Journal: Nature Communications

Article Title: Zfp260 choreographs the early stage osteo-lineage commitment of skeletal stem cells

doi: 10.1038/s41467-024-54640-0

Figure Lengend Snippet: a Workflow of in vivo labeling strategy using TAM and the experimental design. b PCA indicating the variations of transcriptomes among Lin - ZsGreen + cells isolated from BF-Ctrl, BF-cKO, MSFL-Ctrl and MSFL-cKO groups. c GO and KEGG enrichment analysis of FACS-RNA-seq data. d Heatmap of replicate data for H3K4me1 and H3K27ac enrichment as detected by CUT&Tag. n = 3 from 3 biological replicates. e GO-biological process enrichment analysis of differentially enriched super-enhancers. f Heatmap of replicate data for Zfp260-V5 enrichment detected by ChIP-seq. n = 2 from 2 biological replicates. g Top enriched de novo motifs of Zfp260-V5 enriched genes. h Distribution of peaks in the genome. i GO and KEGG enrichment analysis of Zfp260-V5 enriched genes. j Screening strategy for the potential master downstream regulator. k Transcripts Per Kilobase (TPM) of Runx2 expression level from fracture and MSFL derived Lin - ZsGreen + cells. n = 3 from 3 biological replicates of RNA-seq data. l Genome browser view of peaks enriched for H3K4me1, Brd4, H3K27ac, and Zfp260-V5 over the Runx2 gene locus on chromosome 17 (left) with the magnified super-enhancer region displayed on the right. Primers 1 and 2 indicated the primer sets for the subsequent ChIP-qPCR detection. m Co-IP was performed to examine the condensates for the super-enhancer via immortalized PSCs. n = 3 from 3 biological replicates. n , o mIHC co-staining for Zfp260 (purple) with Brd4 (gold), Med1 (cyan), and P300 (gray) in the homeostatic and osteogenic states of PSCs. The yellow dotted line indicated the route for the subsequent fluorescence intensity measurements. n = 3 from 3 biological replicates. p Fluorescence intensity measurements along the route, with black triangles indicating the merged signals of the four channels. q , r ChIP-qPCR assays for H3K27ac and Brd4 binding via immortalized PSCs. n = 6 from 2 biological replicates. Two-way ANOVA. Scalebars: 5 μm. All data in this figure are represented as mean ± SD. Source data and exact p values are provided in the Source Data file.

Article Snippet: The primary antibodies used in mIHC (dilution 1:400 for all antibodies) included goat anti-mouse/human/rat Itgav (AF1219, Novus Biologicals), mouse anti-mouse/rat CD90 (NB100-65543, Novus Biologicals), mouse anti-mouse/human CD105 (NBP2-22122, Novus Biologicals), rabbit anti-human/mouse/rat CD200 (AF2724, Novus Biologicals), rabbit anti-mouse/human/rat Runx2 (ab236639, Abcam), rabbit anti-mouse/human/rat Sox9 (ab185966, Abcam), rabbit anti-mouse/human Alpl (MA5-24845, Invitrogen), rabbit anti-mouse/human/rat Zfp260 (ABE295, Merck), mouse anti-human/mouse/rat p300 (NB100-616, Novus Biologicals), rabbit anti-human/mouse MED1 (NB100-2574, Novus Biologicals), rabbit anti-human/mouse BRD4 (NBP2-76393, Novus Biologicals), mouse anti-human/mouse/rat Prkca (NB600-201, Novus Biologicals), rabbit anti-V5 tag (13202, CST), mouse anti-Collagen type I (67288-1-Ig, proteintech), rabbit anti-Collagen type II (28459-1-AP, proteintech).

