EMBO/soda-nel huggingface dataset:
Cardiac LXRα protects against pathological cardiac hypertrophy and dysfunction by enhancing glucose uptake and utilization. (2015)
Figure 6

LXRα enhances glucose uptake and O-GlcNAc signaling via activation of the hexosamine biosynthetic pathway (HBP) in cultured cardiomyocytes

Neonatal rat ventricular myocytes were transfected with Ad-LXRα or GL2 (Ad-cont), and treatments with phenylephrine (PE) and DON (inhibitor of HBP) were initiated for 24 h.

A Assessment of 2-deoxyglucose (2-DG) uptake from 4 independent experiments. *P = 0.03 versus Ad-cont.

B, C Glut mRNA expression determined by RT–PCR normalized to 36b4, n = 5 per condition in the absence of PE, n = 4 per condition in the presence of PE. *P = 0.02 versus Ad-cont, **P = 0.008 versus Ad-cont, #P = 0.03, ##P = 0.008.

D Western blot indicating Ad-LXRα- and PE-induced increases in global protein O-GlcNAcylation, which was abrogated following inhibition of HBP with DON. LXRα protein expression is shown, and GAPDH served as a loading control.

E, F Modulation of Anp and Bnp mRNA levels by Ad-LXRα-induced O-GlcNAc signaling. Gene expression as determined by RT–PCR normalized to 36b4, n = 5 per condition in the absence of PE, n = 4 per condition in the presence of PE. *P = 0.02 versus Ad-cont, **P = 0.008 versus Ad-cont, #P = 0.03, ##P = 0.02.

G Measurement of cell size, n = 5 per condition. *P = 0.008 versus Ad-cont, #P = 0.02, ##P = 0.03.

H Representative images for the determination of cell size. Cells were stained with an antibody specific for LXRα (green, indicated by arrow), DAPI for nuclei (blue), and rhodamine-phalloidin for F-actin (red); scale bar = 50 μm.

Data information: Data are means ± SEM and are reported as fold change with respect to control group; Kruskal–Wallis test followed by Mann–Whitney U-test was used for group comparisons. Source data are available online for this figure.
Cardiac LXRα protects against pathological cardiac hypertrophy and dysfunction by enhancing glucose uptake and utilization. (2015)
Figure 7

Knockdown of LXRα increases cellular hypertrophy in cultured cardiomyocytes

Cells were transfected with Ad-LXRα, si-LXRα, or Ad-cont, in the absence or presence of phenylephrine (PE).

A Protein synthesis determined via leucine incorporation, n = 2–4 independent experiments. *P = 0.03 versus Ad-cont, §P = 0.06 versus Ad-cont.

B Global O-GlcNAc protein expression with Western blot, GAPDH served as a control.

C–F mRNA expression, normalized to 36b4, n = 6 per condition, except n = 5 for si-LXRα control. Anp: *P = 0.009 versus Ad-cont, #P = 0.004, ##P = 0.009; Bnp: *P = 0.004 versus Ad-cont, **P = 0.002 versus Ad-cont; Acta1: *P = 0.002 versus Ad-cont, §P = 0.06 versus Ad-cont, #P = 0.02; Rcan1: *P = 0.03 versus Ad-cont, **P = 0.004 versus Ad-cont, #P = 0.004.

Data information: Data are means ± SEM and are reported as fold change with respect to control group; Kruskal–Wallis test followed by Mann–Whitney U-test was used for group comparisons. Source data are available online for this figure.
Cardiac LXRα protects against pathological cardiac hypertrophy and dysfunction by enhancing glucose uptake and utilization. (2015)
Figure 8

O-GlcNAcylation is increased with cardiac LXRα overexpression in mice

A, B Western blot analyses of global protein O-GlcNAc levels in left ventricles of mice with either (A) cardiac-specific LXRα overexpression or (B) LXRα deficiency and subjected to 5 weeks TAC.

