Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Acetylated HOXB13 Regulated Super Enhancer Genes Define Therapeutic Vulnerabilities of Castration-Resistant Prostate Cancer

doi: 10.1158/1078-0432.CCR-21-3603

Figure Lengend Snippet: A, Mass spectrometry analysis of immunoaffinity-purified HOXB13. A peptide corresponding to acetylated HOXB13-K13 was detected with a mass-to-charge ratio of 840.74 m/z and molecular weight of 2519.20 Da. Schematic shows the K13 acetylation site at the N-terminus and the DNA binding homeobox domain (216 to 275 aa) of HOXB13. B, HA-tagged WT HOXB13, or HOXB13K13A, or HOXB13G84E mutants were co-expressed with FLAG-tagged CBP in HEK293T cells. Immunoblot analysis with acK13-HOXB13, HA, FLAG tag and Actin antibodies. C, HA-tagged wild type (WT) HOXB13, HOXB13K13A or HOXB13K13R were co-expressed with His-tagged p300 in HEK293T cells. Immunoblot analysis for acK13-HOXB13, HOXB13, H3K27ac, Histidine or Actin. D, Immunoblot analysis of acK13-HOXB13, pan-HOXB13, AR, H3K27ac, and actin in normal and PC cell lines treated with Vehicle or A-485 (5μM). Actin is used as a normalization control. E, Immunofluorescence analysis with anti-acK13-HOXB13 antibody and DAPI nuclear stain; merged image (overlay) is shown. Scale bars are 200 μm. F, C4-2B cells transfected with control or CBP specific siRNAs and cultured for 48 and 72 hrs. Immunoblot analysis with indicated antibodies. G, C4-2B cells transfected with control, and two different CBP specific siRNAs and harvested after 48 hrs. Immunoblot analysis with indicated antibodies. H, C4-2B cells transfected with control, HOXB13, p300, and/or CBP specific siRNAs and cultured for 48 hrs. Immunoblot analysis with indicated antibodies. I, VCaP cells transfected with control, HOXB13, p300, and/or CBP specific siRNAs and cultured for 48 hrs. Immunoblot analysis with indicated antibodies. Quantitation of the ratio of acK13-HOXB13/pan-HOXB13 is shown in F-I. nq: not quantified.

Article Snippet: Antibodies pan-HOXB13 (F-9) monoclonal, SCBT Cat#sc-28333 (RRID: AB_627744), pan-HOXB13 (H-80) polyclonal, SCBT Cat#sc-66923 (RRID: AB_2233136), pan-HOXB13 (D7N8O) CST Cat#90944 (RRID: AB_2734734), AR (F39.4.1), BioGenex Cat#AM256 (RRID: AB_2687514), CBP (D6C5) CST, Cat#7389 (RRID: AB_2616020); p300 (F-4) SCBT Cat# sc-48343 (RRID: AB_ 628075); CTCF Diagenode Cat#C15410210 (RRID: AB_2753160); Total H3 (96C10) CST Cat#3638 (RRID: AB_1642229); H3K27ac CST Cat#4353 (RRID- AB_10949503); H3K27ac Active Motif Cat#39133 (RRID: AB_2561016); Total H4 (L64C1) CST, Cat#2935 (RRID: AB_1147658); H4 pan-acetyl; Active Motif, Cat#39925 (RRID: AB_2687872); HA (C29F4), CST Cat#3724 (RRID: AB_1549585); FLAG (M2); Sigma-Aldrich Cat#F1804 (RRID: AB_262044); Histidine (RM146); Sigma-Aldrich; Cat#SAB5600227 (RRID: AB_2810125); β-Actin (AC-74); Sigma-Aldrich Cat#A2228; (RRID: AB_476697); BRD9, Bethyl laboratories (A303-781A) RRID: AB_11218396; HRP-conjugated anti-mouse; Promega Cat#W4021 (RRID: AB_430834); HRP-conjugated anti-rabbit; Promega Cat#W4011 (RRID: AB_430833).