Techniques: In Vivo, Labeling, Isolation, RNA Sequencing, ChIP-sequencing, Expressing, Derivative Assay, ChIP-qPCR, Co-Immunoprecipitation Assay, Staining, Fluorescence, Binding Assay

Journal: Advanced Science

Article Title: The Long Non‐Coding RNA Obesity‐Related (Obr) Contributes To Lipid Metabolism Through Epigenetic Regulation

doi: 10.1002/advs.202401939

Figure Lengend Snippet: Obr associates with the Creb histone acetyltransferase complex. A) C9 cells stably overexpress, or the expression of Obr downregulated using ASOs showing the expression of Creb and histone acetyltransferases Cbp and p300. No change in the expression was observed by western blot analysis. Gapdh expression was used as the loading control. B) RNA immunoprecipitation (RIP) analysis shows that a significant amount of Obr is immunoprecipitated using an anti‐Creb antibody compared to IgG control, suggesting that Obr associates with Creb. C) Fold enrichment of Obr to that of Gapdh by RIP establishes the specificity of RIP. D) RIP analysis of C9 cells shows that Creb and histone acetyltransferases Cbp and p300 were co‐immunoprecipitated using an anti‐Creb antibody. IgG was used as control, and 10% was used as Input. E) Representative blot of ChRIP analysis of C9 cells with vector control or overexpression of Obr shows that Creb and histone acetyltransferases Cbp and p300 were co‐immunoprecipitated using an Obr antisense probe. Western analysis with pCreb, Cbp, and p300 antibodies shows all were co‐immunoprecipitated, and their level increased with over‐expression. 10% was used as input (lanes 1 and 3). The colocalization of Obr with the pCreb and histone acetyltransferases Cbp and p300 was analyzed using in situ hybridization of Obr followed by pCreb and PI as a nuclear marker F), Obr in situ, followed by pCreb and p300 immuno staining G) and H) Obr in situ followed by pCreb and Cbp immunostaining. Obr was colocalized with pCreb, p300, and Cbp in the nucleus. Scale bar = 50 µm.

Article Snippet: For RNA in situ hybridization followed by immunohistochemistry after the hybridization, the cells were incubated with primary antibodies (CBP, MAB2676, R&D systems; p300, AF3789, R&D systems; and or pCREB, 9198s, Cell Signaling Technology) followed by secondary antibodies conjugated with fluorophores (RRX Donkey Anti‐Mouse IgG, 715‐295‐151; Cy5 Donkey Anti‐Goat IgG, 705‐175‐147; Cy5 Donkey Anti‐Rabbit IgG, 711‐175‐152; RRX Donkey Anti‐Goat IgG Jackson Immuno‐Research).

Techniques: Stable Transfection, Expressing, Western Blot, Control, RNA Immunoprecipitation, Immunoprecipitation, Plasmid Preparation, Over Expression, In Situ Hybridization, Marker, In Situ, Immunostaining

Journal: Advanced Science

Article Title: The Long Non‐Coding RNA Obesity‐Related (Obr) Contributes To Lipid Metabolism Through Epigenetic Regulation

doi: 10.1002/advs.202401939

Figure Lengend Snippet: Obr is a member of the Creb histone acetyltransferase complex in vivo and is altered in diet‐induced obesity. Liver tissues from normal and HFD‐fed Wistar rats, as described in the Experimental Section, were used to assess the expression of the regulatory genes of lipid metabolism, Crebp, C/ebpβ, Pparγ, C/ebpα, Grpr, pCreb, and the acetyltransferases Cbp and P300 by western blotting. Gapdh was used as a loading control. A) The expression of these genes was increased in the livers of HFD‐fed Wistar rats compared to the chow‐fed control rats. B) ChRIP analysis using livers from the control or HFD‐fed Wistar rats shows that Creb and histone acetyltransferases Cbp and p300 were co‐immunoprecipitated using an anti‐Obr probe. Western blot analysis with pCreb, Cbp, and p300 showed all were precipitated, and their level increased in the levers of HFD‐fed animals. ChRIP analysis using a Gapdh probe was used as a negative control. 10% was used as input (lanes 1,2, 5,6, and 9,10). C–F) ChRIP analysis using the liver tissue from rats fed either with normal chow or with HFD showed increased binding to the promoters of C/ebpα, C/ebpβ, Grpr, and Pparγ. The region verified is shown above. The results show increased binding to the promoters of all these genes. Gapdh primers were used as a negative control for the ChRIP analysis.

