were pre-treated with DMSO or cycloheximide (CHX, 1 μg/ml) for 30 min, and then treated with TNF (10 ng/ml) together with CHX for 12 hours. Afterwards, the cells were subjected to Annexin V/7AAD staining followed by flow cytometry analysis, all Annexin V positive cells were counted for analysis, or protein extractions followed by western blotting analysis.

B, C. HeLa (B) or primary MEFs (C) were transfected with control (siCtrl) or two siRNAs against Bclaf1 (siBclaf1-1 and -2) and then treated and analyzed as described in A.

D, E. MEFs transfected with control (siCtrl) or siBclaf1-1 and/or siBclaf1-2 siRNAs were pre-treated with CHX (1 μg/ml)/z-VAD (20 μM) (D) or SM-164 (100 nM)/z-VAD (20 μM) (E) for 30 min, and then added TNF (10 ng/ml) for indicated times. The cells were subjected to Annexin V/7AAD staining after 9 hours followed by flow cytometry analysis for 7AAD positive and Annexin V negative cells (up), or protein extractions and western blotting analysis at indicated time periods (down).

Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; **p<0.01; ****p<0.0001. One-way ANOVA test.
Bclaf1 regulates c-FLIP and protects cells from TNF induced apoptosis and tissue injury (2021)
Figure 2

Figure 2. Bclaf1 deficiency does not affect TNF induced activation of NF-κB and MAPKs.

A, B. Cell lysates of HeLa Bclaf1 WT and KO cells (A), and HeLa cells transfected with siCtrl or siBclaf1-1 (B) treated with TNF (10 ng/ml) for indicated time periods were analyzed by western blotting with the indicated antibodies.

C, D. HeLa Bclaf1 WT and KO cells (C), and HeLa cells transfected with siCtrl or siBclaf1-1 (D) were treated with TNF (10 ng/ml) for the indicated time periods, and then subject to nucleus-cytoplasm fractionation as described in materials and methods followed by western blotting. Tubulin and histone H3 were used as loading controls for cytoplasmic and nuclear fractions, respectively.
Bclaf1 regulates c-FLIP and protects cells from TNF induced apoptosis and tissue injury (2021)
Figure 3

Figure 3. Bclaf1 deficiency inhibits c-FLIP expression and activates caspase 8.

A. HeLa cells transfected with siCtrl or siBclaf1-1 were treated with TNF (10 ng/ml) for the indicated time periods followed by western blotting analysis with the indicated antibodies.

B. MEFs treated with TNF (10 ng/ml) for 4 hours were subject to FADD immunoprecipitation, and then analyzed by western blotting.

C, D. MEFs (C) and HeLa (D) cells pretreated with CHX (1 μg/ml) for 30 min and then stimulated with TNF (10 ng/ml) in the presence of CHX for indicated times were subject to FADD immunoprecipitation, and then analyzed by western blotting.

E, F. Bclaf1 WT, KO-1 and -2 HeLa cells (E) or MEFs transfected with siCtrl or siBclaf1-1 and -2 (F) were pretreated with CHX (1 μg/ml) for 30 min and then stimulated with TNF (10 ng/ml) in the presence of CHX for 12 hours followed by western blotting analysis.

G, H. HeLa cells transfected with siRNAs against Bclaf1 or c-FLIP were pretreated with CHX (1 μg/ml) for 30 min and then stimulated with TNF (10 ng/ml) in the presence of CHX for 12 hours followed by Annexin V/7AAD staining and flow cytometry analysis (G), or protein extractions followed by western blotting analysis (H).

I. After transfection with siBclaf1-1 for 12 hours, the HeLa cells were transfected with Flag-FLIP for 24 hours, and then treated as described in H followed by western blotting analysis.

Data information: Data are shown as mean ± SD. n=3 biological replicates. **p<0.01; ****p<0.0001. One-way ANOVA test.
Bclaf1 regulates c-FLIP and protects cells from TNF induced apoptosis and tissue injury (2021)
Figure 4

Figure 4. Bclaf1 upregulates the transcription of CFLAR.

