EMBO/SourceData huggingface dataset:
Figure 5(A) Immunoblot showing the degradation of MAVS in HEK293T cells subjected to VSV infection (MOI = 1.0) for the indicated times. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.(B) Immunoblot showing the degradation of MAVS in HEK293T cells infected with VSV (MOI = 1.0) for the indicated times in the presence or absence of NH4Cl (3 mM). α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.(C) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing Flag-MAVS or Flag-mMAVS together with Myc-RNF34. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.(D) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing Flag-MAVS or its 4KR mutant together with increasing amounts of Myc-RNF34. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.(E) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing Flag-MAVS with increasing amounts of HA-RNF34 or its H342A mutant. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.(F) SDD-AGE analysis of MAVS aggregates in HEK293T cells expressing Flag-MAVS together with WT Myc-RNF34 or its ligase-dead mutant and cultured in the presence or absence of MG132 (10 μM), 3-MA (1 mM) or NH4Cl (3 mM). SDS-PAGE immunoblotting was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.(G) SDD-AGE analysis of MAVS aggregates in siCtrl- or siRNF34-1-transfected cells using an anti-MAVS antibody. SDS-PAGE immunoblotting was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.(H) Immunoblot showing the levels of the MAVS protein in siCtrl- or siRNF34-1-transfected PBMCs. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.(I) SDD-AGE analysis of MAVS aggregates in THP-1 cells expressing Flag-MAVS or the K311R mutant together with Myc-RNF34 or Myc-TRIM31. SDS-PAGE immunoblotting was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.
Figure 7(A) Immunoblot showing MAVS, IRF3-p, LC3, TOM20 and HSP60 levels in siCtrl- or siRNF34-1 cells infected with VSV and cultured in the presence of Z-VAD (50 μM) for the indicated times. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.(B) Immunoblot showing MAVS, LC3, TOM20 and HSP60 levels in cells transfected with Flag-RNF34 or its H342A mutant and subsequently infected with VSV for the indicated times. Cell-based studies were performed independently at least three times with comparable results.Representative confocal images (C) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl- or shRNF34-1 cells infected with VSV for the indicated times. Red represents MAVS and green represents NDP52. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.quantification (D) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl- or shRNF34-1 cells infected with VSV for the indicated times. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.(E) Transmission electron microscopy images of shCtrl- and shRNF34-1 HEK293T cells cultured in the presence or absence of VSV. Arrows indicate mitochondria engulfed in autophagosomes. Double arrows indicate swollen mitochondria with disrupted cristae. M, mitochondria and N, nucleus. 40000× magnification. Cell-based studies were performed independently at least three times with comparable results.(F) Representative confocal images of the shift in the fluorescence emission of Keima from 458 nm to 543 nm. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.
Figure 1Colony formation assay of miRNA mimic transfected-Dgcr8-/- ESCs. There were 17 miRNAs that decreased the colony forming ability, 15 miRNAs that did not affect colony formation and 8 miRNAs enhanced colony formation of ESCs. Representative pictures are shown. Full data are shown in Supplementary Fig S1.
Figure 2Alkaline phosphatase staining of ESCs after miRNA mimic transfection. There were 21 miRNAs that decreased the AP activity, 11 miRNAs that mildly affected the AP activity and 8 miRNAs boosted the AP activity of ESCs. Representative pictures are shown. Full data are shown in Supplementary Fig S2.Cell cycle distribution in representative miRNAs evoking G1 phase accumulation in miRNA mimic transfected Dgcr8-/- ESCs. Error bars indicate s. d. (n=3). Full data are shown in Supplementary Fig S3A, B.Immunofluorescence staining of Oct4 in miRNA mimic transfected ESCs. 20 miRNAs decreased Oct4 expression and produced small and grossly differentiated cell colonies, 12 miRNAs mildly affected Oct4 expression, and 8 miRNAs improved Oct4 expression and yielded compact and undifferentiated cell colonies. Representative pictures are shown. Full data are shown in Supplementary Fig S3C.AP staining of V6.5 ESCs after mimic transfection (Full data are shown in Supplementary Fig S4B).
