EMBO/soda-nel huggingface dataset:
LSD1 inhibition induces differentiation and cell death in Merkel cell carcinoma (2020)
Figure 7

Figure 7. Pharmacological LSD1 inhibition induces a sustained change in cell fate.

A. Schematic of drug pulse and wash-out experiment. PeTa cells were treated with 100 nM GSK-LSD1 or DMSO. After 24h, a cell sample was harvested (T0, ON-drug) and the remaining cells were washed and changed into fresh medium without drug. Cell samples were taken 24h, 72h, 6 days (D6) and 8 days (D8) after drug wash-out (OFF-drug) for downstream readouts.

B. RT-qPCR quantification of neuronal genes at the different time points and conditions depicted in Fig 7A. Data are relative to the housekeeping gene HPRT1 and normalized to the respective DMSO control. n = 4 technical replicates. Bar graphs represent mean ± SD.

C. Immunoblot probing for sustainability of LSD1 activity inhibition at the different time points and conditions depicted in Fig 7A. α-Tubulin serves as loading control. pSMAD1/5/9, phospho-SMAD1/5/9.

D. Immunoblot probing for LSD1-CoREST complex members at the different time points and conditions depicted in Fig 7A. α-Tubulin serves as loading control.

E. Representative FACS readout of EdU/Propidium iodide cell cycle staining of PeTa cells 8 days after drug wash-out.

F. Quantification of EdU/Propidium iodide staining depicted in Fig 7E. n = 3 biological replicates. Data are represented as means ±SD. *p<0.05, **p<0.01; ***p<0.001 (G1 phase: DMSO vs GSK-LSD1 p = 0.0104; S phase: DMSO vs GSK-LSD1 p = 0.0001; G2/M phase: DMSO vs GSK-LSD1 p = 0.0062; unpaired Student's t-test).

G. Quantification of FACS Annexin V/DAPI staining of PeTa cells 8 days after drug wash-out. n = 3 biological replicates. Data are represented as means ±SD. ****p<0.0001 (unpaired Student's t-test).

H. Schematic depicting the experimental setup of the in vivo tumor growth of pre-treated cells. PeTa cells were pre-treated in vitro with 100 nM GSK-LSD1 for 1, 3 or 6 days, or DMSO treated for 6 days prior injection.

I. Subcutaneous tumor growth of pretreated cells (untreated, n = 8; D1, n = 9; D3, n = 9; D6, n = 4) Data are represented as means ±SEM. *p<0.05, ***p<0.001 (untreated vs D1 p = 0.023; untreated vs D3 p = 0.023; untreated vs D6 p = 0.0007; Kruskal-Wallis test).
RKIP and TBK1 form a positive feedback loop to promote type I interferon production in innate immunity (2016)
Figure 1

Figure 1. RKIP is required for viral infection and poly(I:C)-induced IFN-β and proinflammatory cytokine production in macrophages.

A-C. Enzyme-linked immunosorbent assay (ELISA) of IL-6, TNF-α and IFN-β protein and real-time PCR analysis of IL-6, TNF-α and IFN-β mRNA in peritoneal macrophage isolated from wild type and RKIP-deficient mice treated with VSV (MOI,0.1) (A) and SEV (MOI,10) (B) or transfected with poly(I:C) ( 1 μg/ml) (C).

D. Fluorescence-activated cell sorting (FACS) and TCID50 (50% tissue culture infective dose) assessing the proliferation level of VSV. RAW264.7 cells line stably overexpressed flag-RKIP was infected with VSV-eGFP at MOI of 1 for the indicated time. The fusion protein fluorophore were analyzed by FACS. Then fresh medium with the VSV-eGFP infection was serially diluted on the monolayer of HEK293 cells for 3-7 days and TCID50 was measured.

E. FACS analysis the infection of wild type and RKIP-deficient peritoneal macrophage infected with VSV-eGFP at MOI of 1. The fresh medium with the VSV-eGFP infection 9 hr were collected for TCID50 assessing.

Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***p < 0.001 (unpaired, two-tailed students’s test). Data are representative of three independent experiments with similar results. See also Figure EV1
RKIP and TBK1 form a positive feedback loop to promote type I interferon production in innate immunity (2016)
Figure 2

Figure 2. RKIP is required for anti-virus innate immunity in vivo.