A-F. Bclaf1 WT, KO-1 and -2 HeLa cells (A and D), as well as HeLa (B and E) and primary MEFs (C and F) which were transfected with siCtrl or siBclaf1-1 and -2 were treated with TNF (10 ng/ml) for 6 hours followed by western blotting analysis (A-C), or for 4 hours followed by total RNA extraction and RT-PCR analysis (D-F).

G. CFLAR full length (FL) promoter ranging from -1179 to +281 and three promoter regions P1 (-1179 to -788), P2 (-788 to -288) and P3 (-288 to +281) were fused with a luciferase reporter and then co-transfected with an increase dose of Bclaf1 in HeLa-Bclaf1-KO cells. At 24 hours after transfection, the cells were collected and luciferase activity was measured. The experiments were performed in triplicates with a Renilla reporter in the transfection mixture for normalization and the activity in the control was arbitrarily set to 1.

H, I. CHIP analysis of the binding of Bclaf1 to the promoter of CFLAR was performed in HeLa-Flag-Bclaf1 cells treated without (H) or with TNF (I). The regions amplified by RT-PCR were delineated.

Data information: Data are shown as mean ± SD. n=3 biological replicates. *p<0.05; ***p<0.001; ****p<0.0001. One-way ANOVA test.
Bclaf1 regulates c-FLIP and protects cells from TNF induced apoptosis and tissue injury (2021)
Figure 5

Figure 5. Bclaf1 upregulates CFLAR through p50.

A. HeLa-Bclaf1-KO cells were transfected with the indicated siRNAs, and then transfected with CFLAR promoter and Bclaf1. The luciferase activity was measured as described before.

B. CFLAR promoter were co-transfected with Bclaf1 or/and p50 into HeLa-Bclaf1-KO cells, and the luciferase activity was measured as described before.

C. HeLa cells were transfected with siRNAs against Bclaf1 or p50. 24 hours post transfection, the cells were treated with TNF (10 ng/ml) for indicated hours. The total RNA was extracted and subjected to RT-PCR analysis.

D. HeLa cells were transfected with siCtrl or siRNA against Bclaf1 or p50, treated with TNF (10 ng/ml) for 6 hours followed by western blotting analysis.

E, F. HeLa cells were transfected with siCtrl or siRNA against Bclaf1 or p50 were pre-treated with DMSO or CHX (1 μg/ml) for 30 min, and then treated with TNF (10 ng/ml) for 12 hours. Cells were lysed and then analyzed by western blotting (E) or collected and subjected to Annexin V/7AAD staining followed by flow cytometry analysis, all Annexin V positive cells were counted for analysis (F).

G. CHIP analysis of the CFLAR promoter in HeLa-Flag-Bclaf1 cells. Immunoprecipitation was performed using anti-Flag and control IgG antibodies followed by RT-PCR analysis. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. One-way or Two-way ANOVA test.
Bclaf1 regulates c-FLIP and protects cells from TNF induced apoptosis and tissue injury (2021)
Figure 6

Figure 6. Bclaf1 interacts with p50.

A. HeLa-Flag-Bclaf1 and Bclaf1 WT HeLa cells treated with or without TNF (10 ng/ml) for 2 hours were separated into the cytoplasmic and the nuclear fraction. Endogenous Flag-Bclaf1 complex in each fraction was immunoprecipitated with M2 beads and analyzed by western blotting.

B. HeLa-Flag-Bclaf1 cells transfected with siCtrl or sip65 were treated with TNF (10 ng/ml) for 2 hours. The nuclear fraction was isolated and subjected to immunoprecipitation with an anti-Flag or control IgG antibody, followed by western blotting analysis.

C. 293T cells transfected with the indicated plasmids were immunoprecipitated with the anti-Flag M2 beads followed by western blotting analysis. A diagram of full-length Bclaf1 and its deletion mutants are presented.

D. CFLAR FL promoter were co-transfected with p50 and Bclaf1 FL or its individual deletion mutant into HeLa-Bclaf1-KO cells. The experiments were performed in triplicate with a Renilla reporter in the transfection mixture for normalization.

Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant. One-way ANOVA test.