Figure 3B,C. Northern blot analysis of the expression of miR-23a, miR-27a and miR-24 during EB formation (B) and RA induced differentiation of V6.5 ESCs (C). The heatmap was representive of the results of quantitative analysis.D,E. The expression of primary miR-23a~27a~24-2 transcript during EB formation (D) and RA induced differentiation (E). Error bars indicate s.d. (n=3).D,E. The expression of primary miR-23a~27a~24-2 transcript during EB formation (D) and RA induced differentiation (E). Error bars indicate s.d. (n=3).F. Q-PCR analysis of miR-23a, miR-27a and miR-24 expression in ESCs (JM8A3) and a variety of C57BL/6J adult tissues. Data are shown as mean + s.d. (n=3).G. Relative protein levels of Oct4, Sox2 and Nanog in miRNA mimic transfected Dgcr8-/- ESCs and wild- type ESCs.H,I. Oct4, Nanog, Foxa2, Gata2 and Nestin immunofluorescence staining of mimic transfected Dgcr8-/- (H) and wild- type ESCs(I).
Figure 4Western blot analysis of the expression of target genes in miR-27a or miR-24 over-expressed V6.5 ESCs. Candidate target genes whose expression was decreased were framed.The relative luciferase activity of the reporter constructs co-transfected with scramble or miR-27a and miR-24 mimics. Error bars indicate s.d. (n=3); * indicates P-value < 0.05. Candidate target genes whose luciferase activity was decreased were framed. PC group was positive reporter control which harbored reverse complementary sequence of miR-27a or miR-24.The relative luciferase activity of the wild-type 3′ UTR or mutated 3′ UTR reporter co-transfected with scramble or miR-27a and miR-24 mimics. Data are shown as mean + s.d. (n=3); * indicates P-value < 0.05.F,G RT-PCR and qRT-PCR analysis of in vivo binding of the miRNAs with their targets. Error bars indicate s.d. (n=3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.H. Western blot analysis of c-Myc expression in siRNA treated V6.5 ESCs.I. Q-PCR analysis of primary-23a~27a~24-2, miR-23a, miR-27a and miR-24 expression in c-Myc knocked down ESCs. Error bars indicate s.d. (n=3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.K. ChIP-PCR and ChIP-q-PCR analysis of c-Myc binding to the putative sites in the promoter. Error bars indicate s.d. (n=3); *** indicates P-value < 0.001.L. Relative luciferase activity of the wild-type and -15 binding site mutated promoter in NIH3T3 cells co-transfected with c-Myc or the blank vector (NC). Error bars indicate s.d. (n=3); * indicates P-value < 0.05.M. Relative luciferase activity of wild-type and -15 binding site mutated promoter in V6.5 ESCs. Error bars indicate s.d. (n=3); * indicates P-value < 0.05.
Figure 5Validation of the effect of miRNA inhibition in MEFs by q-PCR. Error bars indicate s.d. (n=3).AP staining of the iPSC colonies in reprogramming with OSK combined with scramble control, inhibitors of miR-23a, miR-27a, miR-24 and let-7c respectively.Fold increase in Oct4-GFP-positive cell colonies in the above cells. Data are shown as mean + s.d. (n=5); ** indicates P-value < 0.01; *** indicates P-value < 0.001.Morphology of typical Oct4-GFP-positive iPSC colonies that were reprogrammed by OSK combined with miRNA inhibitors.Detection of endogenous and exogenous Oct4, Sox2, Klf4 and Nanog expression in iPSCs, ESCs and MEFs by RT-PCR.Immunofluorescence staining of Oct4, Nanog and SSEA1 of the iPSC colonies.Q-PCR analysis of the expression of self-renewal (Oct4, Sox2, Nanog) and differentiation (Cdx2, Foxa2, Brachyury, Hand1, Nestin, FGF5) relative markers in EBs derived from the iPSCs. Error bars indicate s.d. (n=3).Hematoxylin and eosin staining of teratoma derived from the iPSC colonies. Neuronal rosette (Ectoderm), Cartilage (Mesoderm) and respiratory epithelium (Endoderm) are marked with black arrows.
Figure 6Genomic PCR of the specific fragments spanning miR-23~27~24 clusters. WT indicate wild-type ESCs; DKO indicate miR-23a~27a~24-2-/-, miR-23b~27b~24-1-/- ESCs; KO indicate miR-23a~27a~24-2-/- ESCs.Real-time PCR analysis of mature miR-23a/b, miR-27a/b and miR-24 expression in wild-type, DKO and KO ESCs. Data are shown as mean + s.d. (n=3).AP staining of wild-type and miR-23~27~24 cluster knockout ESCs.Real-time PCR analysis of Oct4, Sox2 and Nanog expression in wild-type and miR-23~27~24 cluster knockout ESCs. Data are shown as mean + s.d. (n=3).Oct4, Sox2 and Nanog immunofluorescence staining of wild-type and the knockout ESCs.