C, D Nuclear protein extracts were precipitated with agarose WGA in the absence or presence of GlcNAc, a competitor, and analyzed by Western blot with antibodies against Mef2c, GATA4, or Nkx-2.5, known transcription factors of natriuretic peptides; bands represent 3 pooled hearts per Wt and LXRα-Tg lanes, and (D) quantification is for n = 2, expressed as fold change.

Data information: Data are means ± SEM. IP, immunoprecipitation; WGA, wheat germ agglutinin; GlcNAc, N-acetylglucosamine; WB, Western blot. Source data are available online for this figure.
PP2A-B56 binds to Apc1 and promotes Cdc20 association with the APC/C ubiquitin ligase in mitosis (2019)
Figure 1

Figure 1. Cdk-dependent phosphorylation of Apc1-loop500 ensures PP2A-B56γ recruitment.

A. Multiple alignment of a predicted Apc1-loop500 domain of vertebrate APC/Cs. The sequences corresponding to residues 515-584 in Xenopus tropicalis Apc1 are shown. A putative B56 binding region is coloured in green. Hs; Homo sapiens human, Pt; Pan troglodytes chimpanzee, Mm; Mus musculus mouse, Gg; Gallus gallus chicken, Xt; Xenopus tropicalis frog.

B. Schematic diagrams of Apc1-loop500 constructs. Apc1-loop500 (residues 515-584) was fused with Maltose binding protein (MBP) at N-terminus and 6xHis at C-terminus. Conserved Cdk phosphorylation sites (SP/TP) are shown as S or T, respectively. The putative B56 binding region is shown in green. The 11-residue [LSPVPELRDST] deletion (∆11) and alanine substitution mutations to three Cdk phosphorylation sites (3A) or to B56 binding motif (L557A/V560A) are shown.

C. Binding assay using MBP fused Apc1-loop500 fragments and B56γ. Apc1-loop500 WT or its derivatives (∆11 or 3A) were incubated with the 35S-labelled Flag-B56γ in interphase extract (Inter) or anaphase extract (Ana) supplemented with CycB∆167 at 23˚C for 1 hr. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or coomassie brilliant blue (CBB) staining. The bar graph is quantification of bound B56γ. The intensities of MBP control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.

D. Cdk-dependent in vitro kinase assay of Apc1-loop500. MBP fused WT or 3A Apc1-loop500 fragment was incubated with Cdk2-cyclin A in the presence of [γ-32P]-ATP at 23˚C for 10 min, separated by SDS-PAGE and detected by autoradiography.

E. Cdk can promote Apc1-loop500 and B56 interaction. MBP fused WT or 3A Apc1-loop500 fragment was incubated in the presence or absence of Cdk2-cyclin A at 30˚C for 60 min. MBP-fused peptides (-/+ kinase) were isolated and incubated with purified Flag-B56γ at 23˚C for 30 min. The bound proteins were recovered by amylose beads and analysed by SDS-PAGE and immunoblotting with indicated antibodies.

F. Quantification of E. Bound B56γ to WT Apc1-loop500 control (-Cdk) was arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.
PP2A-B56 binds to Apc1 and promotes Cdc20 association with the APC/C ubiquitin ligase in mitosis (2019)
Figure 2

Figure 2. B56 binding to Apc1-loop500 is important for the formation of the active APC/C-Cdc20 complex.

A. Cdc20 binding assay in Xenopus egg extracts. The purified recombinant wild-type (WT) or B56 binding site mutant APC/C (1-L557A/V560A) was incubated with APC/C-depleted (∆APC) interphase extract (Inter) or ∆APC anaphase extract supplemented with CycB∆167 (Ana) at 23˚C for indicated times. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies. pApc1 (pS314/pS318) is a phospho-site specific antibody that binds only when both S314 and S318 are phosphorylated.

B. Quantification of A. The intensities of WT control in interphase were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.

C. B56 binding site mutant APC/C (1-L557A/V560A) is less active in ubiquitylation assay than WT APC/C. The purified recombinant WT APC/C or B56 binding motif mutant APC/C (1-L557A/V560A) was incubated with ∆APC anaphase extract. The recovered APC/C-Cdc20 complex was subjected to ubiquitylation assay using 35S-labelled cyclin B as a substrate. Samples were taken at indicated time points and analysed by SDS-PAGE and autoradiography. Cdc20 bound to the APC/C is presented in Figure EV2B.