Techniques: Mass Spectrometry, Purification, Molecular Weight, Binding Assay, Western Blot, FLAG-tag, Immunofluorescence, Staining, Transfection, Cell Culture, Quantitation Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Acetylated HOXB13 Regulated Super Enhancer Genes Define Therapeutic Vulnerabilities of Castration-Resistant Prostate Cancer

doi: 10.1158/1078-0432.CCR-21-3603

Figure Lengend Snippet: A, ChIP profile of acK13-HOXB13 binding in human normal (pink) and prostate tumor #1 (purple) and prostate tumor #2 (mustard) within 1 kb of the TSSs. B, ChIP profile of H3K27ac in human normal prostate (pink) and tumor #1 (purple) within 1 kb of the TSSs. C, Heatmap of acK13-HOXB13 binding peaks within 1 kb of TSSs in prostate normal and tumor. D, Heatmap of H3K27ac peaks within 1 kb of TSSs in prostate normal and tumor. E, ChIP peak profile of acK13-HOXB13 and F, H3K27ac signals in normal prostates. G, ChIP peak profile of acK13-HOXB13 binding in prostate tumor #1 and tumor #2 and; H, H3K27ac in prostate tumor #1 within 4 kb of characterized SEs. I-K, ROSE in matched normal and two spatially distinct tumor specimens from a PC patient (age 51 years) who underwent a radical prostatectomy. L, acK13-HOXB13-enriched SE proximal in normal prostate (top panel; NES = −1.563, FDR q = 0.007) and M, Prostate Tumor (bottom panel; NES = 2.695, FDR q=0.000). Homer de novo motifs analysis of acK13-HOXB13 binding sites in normal prostate; N, and prostate tumor, O.

Article Snippet: Antibodies pan-HOXB13 (F-9) monoclonal, SCBT Cat#sc-28333 (RRID: AB_627744), pan-HOXB13 (H-80) polyclonal, SCBT Cat#sc-66923 (RRID: AB_2233136), pan-HOXB13 (D7N8O) CST Cat#90944 (RRID: AB_2734734), AR (F39.4.1), BioGenex Cat#AM256 (RRID: AB_2687514), CBP (D6C5) CST, Cat#7389 (RRID: AB_2616020); p300 (F-4) SCBT Cat# sc-48343 (RRID: AB_ 628075); CTCF Diagenode Cat#C15410210 (RRID: AB_2753160); Total H3 (96C10) CST Cat#3638 (RRID: AB_1642229); H3K27ac CST Cat#4353 (RRID- AB_10949503); H3K27ac Active Motif Cat#39133 (RRID: AB_2561016); Total H4 (L64C1) CST, Cat#2935 (RRID: AB_1147658); H4 pan-acetyl; Active Motif, Cat#39925 (RRID: AB_2687872); HA (C29F4), CST Cat#3724 (RRID: AB_1549585); FLAG (M2); Sigma-Aldrich Cat#F1804 (RRID: AB_262044); Histidine (RM146); Sigma-Aldrich; Cat#SAB5600227 (RRID: AB_2810125); β-Actin (AC-74); Sigma-Aldrich Cat#A2228; (RRID: AB_476697); BRD9, Bethyl laboratories (A303-781A) RRID: AB_11218396; HRP-conjugated anti-mouse; Promega Cat#W4021 (RRID: AB_430834); HRP-conjugated anti-rabbit; Promega Cat#W4011 (RRID: AB_430833).

Techniques: Binding Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Acetylated HOXB13 Regulated Super Enhancer Genes Define Therapeutic Vulnerabilities of Castration-Resistant Prostate Cancer

doi: 10.1158/1078-0432.CCR-21-3603

Figure Lengend Snippet: A, qRT-PCR of acK13-HOXB13 target genes in normal (N) and tumor (T) tissues after RP; Gene expression normalized to actin; ****p<0.0001, ***p<0.001, **p<0.01 (n = 19N and 19T matched specimens: total 38). B, Genome browser view of acK13-HOXB13 and H3K27ac binding at the ACK1 gene locus in the patient specimens after radical prostatectomy (RP). C, Immunoblot analysis of acK13-HOXB13, ACK1, pan-HOXB13, AR, and PSMA in patient specimens; normal (N), PC (T), and benign prostatic hyperplasia (BPH). Actin is a normalization control. Quantitation of acK13-HOXB13 signal in tumors relative to normal. D, Immunofluorescent staining for pY284-ACK1, AR, HOXB13 and DAPI-nucleus (blue) in FFPE specimens. Hematoxylin and Eosin Staining (Last panel). Scale bar =100 μm.