Figure 7Real-time PCR analysis of Oct4 and several lineage differentiation markers expression during EB formation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).Beating EB incidence during cardiac differentiation of wild-type and knockout ESCs. Data are shown as mean + s.d. (n=3). * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.Real-time PCR analysis of several cardiac markers expression during cardiac differentiation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).Actinin, ANP and Troponin Iimmunofluorescence staining of differentiation cultures derived from wild-type and knockout ESCs.Volumes of teratomas at different days after injection. Data are shown as mean + s.d. (n=4). * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.Weight of teratomas derived from wild-type and knockout ESCs. Data are shown as mean ± s.d. ** indicates P-value < 0.01.Oct4 immunohistochemistry staining of teratomas derived from wild-type and knockout ESCs. Representative Oct4 positive regions are shown.Hematoxylin and eosin staining of teratomas derived from wild-type and knockout ESCs. Ectoderm, Mesoderm and Endoderm are marked with different color arrows.
Figure 1(A) The list of co-precipitated proteins as determined by immunoprecipitation followed by mass spectrometry (IP-MS). The SWR1 subunits not shared by the ISWI complexes are shown in blue; The DDT domain-containing ISWI specific subunits are shown in orange; CHR11 and CHR17 are shared subunits of the SWR1 and ISWI complexes and are shown in green. (C) Determination of the interaction of MBD9 with HTA9, HTA11, PIE1, CHR11, and CHR17 by co-immunoprecipitation.
Figure 2 Determination of the interaction of PIE1-Flag, MBD9-Myc, and CHR11-HA by in vitro pull down assays. PIE1-Flag, MBD9-Myc, and CHR11-HA were expressed from yeast and subjected to the pull down assays. Anti-Flag agaroses were independently used in the pull down assays.  C) Determination of the interaction of MBD9-Myc, and CHR11-HA by in vitro pull down assays. MBD9-Myc, and CHR11-HA were expressed from yeast and subjected to the pull down assays. anti-HA (C) agaroses were independently used in the pull down assays.  D) he effect of the pie1 and mbd9-1 mutations on the formation of the SWR1 complex as determined by IP-MS. Transgenic plants expressing Flag-tagged MBD9, PIE1, and CHR11 in the WT and mutant backgrounds were subjected to the IP-MS experiment with anti-Flag agarose.
Figure 3 (A) Morphological phenotypes of the WT, chr11/17, pie1, swc2, and rin2 mutant plants. Shown are 24-day-old plants. The top panel shows the phenotypes of plants in the soil; the bottom panel shows the phenotypes of plants on the black cloth.  (B) Shown are rosette leaves of the WT, pie1, swc2, and rin2 mutant plants. Rosette leaves were cut from bolting plants.  (C) The early-flowering phenotype of the mbd9-1, mbd9-3, mbd9-4 and hta9-1/11-1 mutants compared to the WT. 24-day-old plants in soil are shown.  (D) Numbers of rosette leaves of the WT and mutant plants shortly after bolting. Rosette leaves from at least 20 plants were counted for each genotype. Values are mean ± SD. ** P<0.01, student's t test.  (E) Determination of the effect of the arp4-1, arp6 and swc6-1 mutations on flowering time. The top panel shows the number of rosette leaves in the WT, arp4-1, arp6, and swc6-1 mutant plants; the bottom panel shows a photograph of the plants. Rosette leaves from at least 20 plants were counted for each genotype. Values are mean ± SD. ** P<0.01, student's t test.  (F) Determination of the effect of the tra1a and tra1b mutations on flowering time. The top panel shows numbers of rosette leaves of the WT, tra1a-1, tra1a-2, and tra1b mutant plants; the bottom panel shows a photograph of the plants. Rosette leaves from at least 20 null were counted for each genotype. Values are mean ± SD. ** P<0.01, student's t test.
Figure 4(B) Heat maps showing the effect of indicated mutations on the expression of differentially expressed genes identified in the mbd9-1 mutant. The color bar shows log2(fold change). Up- and down-regulation are shown by red and blue, respectively. (C) Scatter plots showing that the expression pattern in the hta9-1/11-1 mutant is correlated with that in the chr11/17, mbd9-1, and pie1 mutants. The differentially expressed genes identified in the hta9-1/11-1 mutant were subjected to the analysis.