A. Wide-type (RKIP+/+) and RKIP deficiency (RKIP-/-) chimera were intraperitoneally injected with VSV (1x107 pfu/g), 12 hours later, ELISA of IFN-β and IL-6 in serum (n=6 per group).

B. Determination of VSV loads in organs by TCID50 assay 12 hours later after RKIP+/+ and RKIP-/- chimera mice (n = 6 per group) intraperitoneally injected with VSV (1x107 pfu/g).

C. Real-time PCR analysis of IFN-β mRNA and VSV expression in liver from RKIP+/+ and RKIP-/- chimera mice 12 hours later intraperitoneally injected with VSV (1x107 pfu/g) (n = 6 per group).

D. Pathology of wild type and RKIP deficient chimeric mice in response to VSV infection. Hematoxylin and eosin staining of liver and lung sections from mice in (A). Scale bar, 100 μm (left) or 50 μm (right).

E. Real-time PCR analysis of IFN-β mRNA expression in peritoneal macrophages from RKIP+/+ and RKIP -/- chimera mice 12 hours after intraperitoneally injected with VSV (1x107 pfu/g) (n = 6 per group).

F. Survival curve for 8 weeks-old wild type and RKIP deficient chimeras challenged with VSV (1x108 pfu/g, i.v.) (n=10 per group). *p<0.05（Wilcoxon Test）.

Data are means ± SEM. *p < 0.05, ***p < 0.001 (unpaired, two-tailed students’s test). Data are representative of two independent experiments. See also Figure EV2
RKIP and TBK1 form a positive feedback loop to promote type I interferon production in innate immunity (2016)
Figure 3

Figure 3. RKIP is required for VSV and poly(I:C)-induced TBK1, IRF3 and p38 phosphorylation.

A. Immunoblot analysis of p-TBK1, -IKKi, -IRF3, -P38, -JNK1/2, -ERK1/2 and -IκBα in lysates of RKIP+/+, RKIP+/- and RKIP -/- peritoneal macrophages stimulated with VSV (MOI, 0.1) for the indicated time.

B. Immunoblot assay of RKIP+/+, RKIP+/- and RKIP -/- peritoneal macrophage cells treated with intracellular poly(I:C) (1 μg/ml).

C. Immunoblot analysis of phosphorylated (p-) TBK1, IKKi, IRF3, P38, JNK1/2, ERK1/2 in peritoneal macrophage transfected with RKIP-specific or scrambled siRNA, followed by infection with VSV (MOI, 0.1). β-actin was used as a loading control.

D. Immunoblot analysis of p- TBK1, -IKKi, -IRF3, -P38, -JNK1/2, -ERK1/2 and -IκBα in lysates of VSV-treated RAW264.7 cells stably over-expressing RKIP.

E. Immunoblot analysis in lysates of RAW264.7 cells stably overexpressing RKIP treated with intracellular poly(I:C) (1 μg/ml).

F. Luciferase activity in 293T cells transfected with plasmid encoding a luciferase reporter for IFN-β (IFN-β-luc) or ISRE (ISRE-luc), and an expression vector for RKIP (0, 50, 100, 200 ng each), together with plasmids encoding RIG-I CARD, TBK1, IKKi and IRF3 5D.

Data are shown as mean ± SEM. *P<0.05, **P<0.01,***P<0.001 and ns, not significant (unpaired, two-tailed students’s test). Data are representative of at least three independent experiments. See also Figure EV3
RKIP and TBK1 form a positive feedback loop to promote type I interferon production in innate immunity (2016)
Figure 4

Figure 4. RKIP interacts with TBK1 and promotes TBK1 autophosphorylation.

A. Immunoblot of extracts of RAW264.7 cells stably overexpressing flag-RKIP infected with VSV (MOI, 0.1) for the indicated times followed by immunoprecipitation (IP). WCL, whole cell lysates.

B. Coimmunoprecipitation and immunoblot analysis of 293T cells transfected with vector for myc-RKIP alone or with the deletion mutants of TBK1. Numbers in parentheses indicate amino acids included in construct. FL, full length; KD, kinase domain; ULD, ubiquitin-like domain; SDD, α-helical scaffold domain; NBD, C-terminal domain.* indicates targeted band.