Article Snippet: Antibodies pan-HOXB13 (F-9) monoclonal, SCBT Cat#sc-28333 (RRID: AB_627744), pan-HOXB13 (H-80) polyclonal, SCBT Cat#sc-66923 (RRID: AB_2233136), pan-HOXB13 (D7N8O) CST Cat#90944 (RRID: AB_2734734), AR (F39.4.1), BioGenex Cat#AM256 (RRID: AB_2687514), CBP (D6C5) CST, Cat#7389 (RRID: AB_2616020); p300 (F-4) SCBT Cat# sc-48343 (RRID: AB_ 628075); CTCF Diagenode Cat#C15410210 (RRID: AB_2753160); Total H3 (96C10) CST Cat#3638 (RRID: AB_1642229); H3K27ac CST Cat#4353 (RRID- AB_10949503); H3K27ac Active Motif Cat#39133 (RRID: AB_2561016); Total H4 (L64C1) CST, Cat#2935 (RRID: AB_1147658); H4 pan-acetyl; Active Motif, Cat#39925 (RRID: AB_2687872); HA (C29F4), CST Cat#3724 (RRID: AB_1549585); FLAG (M2); Sigma-Aldrich Cat#F1804 (RRID: AB_262044); Histidine (RM146); Sigma-Aldrich; Cat#SAB5600227 (RRID: AB_2810125); β-Actin (AC-74); Sigma-Aldrich Cat#A2228; (RRID: AB_476697); BRD9, Bethyl laboratories (A303-781A) RRID: AB_11218396; HRP-conjugated anti-mouse; Promega Cat#W4021 (RRID: AB_430834); HRP-conjugated anti-rabbit; Promega Cat#W4011 (RRID: AB_430833).

Techniques: Quantitative RT-PCR, Expressing, Binding Assay, Western Blot, Quantitation Assay, Staining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Acetylated HOXB13 Regulated Super Enhancer Genes Define Therapeutic Vulnerabilities of Castration-Resistant Prostate Cancer

doi: 10.1158/1078-0432.CCR-21-3603

Figure Lengend Snippet: A, Cross-linked chromatin extracts from androgen-deprived C4-2B were immunoprecipitated with acK13-HOXB13 or IgG. The pie-chart shows the distribution of acK13-HOXB13 genome-wide signals. B, Binding profile of acK13-HOXB13 −/+ 3 kb of the TSS. C, acK13-HOXB13 binding is enriched for GTAAACA motifs. The other top binding motifs are shown in the table. D, Genome browser view of acK13-HOXB13 peaks at HOXB13 (top panel) in C4-2B CRPCs. Peaks were normalized to input or control IgG. H3K27ac, H3K4me2, and H3K4me3 peaks correspond to ChIP-sequencing data from C4-2B (GSE72714). E-G, ROSE analysis enrichment for selection of acK13-HOXB13 peak-enriched SEs. H, GSEA of acK13-HOXB13 signal marked SE proximal genes in mCRPCs relative to primary PCs (NES = 1.392; FDR q = 0.022) and in primary PC relative to normal (NES= 1.955; FDR q=0.000). I, Integrative GSEA analysis of HOXB13 DEGs proximal to acK13-HOXB13 signal SEs. Left panel: NES = 1.998; FDR q = 0.000. Right panel: NES = 2.301; FDR q = 0.000). J, acK13-HOXB13 tracks at the ACK1/TNK2 genomic locus (>chr3:195635389–195636094; peaks 1 [P1] and 3 [P3]). K, Direct ChIP-qPCR of acK13-HOXB13, RNA Pol II or IgG at the ACK1 locus in C4-2B cells; n = 3 technical replicates. IGX1A, control for non-specific binding; ****p<0.0001 (one-way ANOVA). Data are represented as mean ± SEM. Data are representative of two independent biological replicates.