Figure 5 (A) The effect of indicated mutations on H2A.Z deposition as determined by immunoblotting. Chromatin-associated proteins were extracted and subjected to immunoblotting by the anti-H2A.Z antibody. The histone H4 level was detected by the anti-H4 antibody and indicated as a loading control.  (B) Meta plots showing H2A.Z occupancy on H2A.Z target genes. TSS, transcription start site; TTS, transcription termination site. RPKM, reads per kilobase per million. The H2A.Z ChIP-seq results were generated from two independent biological replicates.  (D) Heat maps showing H2A.Z enrichment in the WT, pie1, chr11/17, and mbd9-1 mutants over the PIE1  (E) Shown are H2A.Z changes in the pie1, chr11/17, and mbd9-1 mutants relative to the WT. PIE1-dependent H2A.Z target genes were subjected to the analysis and sorted by H2A.Z density in the WT. The color bar shows log2(fold change).  (F) Meta plots showing CHR11 and PIE1 occupancy on PIE1-dependent H2A.Z target genes. TSS, transcription start site; TTS, transcription termination site. The CHR11-Flag and PIE1-Flag ChIP-seq data are from two independent biological replicates. (G) Heat maps showing the enrichment of PIE1-Flag and CHR11-Flag over the PIE1-dependent H2A.Z target genes. The peaks were sorted by H2A.Z density in the WT. (H) Box plot showing the enrichment of CHR11 and PIE1 over H2A.Z target genes and non-H2A.Z target genes. The log2(fold change) values of RPKM between the CHR11-Flag or PIE1-Flag signals and the WT signals are shown. Center lines and box edges represent medians and the interquartile range (IQR), respectively. Whiskers extend to minimum and maximum values within 1.5 times of the IQR. The data shown are from two independent biological replicates. The difference between the CHR11-Flag and PIE1-Flag ChIP-seq levels was determined by student's t test. ** P<0.01.
Figure 6(A) Increasing amounts of CHR11 (left panel) or PIE1 (right panel) were incubated with end-positioned (lane 1-4 and 9-12) or center-positioned (lane 5-8 and 13-16) mono-nucleosomes in the presence of ATP. The sliding results were then visualized on native PAGE gels by EB staining.(B) Distribution of nucleosomes over TSS of all protein-coding genes in the WT, chr11/17, pie1, and mbd9-1 mutants. The MNase-seq results were generated from two independent biological replicates.
Figure 1(E) Representative immunoblot for key lipolytic enzymes HSL and ATGL protein expression and quantification (n = 5-6/group). Data are represented as mean ± s.e.m. E, Unpaired Student's t-test.(F) Immunoblot analysis of autophagic flux in mWAT (upper panel) and gWAT (lower panel) adipose tissue stimulated ex vivo with lysosomal inhibitors 100nM Bafilomycin A1 and 20mM NH4Cl for 4 hours or DMSO (Vehicle) (n = 3-4/group). Data are represented as mean ± s.e.m. Unpaired Student's t-test.(G) Representative transmission electron microscopy images from mesenteric adipose tissue 7 days post DSS-induced colitis induction. Lower panel is showing magnification of selected area. White arrows show autophagosomal structures.(J) Representative immunoblot for LC3-I/-II and ACTIN protein expression and quantification of autophagic flux in creeping fat tissues (CrF) and adjacent mesenteric adipose tissues (Ad. MAT) of Crohn's disease patients (n = 3/group). Additionally, autophagic flux was determined in the mesenteric adipose tissue (MAT) of a colorectal cancer patient as control (dotted line). Data are represented as mean ± s.e.m. (J) Paired Student's t-test.
Figure 2(C) Representative immunoblot for LC3-I and LC3-II protein expression and quantification of LC3 conversion ratio (LC3-II/LC3-I) (n = 3/group). Data are represented as mean ± s.e.m. C, Unpaired Student's t-test.(F) Colon length after two weeks post-deletion (steady state; n = 14/group) and after DSS at day 7 (n = 18-22/group). Data are represented as mean ± s.e.m. , F, Unpaired Student's t-test.(G) Representative H&E staining images (10x magnification) of colon sections and quantification of histological score at steady state (n = 9/group) and DSS colitis (n = 18-22/group). Data are represented as mean ± s.e.m. G, Unpaired Student's t-test.(I) Absolute number CD45+ immune cells from colons at steady state (n = 13-14/group) or at 7 days post-DSS induction (n = 18-22/group). (J) Frequency of myeloid cell population in colon at day 7 post-DSS induction (n = 18-25/group). (K) Absolute number of Ly6C+ monocytes discriminated by the absence or presence of MHCII for infiltrating and inflammatory monocytes respectively (n = 18-25/group). Data are represented as mean ± s.e.m. I) Unpaired Student's t-test. (J, K) Two-Way ANOVA.