C. Immunoblot of 293T cells transfected with vector for myc-TBK1 together with plasmid for HA-GSK3β or HA-RKIP, followed by immunoprecipitation. * indicates targeted band.

D. In vitro kinase assay was carried out in the reaction mixture containing 2 mM ATP, 100 μM GST-RKIP or 100 μM GST-GFP and 5 μM flag-TBK1 at 30 °C for 30 min and then followed by immunoblotting with the indicated Antibody.

Data are from one experiments representative of three independent experiments with similar results. See also Figure EV4
RKIP and TBK1 form a positive feedback loop to promote type I interferon production in innate immunity (2016)
Figure 5

Figure 5. RKIP promotes anti-viral immune response in a S109 phosphorylation-dependent manner.

A. Real-time PCR analysis of IL-6, TNF-α and IFN-β mRNA in RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D, followed by infection with VSV (MOI, 0.1).

B. Immunoblot analysis of p- TBK1, -IRF3, -P38 of cells as in (A).

C. FACS analysis of RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D infected with VSV-eGFP at MOI of 1.

D. Immunoblot analysis of phosphorylated-TBK1, -IRF3, -P38 in lysates of RKIP+/+ or RKIP-/- bone marrow derived macrophages (BMDM) transfected with flag-RKIP, flag-S109A and flag-S109D, and then stimulated with VSV (MOI, 0.1).

E. ELISA of IFN-β and TNF-α of cells in (D).

Data are mean ± SEM. *p < 0.05, **P<0.01,***P<0.001 and ns, not significant (unpaired, two-tailed students’s test). Data are representative of three (A-C) or two (D-E) independent experiments. See also Figure EV5
RKIP and TBK1 form a positive feedback loop to promote type I interferon production in innate immunity (2016)
Figure 6

Figure 6. TBK1 phosphorylates RKIP at serine109.

A. BMDM cells were infected with VSV (MOI, 0.1) or HSV (MOI, 5) for the indicated times. Cell lysates were analyzed by immunoblotting with the indicated antibodies.

B. BMDM cells were transfected with control or TBK1 siRNA. 72 hours after transfection, cells were analyzed by immunoblotting with antibody for phospho-RKIP or TBK1.

C. Immunoblot analysis of RKIP phosphorylation in control or TBK1-/- mouse embryonic fibroblasts after VSV (MOI, 0.1) infection for the indicated times.

D. Recombinant flag-tagged TBK1 was immunoprecipitated with anti-flag M2 beads in flag-TBK1 transfected 293T cells. Followed by in vitro kinase assay, which was carried out in the reaction mixture containing 2 mM ATP, 100 μM GST-RKIP or 100 μM GST-GFP together with 5 μM flag-TBK1 at 30 °C for 30 min. Assay mixtures were immunoblotted with the indicated antibody for phospho-RKIP, flag, GST or his.

E. Immunoblot analysis of extracts of RAW264.7 cells stably overexpressing flag-RKIP, flag-S109A and flag-S109D, infected with VSV(MOI, 0.1) for the indicated times, followed by immunoprecipitation with anti-flag beads.

F. BMDM cells were infected with VSV (MOI, 0.1) for the indicated times. The cell lysates were immunoprecipitated with anti-TBK1 (left) or anti-RKIP (right). The immunoprecipitates were analyzed by immunoblot with anti-RKIP or anti-TBK1 antibody. The levels of the endogenous RKIP, TBK1, and p-RKIP were detected by immunoblot analysis.

G. Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with myc-TBK1 alone or with flag-RKIP, flag-S109A or flag-S109D.

H. GST-pull down experiment was performed with 1 μg fusion protein GST-GFP, GST-his-RKIP or GST-his-S109D mixed with 20 μl precleared agarose beads in 800 μl reaction medium, followed by adding 1 μg flag-TBK1 and incubating at 4°C for 3 hours with gentle rotation. Pull down (1st-3th lane) and input samples (4th lane) were separated by SDS-PAGE followed by immunoblotting with anti-GST or anti-flag antibody.