D. Quantification of C. Error bars, s.e.m. from three independent experiments.
PP2A-B56 binds to Apc1 and promotes Cdc20 association with the APC/C ubiquitin ligase in mitosis (2019)
Figure 3

Figure 3. Apc1-loop500 regulates APC/C activation independently of phosphorylation of Apc1-loop300.

A. (left panel) Cdc20 binding to APC/C loop domain mutants in interphase extract. The purified recombinant WT or Apc1 mutant APC/Cs (1-∆L300, 1-∆L300/∆11 or 1-∆L300/L557A/V560A) were incubated with ∆APC interphase extract at 23˚C for 1 hr. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies. (right panel) Quantification of bound Cdc20. The intensities of 1-∆L300 control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.

B. (left panel) Cdc20 binding to APC/C loop domain mutants in anaphase extracts. Same as A, but ∆APC anaphase extract was used and incubated at 23˚C for 50 min in anaphase extract. (right panel) Quantification of bound Cdc20. The intensities of 1-∆L300 control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.
PP2A-B56 binds to Apc1 and promotes Cdc20 association with the APC/C ubiquitin ligase in mitosis (2019)
Figure 4

Figure 4. PP2A-B56 bound to Apc1-loop500 promotes APC/C-Cdc20 complex formation through dephosphorylation of N-terminal Cdc20.

A. (left panel) Cdk-dependent in vitro kinase assay of Apc1-loop300. 3xFlag-tagged WT or 7T Apc1-loop300 fragment was incubated with Cdk2-cyclin A in the presence of [γ-32P]-ATP at 30˚C for 30 min, and separated by SDS-PAGE and detected by autoradiography. (right panel) 32P-phosphorylated WT or 7T Apc1-loop300 fragment was incubated in anaphase extract and removal of radioactivity was analysed by SDS-PAGE followed by autoradiography.

B. Quantification of removal of radioactivity in A. Error bars, s.e.m. from three independent experiments.

C. Cdc20-5A efficiently binds to the B56 binding site mutant APC/C. The purified recombinant WT APC/C or Apc1-loop500 mutant APC/C (1-L557A/V560A) was incubated with WT Cdc20 or non-phosphorylatable Cdc20 mutant (5A) in ∆APC∆Cdc20 anaphase extract at 23˚C for 55 min. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies.

D. Quantification of C. The intensities of WT APC/C control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.

E. The APC/C and Cdc20 immunoprecipitated from CSF extract were incubated in the presence of a range of concentrations or absence of PP2A-B56γ at 23˚C. Samples were taken at the indicated time points and analysed by SDS-PAGE and immunoblotting with antibodies, including phospho-site specific antibodies for pCdc20 (pT79) and pApc1 (pS314/pS318).

F. A model for Apc1-loop500-mediated Cdc20-APC/C complex formation in mitosis. Apc1-loop500 is phosphorylated by Cdk1 and binds to PP2A-B56 in anaphase. PP2A-B56 dephosphorylates inhibitory phosphorylation sites in N-Cdc20 and promotes the formation of active APC/C-Cdc20 complex. Apc1-loop500 may control phosphorylation of other APC/C subunits and possibly interacting proteins although the mechanisms involved remain elusive. The indicated numbers on the schematic view of the APC/C (the back view is rotated by 180˚ around the vertical axis from the front view) represent APC/C subunits.

Expanded View Figure legends
Ageing induces tissue specific transcriptomic changes in Caenorhabditis elegans (2022)
Figure 6

Figure 6. irx-1 promotes the breakdown of intestinal barrier in aged worms.

A The Age-BPs under irx-1 control in intestine. Age-BPs with more than 5 regulated genes were listed. Categories 1 and 2 are differentiated by capitalisation and bold fonts. Category 2 are shown below the corresponding Category 1.

B, C The intestine-specific RNAi against irx-1 reduces the Smurf phenotype at day 8 of adulthood. Mean ± SD. Scale bar: 100 μm. Unpaired t-test, two-tailed. More than 30 worms analysed in each of the three biological replicates.