Article Snippet: Antibodies pan-HOXB13 (F-9) monoclonal, SCBT Cat#sc-28333 (RRID: AB_627744), pan-HOXB13 (H-80) polyclonal, SCBT Cat#sc-66923 (RRID: AB_2233136), pan-HOXB13 (D7N8O) CST Cat#90944 (RRID: AB_2734734), AR (F39.4.1), BioGenex Cat#AM256 (RRID: AB_2687514), CBP (D6C5) CST, Cat#7389 (RRID: AB_2616020); p300 (F-4) SCBT Cat# sc-48343 (RRID: AB_ 628075); CTCF Diagenode Cat#C15410210 (RRID: AB_2753160); Total H3 (96C10) CST Cat#3638 (RRID: AB_1642229); H3K27ac CST Cat#4353 (RRID- AB_10949503); H3K27ac Active Motif Cat#39133 (RRID: AB_2561016); Total H4 (L64C1) CST, Cat#2935 (RRID: AB_1147658); H4 pan-acetyl; Active Motif, Cat#39925 (RRID: AB_2687872); HA (C29F4), CST Cat#3724 (RRID: AB_1549585); FLAG (M2); Sigma-Aldrich Cat#F1804 (RRID: AB_262044); Histidine (RM146); Sigma-Aldrich; Cat#SAB5600227 (RRID: AB_2810125); β-Actin (AC-74); Sigma-Aldrich Cat#A2228; (RRID: AB_476697); BRD9, Bethyl laboratories (A303-781A) RRID: AB_11218396; HRP-conjugated anti-mouse; Promega Cat#W4021 (RRID: AB_430834); HRP-conjugated anti-rabbit; Promega Cat#W4011 (RRID: AB_430833).

Techniques: Immunoprecipitation, Genome Wide, Binding Assay, ChIP-sequencing, Selection

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Acetylated HOXB13 Regulated Super Enhancer Genes Define Therapeutic Vulnerabilities of Castration-Resistant Prostate Cancer

doi: 10.1158/1078-0432.CCR-21-3603

Figure Lengend Snippet: A, Genome browser view of acK13-HOXB13, H3K27ac, H3K4me2, H3K4me3, RNA Pol II, and AR binding at SPON2, ACK1, SLC45A3/Prostein, and VEGFA in C4-2B. B-D, ChIP-qPCR to detect acK13-HOXB13 and H3K27ac binding at the B, SPON2 C, ACK1 and IGX1A - control and D,VEGFA genes in C4-2B treated with vehicle alone (DMSO) or ENZ (5 μM). EP, Enhancer primer. ARBS AR binding site; DIS-TSS Distal to TSS. DEP, Distal Enhancer; PP, Proximal Enhancer. n = 3 technical replicates, ****p < 0.0001, ns = not significant (one-way ANOVA). E-G, t-distributed stochastic neighbor embedding (t-SNE) analysis of single-cell RNA sequencing data of normal human prostate organoid cultured for 14 days (HOXB13, AR and ACK1 expression in individual clusters is shown). H, Heatmap of cell-lineage specific markers and ACK1(TNK2) is shown. I, qRT-PCR for PC marker expression in Normal (N) and tumor (T) PDOs (n=4 each). ****p < 0.0001, ns = not significant (one-way ANOVA). J, Organoids were cultured in the presence of the vehicle, (R)-9b, or anti-androgens ENZ or abiraterone. Phase-contrast microscopy images are as shown; scale bar 50 μm. K, Normal (N) and tumor (T) PDOs were treated with anti-androgens or ACK1 inhibitor (R)-9b. Viability was measured with Cell-titer Glo assays of organoid lysates. Data are mean ± SEM.

Article Snippet: Antibodies pan-HOXB13 (F-9) monoclonal, SCBT Cat#sc-28333 (RRID: AB_627744), pan-HOXB13 (H-80) polyclonal, SCBT Cat#sc-66923 (RRID: AB_2233136), pan-HOXB13 (D7N8O) CST Cat#90944 (RRID: AB_2734734), AR (F39.4.1), BioGenex Cat#AM256 (RRID: AB_2687514), CBP (D6C5) CST, Cat#7389 (RRID: AB_2616020); p300 (F-4) SCBT Cat# sc-48343 (RRID: AB_ 628075); CTCF Diagenode Cat#C15410210 (RRID: AB_2753160); Total H3 (96C10) CST Cat#3638 (RRID: AB_1642229); H3K27ac CST Cat#4353 (RRID- AB_10949503); H3K27ac Active Motif Cat#39133 (RRID: AB_2561016); Total H4 (L64C1) CST, Cat#2935 (RRID: AB_1147658); H4 pan-acetyl; Active Motif, Cat#39925 (RRID: AB_2687872); HA (C29F4), CST Cat#3724 (RRID: AB_1549585); FLAG (M2); Sigma-Aldrich Cat#F1804 (RRID: AB_262044); Histidine (RM146); Sigma-Aldrich; Cat#SAB5600227 (RRID: AB_2810125); β-Actin (AC-74); Sigma-Aldrich Cat#A2228; (RRID: AB_476697); BRD9, Bethyl laboratories (A303-781A) RRID: AB_11218396; HRP-conjugated anti-mouse; Promega Cat#W4021 (RRID: AB_430834); HRP-conjugated anti-rabbit; Promega Cat#W4011 (RRID: AB_430833).