Article Snippet: For RNA in situ hybridization followed by immunohistochemistry after the hybridization, the cells were incubated with primary antibodies (CBP, MAB2676, R&D systems; p300, AF3789, R&D systems; and or pCREB, 9198s, Cell Signaling Technology) followed by secondary antibodies conjugated with fluorophores (RRX Donkey Anti‐Mouse IgG, 715‐295‐151; Cy5 Donkey Anti‐Goat IgG, 705‐175‐147; Cy5 Donkey Anti‐Rabbit IgG, 711‐175‐152; RRX Donkey Anti‐Goat IgG Jackson Immuno‐Research).

Techniques: In Vivo, Expressing, Western Blot, Control, Immunoprecipitation, Negative Control, Binding Assay

Journal: PLOS ONE

Article Title: Acss2/HIF-2 signaling facilitates colon cancer growth and metastasis

doi: 10.1371/journal.pone.0282223

Figure Lengend Snippet: (A) Detection of endogenous HIF-1α or HIF-2α in HCT116 whole cell extracts by immunoblotting after the indicated period of hypoxia exposure or glucose deprivation. Alpha-tubulin levels for each immunoblot are also shown. (B) Cellular acetate levels generated in HCT116 cells after the indicated period of hypoxia exposure (left side) or glucose deprivation (right side) ( n = 3 biological replicates/time-point; single measurement/replicate; mean/SD) differed as a function of time (P<0.001, one-way ANOVA). (C) Acetylation of endogenous HIF-2α in HCT116 cells after immunoprecipitation (IP) and detection by immunoblotting (IB) after the indicated period of hypoxia exposure (left side) or glucose deprivation (right side) with pharmacological inhibition of Sirt1 by sirtinol and nicotinamide (NAM). (D) Detection of endogenous CBP/HIF-2α or p300/HIF-2α complexes in HCT116 cells by immunoblotting (IB) after early (4 hr) and late (16 hr) hypoxia exposure (left side), or after early (2 hr) and late (24 hr) low glucose exposure (right side).

Article Snippet: After binding, beads were pelleted by centrifugation and washed with buffer, then immunoprecipitated materials were eluted and immunoblotted with anti-human p300 (1:500 dilution; Cat. No. sc-584, Santa Cruz Biotechnology, Santa Cruz, CA), anti-human CBP (1:500 dilution; Cat. No. 4772, Cell Signaling Technology, Danvers, MA), or anti-HIF-2α (1:1,000 dilution; Cat. No. NB100-132, Novus Biologicals) primary antibodies.

Techniques: Western Blot, Generated, Immunoprecipitation, Inhibition

Journal: PLOS ONE

Article Title: Acss2/HIF-2 signaling facilitates colon cancer growth and metastasis

doi: 10.1371/journal.pone.0282223

Figure Lengend Snippet: (A) Detection of endogenous HIF-1α or HIF-2α in HT-29 whole cell extracts by immunoblotting after the indicated period of hypoxia exposure or glucose deprivation. Alpha-tubulin levels for each immunoblot are also shown. (B) Cellular acetate levels generated in HT29 cells after the indicated period of hypoxia exposure (left side) or glucose deprivation (right side) ( n = 3 biological replicates/time-point; single measurement/replicate; mean/SD) differed as a function of time (P<0.001, one-way ANOVA). (C) Acetylation of endogenous HIF-2α in HT29 cells after immunoprecipitation (IP) and detection by immunoblotting (IB) after the indicated period of hypoxia exposure (left side) or glucose deprivation (right side) with pharmacological inhibition of Sirt1 by sirtinol and nicotinamide (NAM). (D) Detection of endogenous CBP/HIF-2α or p300/HIF-2α complexes in HT29 cells by immunoblotting (IB) after early (4 hr) and late (16 hr) hypoxia exposure (left side), or after early (2 hr) and late (24 hr) low glucose exposure (right side).