I. Immunoprecipitation and immunoblot analysis of 293T cells transfected with indicated combinations of vector for myc-TBK1, flag-RKIP, flag-S109A, flag-S109D and HA-GSK3β or flag-GFP.

J. In vitro assay was carried out in the reaction mixture containing 2 mM ATP, 50 μM GST-TBK1 together with 100 μM GST-his-RKIP, 100 μM GST-his-S109D, 100 μM GST-his-S109A or 100 μM GST, respectively. React at 30 °C for 30 min. Then followed by immunoblotting with anti-p-TBK1or anti-GST antibody.

Data are representative of at least three independent experiments with similar results. See also Appendix Figure S1, S2
Cardiac-specific Succinate Dehydrogenase Deficiency in Barth Syndrome (2015)
Figure 1

Figure 1. Cardiolipin species composition and effects on cardiac function in Tafazzin deficient mice (shTAZ) compared to control (WT).

A-E Echocardiographic analysis of (A) posterior wall thickness, (B) septum thickness, (C) left ventricle end diastolic diameter, (D) fractional area change and (E) ejection fraction. n= 8 per genotype.

F Comparison of TAZ gene expression, normalized to S12, in heart, liver, and kidney, analyzed in triplicates by quantitative PCR. n= 5 per genotype.

G TAZ gene expression in percent. Wild-type is set to 100%.

H, I Lipid species profiles of cardiolipin (H) and monolysocardiolipin (I) in isolated cardiac mitochondria analyzed by mass spectrometry. n= 4 per genotype.

J Comparison of ratios of total monolysocardiolipin and cardiolipin content in indicated tissues.
Cardiac-specific Succinate Dehydrogenase Deficiency in Barth Syndrome (2015)
Figure 4

Figure 4. Cardiolipin deficiency differently affects individual respiratory chain complexes.

A Enzymatic activity of ubiquinone-cytochrome c oxidase in mitochondria isolated from cardiac tissue. The ubiquinone analogue decylubiquinone was used in this assay. Data were standardized to malate-dehydrogenase activity and presented as percent of wild-type, n= 4 per genotype.

B Cytochrome c reductase activity, standardized to malate-dehydrogenase activity in cardiac mitochondria, were plotted as % of wild-type, n= 3 per genotype.

C Protein levels of Cox1 and Rieske compared to VDAC3 in three different dilutions of isolated mitochondria, separated on SDS gel and visualized by Western-blotting.

D Respiratory chain complexes in cardiac mitochondria were separated on BN-PAGE and stained for activity of complex I, II, IV and F1FO- ATPase. sh, shTAZ; Q, ubiquinone.
Cardiac-specific Succinate Dehydrogenase Deficiency in Barth Syndrome (2015)
Figure 5

Figure 5. Structural and functional defects on the succinate dehydrogenase in the heart of shTAZ mice.

A Enzymatic activity of the succinate dehydrogenase in isolated cardiac mitochondria from 3 animals per genotype.

B Mitochondrial membranes were solubilized in 1% digitonin and analyzed by BN-PAGE and Western-blotting using antibodies against SDH components SDHA and SDHB (upper panel). For a loading control, samples were separated on SDS gel and analyzed by Western-blotting (lower panel).

C Steady state protein levels analyzed by SDS gel separation of indicated amounts of mitochondrial protein and subsequent Western- blotting with antibodies against SDHA and VDAC3 or visualizing of Flavin fluorescence after excitation with 488 nm. Lower panel: quantification of signal ratio SDHA/VDAC3 in shTAZ and control (set to 100%).

D Analysis of gene expression in cardiac tissue analyzed in (C) by qPCR using primers against indicated mRNA. Data of one representative measurement, normalized to β-actin with WT set to 100% are shown. Identical results were obtained in further experiments with two different pairs of animals.

E Upper panel: SDH complex of cardiac, liver and kidney were analyzed by BN-PAGE as in (B); lower panel: the same samples were analyzed by SDS gel and Western-blotting.

F, G Analysis of enzymatic activity of the succinate dehydrogenase related to the malate dehydrogenase activity in mitochondria isolated from kidney (F) and liver (G). Values of 3 animals per genotype are plotted as percent of wild-type. sh, shTAZ.
Cardiac-specific Succinate Dehydrogenase Deficiency in Barth Syndrome (2015)
Figure 6

Figure 6. Irregularities in the sarcomeric structure in patient iPSC derived cardiomyocytes.