Techniques: Binding Assay, RNA Sequencing Assay, Cell Culture, Expressing, Quantitative RT-PCR, Marker, Microscopy

Journal: Experimental and Therapeutic Medicine

Article Title: Metformin reduces chondrocyte pyroptosis in an osteoarthritis mouse model by inhibiting NLRP3 inflammasome activation

doi: 10.3892/etm.2022.11146

Figure Lengend Snippet: Metformin can decrease chondrocyte pyroptosis in a destabilization of the medial meniscus model at the protein level. (A) NLRP3, (B) caspase-1, (C) GSDMD and (D) IL-1β immunohistochemistry test results (magnification, x400). Scale bar=100 µm. Black arrows indicate the positive cells. The ratio of positive cells immunoreactive for (E) NLRP3, (F) caspase-1, (G) GSDMD and (H) IL-1β. One-way ANOVA followed by Tukey's multiple comparison test was used to compare data among groups after testing the data for homogeneity of variance. *** P<0.001; ### P<0.001. NLRP3, NOD-like receptor protein 3; GSDMD, gasdermin D.

Article Snippet: Next, the sections were blocked with 5% normal goat serum (OriGene Technologies, Inc.) at 37˚C for 30 min and incubated overnight at 4˚C with the following primary antibodies: Anti-MMP-13 (cat. no. ab39012; 1:300 dilution; Abcam), anti-Col II (cat. no. ab34712; 1:300 dilution; Abcam), anti-NLRP3 (cat. no. ab214185; 1:200 dilution; Abcam), anti-caspase-1 (cat. no. 22915-1-AP; 1:200 dilution; ProteinTech Group, Inc.), anti-GSDMD (cat. no. ab219800; 1:200 dilution; Abcam) and anti-IL-1β (cat. no. ab205924; 1:300 dilution; Abcam).

Techniques: Immunohistochemistry

Journal: Experimental and Therapeutic Medicine

Article Title: Metformin reduces chondrocyte pyroptosis in an osteoarthritis mouse model by inhibiting NLRP3 inflammasome activation

doi: 10.3892/etm.2022.11146

Figure Lengend Snippet: Metformin can decrease chondrocyte pyroptosis in a destabilization of the medial meniscus model at gene level. Relative expression levels of (A) NLRP3, (B) caspase-1, (C) GSDMD and (D) IL-1β mRNA. One-way ANOVA followed by Tukey's multiple comparison test was used to compare data among groups after testing the data for homogeneity of variance. * P<0.05 and *** P<0.001; # P<0.05; ## P<0.01; ### P<0.001. NLRP3, NOD-like receptor protein 3; GSDMD, gasdermin D.

Article Snippet: Next, the sections were blocked with 5% normal goat serum (OriGene Technologies, Inc.) at 37˚C for 30 min and incubated overnight at 4˚C with the following primary antibodies: Anti-MMP-13 (cat. no. ab39012; 1:300 dilution; Abcam), anti-Col II (cat. no. ab34712; 1:300 dilution; Abcam), anti-NLRP3 (cat. no. ab214185; 1:200 dilution; Abcam), anti-caspase-1 (cat. no. 22915-1-AP; 1:200 dilution; ProteinTech Group, Inc.), anti-GSDMD (cat. no. ab219800; 1:200 dilution; Abcam) and anti-IL-1β (cat. no. ab205924; 1:300 dilution; Abcam).

Techniques: Expressing