Article Snippet: After binding, beads were pelleted by centrifugation and washed with buffer, then immunoprecipitated materials were eluted and immunoblotted with anti-human p300 (1:500 dilution; Cat. No. sc-584, Santa Cruz Biotechnology, Santa Cruz, CA), anti-human CBP (1:500 dilution; Cat. No. 4772, Cell Signaling Technology, Danvers, MA), or anti-HIF-2α (1:1,000 dilution; Cat. No. NB100-132, Novus Biologicals) primary antibodies.

Techniques: Western Blot, Generated, Immunoprecipitation, Inhibition

Journal: PLOS ONE

Article Title: Acss2/HIF-2 signaling facilitates colon cancer growth and metastasis

doi: 10.1371/journal.pone.0282223

Figure Lengend Snippet: (A) Acetylation detected by immunoblotting (IB) of ectopic HIF-2α in HIF-2α knockdown HCT116 cells rescued with wild-type (K3) or arginine-lysine substituted mutant (R3) HIF-2α (upper panel), or of endogenous HIF-2α in Acss2 knockdown HCT116 cells rescued with wild-type (WT) or cytosol-restricted mutant (ED) Acss2 (lower panel), following immunoprecipitation (IP) after the indicated period of hypoxia exposure (left side) or glucose deprivation (right side). (B) Interactions of endogenous Cbp or p300 with ectopic HIF-2α in HIF-2α knockdown HCT116 cells rescued with wild-type (K3) or arginine-lysine substituted mutant (R3) HIF-2α (upper panel), or with endogenous HIF-2α in Acss2 knockdown HCT116 cells rescued with wild-type (WT) or cytosol-restricted mutant (ED) Acss2 (lower panel), detected by immunoblotting following immunoprecipitation after hypoxia exposure (left side) or glucose deprivation (right side).

Article Snippet: After binding, beads were pelleted by centrifugation and washed with buffer, then immunoprecipitated materials were eluted and immunoblotted with anti-human p300 (1:500 dilution; Cat. No. sc-584, Santa Cruz Biotechnology, Santa Cruz, CA), anti-human CBP (1:500 dilution; Cat. No. 4772, Cell Signaling Technology, Danvers, MA), or anti-HIF-2α (1:1,000 dilution; Cat. No. NB100-132, Novus Biologicals) primary antibodies.

Techniques: Western Blot, Knockdown, Mutagenesis, Immunoprecipitation

Journal: PLOS ONE

Article Title: Acss2/HIF-2 signaling facilitates colon cancer growth and metastasis

doi: 10.1371/journal.pone.0282223

Figure Lengend Snippet: (A) Acetylation detected by immunoblotting (IB) of ectopic HIF-2α in HIF-2α knockdown HT-29 cells rescued with wild-type (K3) or arginine-lysine substituted mutant (R3) HIF-2α (upper panel), or of endogenous HIF-2α in Acss2 knockdown HT29 cells rescued with wild-type (WT) or cytosol-restricted mutant (ED) Acss2 (lower panel), following immunoprecipitation (IP) after the indicated period of hypoxia exposure (left side) or glucose deprivation (right side). (B) Interactions of endogenous Cbp or p300 with ectopic HIF-2α in HIF-2α knockdown HT29 cells rescued with wild-type (K3) or arginine-lysine substituted mutant (R3) HIF-2α (upper panel), or with endogenous HIF-2α in Acss2 knockdown HT29 cells rescued with wild-type (WT) or cytosol-restricted mutant (ED) Acss2 (lower panel), detected by immunoblotting following immunoprecipitation after hypoxia exposure (left side) or glucose deprivation (right side).

Article Snippet: After binding, beads were pelleted by centrifugation and washed with buffer, then immunoprecipitated materials were eluted and immunoblotted with anti-human p300 (1:500 dilution; Cat. No. sc-584, Santa Cruz Biotechnology, Santa Cruz, CA), anti-human CBP (1:500 dilution; Cat. No. 4772, Cell Signaling Technology, Danvers, MA), or anti-HIF-2α (1:1,000 dilution; Cat. No. NB100-132, Novus Biologicals) primary antibodies.

Techniques: Western Blot, Knockdown, Mutagenesis, Immunoprecipitation