A Flow cytometry analysis of cardiac troponinT (cTNT) expression was performed at day 60 post cardiac differentiation. 98% of cTNT+ cardiomyocytes (CM) were obtained after metabolic selection.

B RT-PCR showing the expression of indicated genes in undifferentiated iPSCs and differentiated iPSC-CMs of BTHS patient TAZ10 and a healthy control.

C Immunostaining of control (left) and BTHS iPSC-CMs at day 60 post differentiation for cardiac troponin T (cTNT) and Connexin 43 (Cx43). Scale bars: 50µm.

D Quantitation of sarcomere organization. Left panel: Immunostaining of cTNT, MLC (myosin light chain) 2a and α-actinin at day 60 post cardiac differentiation. Right panel: The bar graphs at the right panel represent the sarcomere regularity from 3 independent experiments. Error bars represent standard deviation for the comparison between control and BTHS patient TAZ10. Significance was analyzed using Student's T-TEST **P = 0.002 and ***P = 0.0006. Scale bars: 50 µm.

E Measurement of cell surface areas. Cell surface area was measured in single cardiomyocytes (n = at least 30 measurements each group from N = 3 independent experiments each group) at day 60 post cardiac differentiation after immunostaining of sarcomeric α-actinin. Scale bars: 50 µm.
Cardiac-specific Succinate Dehydrogenase Deficiency in Barth Syndrome (2015)
Figure 7

Figure 7. Succinate dehydrogenase deficiency in BTHS cardiomyocytes.

A Oxygen consumption rate (OCR) of cardiomyocytes from patient TAZ10 and control at basal conditions and after the administration of the indicated compounds.

B Mitochondrial membranes analyzed by BN-PAGE and Western-blotting using indicated antibodies. For a loading control samples were separated on SDS gel and analyzed by Western-blotting (lower panel).

C Western blot analysis of SDHA protein levels compared to VDAC3. Quantification of SDHA relative to VDAC3 in percent with control set to 100%. Error bars indicate range of values of two independent experiments.

D Analysis of SDHA gene expression in triplicates by qPCR in cardiomyocytes, analyzed in (C).
MYC Promotes Cancer Progression by Modulating m6A Modifications to Suppress Target Gene Translation (2020)
Figure 1

Figure 1. MYC down-regulates mRNA m6A levels and inhibits protein expression of selected MRGs.

(A, B) m6A dot blot of the P493-6 cells treated with Tet for 0h or 72h (A) or Raji cells that expressed NTC or MYC shRNAs (B); equal mRNA loading was verified by methylene blue staining. The shown data are representative of at least three independent experiments.

(C) Quantification of m6A abundance in (A) by HPLC-MS. *** P<0.001 as compared to corresponding high MYC group (mean ± SD, n = 3 biological replicates, Student's t‐test).

(D) Metagene profiles of m6A peak distribution along a normalized transcript composed of three rescaled non-overlapping segments: 5′UTR, CDS, and 3′UTR in P493-6 cells treated with Tet for 0h or 72h.

(E) Representative GO terms analysis of transcripts with significantly up-regulated m6A peaks in P493-6 cells treated with Tet for 72h.

(F) IGV graph showing location of m6A peaks on representative genes.

(G) m6A-RIP assay in P493-6 cells treated with Tet for 0h or 72h. HPRT1 serves as negative control. *** P<0.001 as compared to corresponding high MYC group, ns, not significant (mean ± SD, n = 3 biological replicates, Student's t‐test).

(H, I) Western blot analysis for protein levels in P493-6 cells treated with Tet for 0h, 24h, 48h and 72h (H) or in Raji cells that expressed NTC or MYC shRNAs (I). HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.
MYC Promotes Cancer Progression by Modulating m6A Modifications to Suppress Target Gene Translation (2020)
Figure 2

Figure 2. ALKBH5 removes m6A from mRNA and inhibits the protein expression of SPI1/PHF12.