D The survival curves of the worms under indicated RNAi treatments.
Ageing induces tissue specific transcriptomic changes in Caenorhabditis elegans (2022)
Figure 7

Figure 7. myrf-2 is a master regulator of the ageing in body wall muscle.

A The Age-BPs regulated by myrf-2 in body wall muscle (BWM). Age-BPs with more than 5 regulated genes were listed. Categories 1 and 2 are differentiated by capitalisation and bold fonts. Category 2 are shown below the corresponding Category 1.

B, C The BWM-specific RNAi against myrf-2 improves sarcomere in the worms at day 8 of adulthood. Arrowheads and arrows respectively denote the gaps of myofilaments and the disorganised myofilaments with GFP aggregations. Scale Bar: 10 μm. Mean ± SD. Unpaired t-test, two-tailed. n>=50.

D, E Suppressing myrf-2 in BWM increases worms' motility at day 5 of adulthood. The tracks were from worms' movement in 30 sec (D). The speed of worm movement was measured by the ratio of track length versus body length (E). Mean ± SD. n>=120. Unpaired t-test, two-tailed.

F BWM-specific RNAi against myrf-2 extends worms' lifespan.

Expanded View

The expanded view contains 5 figures, 2 tables, 10 datasets, and 1 movie.
Autoimmunity Gene IRGM Suppresses cGAS-STING and RIG-I-MAVS Signaling to control Interferon Response (2020)
Figure 1

Figure 1. IRGM is a master negative regulator of the interferon response.

(A) The bar graph represents top 10 biological pathways upregulated in gene ontology (GO) based Reactome pathway analysis using a set of genes induced (1.5 fold, p<0.05 (Wald Test), 3 biological replicates) in RNA-seq analysis in IRGM shRNA knockdown HT29 cells compared to control shRNA cells. Heatmaps were generated for sentinel interferon-regulated genes (three biological replicates) using 'ComplexHeatmap' library using 'R' Bioconductor package where the gene expression matrix was transformed into z-score. The heat map was generated from the common genes present in the three GO terms indicated by the three black lines. The numbers on the bars indicate the p-value of that particular GO term.

(B) Interferome database analysis with a set of genes induced (1.5 fold, p<0.05 (Wald Test), 3 biological replicates) in IRGM shRNA knockdown HT29 cells compared to control shRNA cells. The Venn diagram depicts the total number of upregulated type I and type II IFN-regulated genes in IRGM KD cells.

(C) Network pathway analysis using IPA. The molecular network of genes connected with the top 4 functions associated genes (1.5 fold, p<0.05 Wald Test, 3 biological replicates) upregulated in IRGM knockdown HT29 cells. The complete list is documented in Dataset EV2.

(D) The qRT-PCR validation of RNA- seq data in control and IRGM KD HT29 cells. Mean ± SD, n = 3 (biological replicates), *P < 0.05, **P < 0.005, ***P < 0.0005, Student's unpaired t-test.

(E) The bar graph represents the top pathways upregulated in GO-based Reactome pathway analysis using set of genes induced (1.5 fold, p<0.05 (Wald Test), 3 mice each group) in the brain of Irgm1-/- mice compared to Irgm1+/+ wild type mice. Heatmaps were generated for sentinel interferon-regulated genes (three biological replicates) using 'ComplexHeatmap' library using 'R' Bioconductor package where the gene expression matrix was transformed into z-score. The heat map was generated from the common genes present in the three GO terms indicated by the three black lines. The numbers on the bars indicate the p-value of that particular GO term.

(F) Interferome database analysis with a set of genes induced (1.5 fold, p<0.05, 3 biological replicates) in the brain of Irgm1-/- mice compared to Irgm1+/+ wild type mice. The Venn diagram depicts the total number of upregulated type I and type 2 IFN-regulated genes in Irgm1-/- mouse brain.