Figure 3(A) Ex vivo lipolysis measured by released free fatty acid (left, n = 4-5/group) and glycerol (right, n = 7-8/group) in culture supernatant of adipose tissue explants simulated with isoproterenol (10µM) for 1-2h. (B) Ex vivo lipolysis measured by released free fatty acid (left, n = 4/group) and glycerol (right, n = 7/group) adipose tissue explants simulated with TNFα (100ng/mL) for 24h before replacing with fresh medium in the absence of TNFα for 3h. Data are represented as mean ± s.e.m. (A, B, Two-Way ANOVA. (B, Unpaired Student's t-test.(E) Concentration of individual FFA species in serum in water-treated and DSS-treated mice as measured by FID-GC (n = 12-14/group). Data are represented as mean ± s.e.m. E) Two-Way ANOVA.
Figure 4(C) Ex vivo lipolysis assays on Atg7-deficient adipose tissue explants simulated with isoproterenol (10µM) for 1-2h (n = 5-6/group). Data are represented as mean ± s.e.m. (C, Two-Way ANOVA.(H) Expression of pro-inflammatory cytokines in colon tissues at 7 days post-DSS induction (n = 8/group). Dotted line represents non-inflamed controls. Data are represented as mean ± s.e.m. Unpaired Student's t-test.(K) Absolute number of Ly6C+ monocytes discriminated by the absence or presence of MHCII for infiltrating and inflammatory monocytes respectively (n = 8/group). Data are represented as mean ± s.e.m. K) Unpaired Student's t-test.
Figure 5(E) Fold change expression of NRF2-target genes in primary visceral adipocytes at day 7 after DSS induction from normalized counts of RNAseq dataset (n = 6/group). Data are represented as mean ± s.e.m. Unpaired Student's t-test.(H) Representative immunoblot of EPHX1 and EPHX2 in gonadal adipose tissues at day 7 after DSS induction. Asterix indicating non-specific band (n = 14-18/group). Data are represented as mean ± s.e.m. Unpaired Student's t-test.
Figure 6(B) Colitis was induced in mice for 7 days and adipose tissues were extracted and cultured for 6 hours in serum-starved medium. Secretion of IL-10 and from mesenteric (left panel) and gonadal adipose tissues (right panel) was measured by ELISA. Shapes identify individual experiments (n = 5-15/group). Data are represented as mean ± s.e.m. (B) Two-Way ANOVA with regression for experiment.(F) Serum cytokines upon DSS-induced colitis at day 7 post-induction (n = 17-23/group). Data are represented as mean ± s.e.m. Two-Way ANOVA.
Figure 7(A) Quantification of Il10 transcript levels in RAW264.7 upon stimulation to epoxy fatty acids (n = 3/group). (B) Quantification of IL-10 protein levels in the supernatant of RAW264.7 upon stimulation to epoxy fatty acids (n = 3/group). Data are represented as mean ± s.e.m. (A,B) One-Way ANOVA.
Figure 1(C-G) ssDNA repair efficiency of the indicated TS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course (right panels). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour (60+), treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. They were also released into medium without MMS for 1 hour to control the formation of spontaneous Rad52-YFP foci (60-). The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panels). Cell cycle progression was determined by cell sorting. The mean and SEM of 3 (wild type, rad18∆, mms2∆, rad51∆, rad57∆, rad54∆), 4 (pol30-K164R) and 5 (rad5∆) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where three asterisks represent p-values <0.001.
Figure 2(C-E) ssDNA repair efficiency of the indicated TLS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course (D and E, right panel). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (C and E, left panel). Data information: The mean and SEM of and 3-12 12 (wild type), 6 (rev1∆, rev3∆, rad30∆, rev1∆ rad30∆, rev3∆ rad30∆), and 3 (rev1∆ rev3∆ and rev1∆ rev3∆ rad30∆) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.