(A, B) Western blot analysis for protein levels of m6A methyltransferases (METTL3 and METTL14) and demethylases (ALKBH5 and FTO) in P493-6 cells treated with Tet for 0h, 24h, 48h and 72h (A) or in Raji cells that expressed NTC or MYC shRNAs (B). β-actin serves as loading controls. Data are representative of at least three independent experiments.

(C) RT-qPCR analysis of the mRNA levels of methyltransferases and demethylases in P493-6 cells treated with Tet for 0h or 24h. Data were presented as mean (±SD), n = 3 biological replicates. * P<0.05 relative to corresponding -Tet group (Student's t‐test).

(D) IGV graph showing location of MYC binding peaks on ALKBH5 and FTO from published ChIP-seq datasets (Walz et al, 2014; Data ref: Eilers M, 2014). In this dataset, high levels of MYC were induced with 1µg/ml doxycycline for 30h and ChIPed by MYC antibody. Red triangle indicates the E-box.

(E) RIP assay, using ALKBH5, FTO or IgG antibody to detect the binding to MRGs (SPI1 and PHF12) in P493-6 cells treated with Tet or not. HPRT1 serves as negative control. *** P<0.001 as compared to corresponding IgG group (mean ± SD, n = 3 biological replicates, Student's t‐test).

(F, G) Western blot analysis for protein levels in P493-6 cells that overexpressed empty vector (EV), ALKBH5, or FTO and were then treated with Tet or not (F) or in Raji cells expressed NTC or MYC shRNAs (G). HPRT1 and β-actin served as negative and loading controls, respectively. Data are representative of at least three independent experiments.

(H) m6A RIP assay in (F) that P493-6 cells that overexpressed EV, ALKBH5, or FTO and were then treated with Tet or not. HPRT1 serves as negative control. *** P<0.001 as compared between indicated groups. ns, not significant (mean ± SD, n = 3 biological replicates, Student's t‐test).

(I, J) Western blot analysis for protein levels in P493-6 cells that expressed NTC or ALKBH5 shRNAs and were then treated with Tet or not (I) or in Raji cells expressed NTC or ALKBH5 shRNAs and knocked down MYC or not (J). HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.

(K) m6A RIP assay in (I). HPRT1 serves as negative control. *** P<0.001 as compared between indicated groups (mean ± SD, n = 3 biological replicates, Student's t‐test).
MYC Promotes Cancer Progression by Modulating m6A Modifications to Suppress Target Gene Translation (2020)
Figure 3

Figure 3. YTHDF3 binds m6A-modified mRNAs and facilitates translation of selected MRG transcripts.

(A) RIP assay in P493-6 cells treated with Tet for 0h or 72h. HPRT1 serves as negative control. *** P<0.001 relative to indicating groups (mean ± SD, n = 3 biological replicates, Student's t‐test).

(B, C) Western blot analysis for protein levels in P493-6 cells that expressed NTC or YTHDF3 shRNAs and were then treated with Tet or not (B) or in Raji cells that expressed NTC or YTHDF3 shRNAs and knocked down MYC or not (C). HPRT1 and β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.

(D) Polysomes profiling assay in P493-6 cells that expressed NTC or YTHDF3 shRNA.

(E) RT-qPCR assay for mRNA levels of MRGs (SPI1 and PHF12) in different fractions in (D). HPRT1 serves as negative control. Data were presented as mean (±SD), n = 3 biological replicates. ** P<0.01 and *** P<0.001 as compared to NTC group (Student's t‐test).

(F) Click-iT AHA (L-azidohomoalaine) experiments were performed using IgG, anti-SPI1 or anti-PHF12 antibody. P493-6 cells expressing NTC or YTHDF3 shRNAs were incubated for 1 hour in medium containing 100 ug/mL AHA. The translated proteins were detected by Western blot. Arrow indicates translated MRGs.

(G, H) Western blot analysis for protein levels in P493-6 cells (G) or in Raji cells (H) that expressed NTC, or ALKBH5 shRNA, or YTHDF3 shRNA. β-actin serve as negative and loading controls, respectively. Data are representative of at least three independent experiments.

(I) m6A RIP assay in (G). HPRT1 serves as negative control. *** P<0.001 as compared between indicated groups, ns, not significant (mean ± SD, n = 3 biological replicates, Student's t‐test).