Journal: Frontiers in Oncology

Article Title: Promotion of colorectal cancer by transcription factor BHLHE40 involves upregulation of ADAM19 and KLF7

doi: 10.3389/fonc.2023.1122238

Figure Lengend Snippet: Regulation of BHLHE40 expression by ETV1 and associated JMJD cofactors. (A) Downregulation of ETV1, JMJD1A or JMJD2A with respective shRNAs in HCT116 cells reduced BHLHE40 protein levels. Asterisks mark BHLHE40 or ETV1, while other bands are unspecific in the respective blots. (B) Corresponding quantitative RT-PCR examining BHLHE40 mRNA levels. Shown are means with standard deviations (n=3). One-way ANOVA with post hoc Dunnett’s multiple comparisons test. *P < 0.05; ***P < 0.001; ****P < 0.0001.

Article Snippet: Rabbit polyclonal antibodies against BHLHE40 [Novus, NB100-1800), ETV1 (#959 generated by our laboratory ( )], JMJD1A (Novus, NB100-77282), JMJD2A (Bethyl, A300-861A) and actin (Sigma, A2066) were used as primary antibodies.

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: Promotion of colorectal cancer by transcription factor BHLHE40 involves upregulation of ADAM19 and KLF7

doi: 10.3389/fonc.2023.1122238

Figure Lengend Snippet: Analysis of the BHLHE40 gene promoter in HCT116 cells. (A) Chromatin immunoprecipitation assay. Four different regions (see scheme on top) of the BHLHE40 promoter were amplified after immunoprecipitation with indicated antibodies. (B) Activation of the (-2000/+50) BHLHE40 promoter luciferase reporter construct or the parental vector pGL2-Basic upon ETV1 overexpression. Shown are means with standard deviations (n=4). One-way ANOVA with post hoc Tukey’s multiple comparisons test. (C) Similar, joint stimulation of the (-2000/+50) BHLHE40 promoter luciferase activity by ETV1 and either JMJD1A or JMJD2A; “mut” indicates catalytically inactive mutants of JMJD1A (H1120A/D1122G) or JMJD2A (H188A). Shown are means with standard deviations (n=6). Statistical significance was determined by one-way ANOVA with post hoc Tukey’s multiple comparisons test. *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant.

Article Snippet: Rabbit polyclonal antibodies against BHLHE40 [Novus, NB100-1800), ETV1 (#959 generated by our laboratory ( )], JMJD1A (Novus, NB100-77282), JMJD2A (Bethyl, A300-861A) and actin (Sigma, A2066) were used as primary antibodies.

Techniques: Chromatin Immunoprecipitation, Amplification, Immunoprecipitation, Activation Assay, Luciferase, Construct, Plasmid Preparation, Over Expression, Activity Assay

Journal: Frontiers in Oncology

Article Title: Promotion of colorectal cancer by transcription factor BHLHE40 involves upregulation of ADAM19 and KLF7

doi: 10.3389/fonc.2023.1122238

Figure Lengend Snippet: BHLHE40 promotes HCT116 growth. (A) Downregulation of BHLHE40 with three different shRNAs. Shown are BHLHE40 and actin Western blots. Asterisk marks BHLHE40. (B) Cell growth assay. Shown are means with standard deviations (n=3). Two-way ANOVA with post hoc Tukey’s multiple comparisons test. (C) Corresponding clonogenic assays with representative images.

Article Snippet: Rabbit polyclonal antibodies against BHLHE40 [Novus, NB100-1800), ETV1 (#959 generated by our laboratory ( )], JMJD1A (Novus, NB100-77282), JMJD2A (Bethyl, A300-861A) and actin (Sigma, A2066) were used as primary antibodies.

Techniques: Western Blot, Growth Assay

Journal: Frontiers in Oncology

Article Title: Promotion of colorectal cancer by transcription factor BHLHE40 involves upregulation of ADAM19 and KLF7

doi: 10.3389/fonc.2023.1122238

Figure Lengend Snippet: Expression of BHLHE40 in colorectal tumors. (A) BHLHE40 mRNA levels (reporter A_23_P57838; median-centered ratio) in TCGA microarray experiments . One-way ANOVA (Dunnett’s multiple comparisons test); ****P < 0.0001; ns, not significant. (B) Association of BHLHE40 mRNA levels with recurrence of colorectal carcinomas 5 years after diagnosis (data from reference ; reporter 201169_s_at; median-centered ratio); unpaired, two-tailed t test. (C) Kaplan-Meier survival curve for patients with colorectal adenocarcinoma comparing high (90% cutoff, n=27) with low (50% cutoff, n=135) BHLHE40 mRNA expression. Analysis was done with GEPIA ( http://gepia.cancer-pku.cn/index.html ). P=0.028 (Logrank test).

Article Snippet: Rabbit polyclonal antibodies against BHLHE40 [Novus, NB100-1800), ETV1 (#959 generated by our laboratory ( )], JMJD1A (Novus, NB100-77282), JMJD2A (Bethyl, A300-861A) and actin (Sigma, A2066) were used as primary antibodies.

Techniques: Expressing, Microarray, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: Promotion of colorectal cancer by transcription factor BHLHE40 involves upregulation of ADAM19 and KLF7

doi: 10.3389/fonc.2023.1122238

Figure Lengend Snippet: Transcriptome changes upon BHLHE40 downregulation. (A) Heat map of transcript changes between control HCT116 cells and those with downregulation of BHLHE40. (B) Volcano plot showing average P values versus average fold changes. Only genes with absolute transcript level changes of greater than 2 and P<0.05 were considered up- or downregulated. (C) Validation by quantitative RT-PCR for indicated transcripts that were found downregulated upon expression of BHLHE40 shRNAs. All mRNA levels were normalized to GAPDH . One-way ANOVA (Dunnett’s multiple comparisons test; n=3); **P < 0.01; ***P < 0.001; ****P < 0.0001. (D) Correlation of BHLHE40 mRNA levels with those of ADAM19 in 592 colorectal adenocarcinomas. Data were derived from the TCGA PanCancer Atlas (RSEM values; batch normalized from Illumina HiSeq_RNASeqV2); r=Spearman correlation coefficient. (E) Analogous for KLF7 .

Article Snippet: Rabbit polyclonal antibodies against BHLHE40 [Novus, NB100-1800), ETV1 (#959 generated by our laboratory ( )], JMJD1A (Novus, NB100-77282), JMJD2A (Bethyl, A300-861A) and actin (Sigma, A2066) were used as primary antibodies.

Techniques: Control, Quantitative RT-PCR, Expressing, Derivative Assay