(G) Network pathway analysis using IPA. The molecular network of genes connected with the top 5 functions/diseases associated with genes (1.5 fold, p<0.05, Wald Test, 3 biological replicates) upregulated in Irgm1 knockout mouse brain. The complete list is documented in Dataset EV2.

(H) The qRT-PCR validation of RNA- seq data in Irgm1+/+ and Irgm1-/- mouse brain. Mean ± SD, n = 3 (biological replicates), *P < 0.05, **P < 0.005, ***P < 0.0005, Student's unpaired t-test.
Autoimmunity Gene IRGM Suppresses cGAS-STING and RIG-I-MAVS Signaling to control Interferon Response (2020)
Figure 2

Figure 2. The nucleic acid-sensing and ISG production pathways are constitutively active in IRGM-depleted cells and mice.

(A) Heat map of nucleic acid sensor proteins upregulated in IRGM KD HT29 cells and Irgm1-/- mouse brain and BMDMs.

(B-D) Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of (B) HT29 control (henceforth IRGM+/+) and single allele CRISPR knockout IRGM cells (henceforth IRGM+/-), (C) HT29 cells stably expressing control shRNA or IRGM shRNA, (D) Irgm1+/+ and Irgm1-/- mouse brain (n=3 mice).

(E) Western blot analysis to determine levels of adaptor proteins in control and IRGM+/- HT29 cells.

(F) SDD-AGE followed by western blot analysis with a mitochondrial fraction from control and IRGM siRNA knockdown THP1 cells. Western blot analysis with cytoplasmic fraction was also performed.

(G-I) Western blot analysis performed with lysates of (G) control and stable IRGM shRNA knockdown HT29 cells (H) IRGM+/+ and IRGM+/- HT29 cells, and (I) Irgm1+/+ and Irgm1-/- mouse brain to determine levels of TBK1 protein.

(J-L) Western blot analysis performed with lysates of (J) IRGM+/+ and IRGM+/- HT29 cells (K-L) Irgm1+/+ and Irgm1-/- mouse brain to determine levels of IRF proteins.

(M-N) Western blot analysis performed with lysates of (M) IRGM+/+ and IRGM+/- HT29 cells, (N) Irgm1+/+ and Irgm1-/- mouse brain to determine levels of STAT proteins (n=3 mice).

(O-P) Western blot analysis performed with lysates of (O) IRGM+/+ and IRGM+/- HT29 cells. 2 biological replicates are shown. (P) Irgm1+/+ and Irgm1-/- mouse brain to determine levels of ISG proteins (n=2 mice).

(Q) Representative confocal image of immunofluorescence assay performed with IRGM+/+ and IRGM+/- HT29 cells stained with MX1 antibodies. Scale bar, 10 µm.
Autoimmunity Gene IRGM Suppresses cGAS-STING and RIG-I-MAVS Signaling to control Interferon Response (2020)
Figure 3

Figure 3. IRGM interacts with DNA/RNA sensors and utilizes autophagy to degrade them so as to constrain IFN response.

(A) Immunoprecipitation (IP) analysis of the interaction between endogenous IRGM and endogenous RIG-I, cGAS, MDA5, TLR3, TLR4, TLR7, NLRC4, and AIM2 in THP-1 cell lysates. IgG (H), IgG heavy chain.

(B-D) Co-immunoprecipitation (Co-IP) analysis of the interaction between (B) GFP-IRGM and Flag-RIG-I, (C) GFP-IRGM, and Flag-cGAS, (D) GFP-IRGM and Flag-TLR3 in HEK293T cell lysates. L.E, Long exposure of input blot. IgG (L), IgG Light chain; IgG (H), IgG heavy chain.

(E) GST pull-down assay of in-vitro translated and radiolabelled myc-tagged cGAS, RIG-I, and TLR3 with GST or GST-tagged IRGM.

(F) Western blot analysis with the lysates of HT-29 cells stably transfected with control or Flag-IRGM plasmid and probed with the indicated antibodies.

(G-I) Western blot analysis with lysates of HEK293T cells transiently expressing GFP or GFP-IRGM and (G) Flag-RIG-I or (H) Flag-cGAS or (I) or Flag-TLR3. 2 biological replicates are shown.

(J-K) The qRT-PCR analysis with RNA isolated from (J) control and Flag-IRGM stable HT-29 cells (K) THP-1 cells transiently transfected with control or Flag-IRGM plasmid. n=3 (biological replicates), Mean ± SD, *P < 0.05, Student's unpaired t-test.

(L-N) Luciferase reporter assays in HEK293T cells transiently transfected with ISRE reporter plasmid and other plasmids as indicated. Mean ± SD, n = 3 (biological repeats), **P < 0.005, ***P < 0.0005, Student's unpaired t-test.

(O) Western blotting analysis with lysates of control and untreated or Bafilomycin A1 (300 nM; 4 h and 8 h) treated Flag-IRGM expressing HT29 stable cell lines.

(P) Western blotting analysis with lysates of control and untreated or chloroquine (50 µM; 4 h and 8 h) treated Flag-IRGM expressing HT29 stable cell lines.

(Q) The qRT-PCR analysis with RNA isolated from untreated or Bafilomycin A1 (300 nM; 4 h, 8 h, and 12 h) treated control or Flag-IRGM expressing HT-29 stable cells as indicated. Mean ± SD, n = 3 (biological replicates), *P < 0.05, **P < 0.005 Student's unpaired t-test.

Data information : *Nonspecific, #degradation products of full-length GFP-IRGM
Autoimmunity Gene IRGM Suppresses cGAS-STING and RIG-I-MAVS Signaling to control Interferon Response (2020)
Figure 4

Figure 4. IRGM utilizes p62-dependent autophagy to control the levels of nucleic acid sensors and ISGs.

(A) The cell lysates of control or BECLIN1 siRNA transfected THP-1 cells expressing GFP or GFP-IRGM were subjected to immunoblotting with indicated antibodies.

(B) The cell lysates of control or ATG5 siRNA transfected THP-1 cells expressing GFP or GFP-IRGM were subjected to immunoblotting with indicated antibodies.

(C-D) qRT-PCR analysis with RNA isolated from control or BECLIN1 siRNA transfected THP-1 cells expressing 3X-Flag epitope or Flag-IRGM as indicated. Mean ± SD, n = 3 (biological replicates), ***P < 0.0005, Student's unpaired t-test.

(E-F) qRT-PCR analysis with RNA isolated from control or ATG5 siRNA transfected THP-1 cells expressing 3X-Flag epitope or Flag-IRGM as indicated. Mean ± SD, n = 3 (biological replicates), ***P < 0.0005, Student's unpaired t-test.

(G-H) qRT-PCR analysis with RNA isolated from control or p62 siRNA or BECLIN1 siRNA transfected HT29 cells expressing 3X-Flag epitope or Flag-IRGM as indicated. Mean ± SD, n = 3 (biological replicates), ***P < 0.0005, Student's unpaired t-test.

(I-J) Co-IP analysis of the interaction between GFP IRGM with (I) Flag-p62 or Flag-NDP52 or Flag-NBR1 or (J) Flag-Optineurin or Flag-TAX1BP1 in HEK293T cell lysates.

(K-M) Co-IP analysis of the interaction between GFP IRGM with (K) Flag-cGAS, (L) Flag-RIG-I, (M) Flag-TLR3 in control siRNA or p62 siRNA transfected HEK293T cell lysates.

(N) The cell lysates of control or p62 siRNA transfected THP-1 cells expressing GFP or GFP- IRGM (as indicate) were subjected to immunoblotting with indicated antibodies.

(O) The qRT-PCR analysis with RNA isolated from control or p62 siRNA transfected THP-1 cells expressing 3X-Flag epitope or Flag-IRGM as indicated. Mean ± SD, n = 3 (biological replicates), ***P < 0.0005, Student's unpaired t-test.

(P-R) Co-IP analysis of the interaction between HA-p62 and (P) Flag-RIG-I, (Q) Flag-TLR3, (R) Flag-cGAS in the absence and presence of GFP or GFP-IRGM in HEK293T cell lysates.

Data information: #degradation products of full-length GFP-IRGM