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bhlhe40 (Novus Biologicals)

Novus Biologicals bhlhe40

<t>Bhlhe40</t> is upregulated in lung tissues and macrophages in LPS-induced ALI. ( A ) Representative Hematoxylin-eosin staining (H&E) of lung tissues in mice stimulated by LPS for different times. Scale bar = 100 μm. ( B ) Lung injury score based on H&E staining. ( C - I ) Relative mRNA levels of inflammatory cytokines ( Il-1β, Il-6, Tnf-α, Ifn-γ , Cxcl10, Mcp-1, Il-10 ) in the lungs of mice after LPS induction for different times. ( J - L ) qRT-PCR and Western blots and statistical analyses of Bhlhe40 in the lungs of mice after LPS induction for different times. ( M ) Representative images of immunohistochemistry for Bhlhe40 expression in the lungs of mice. Scale bar = 50 μm and 25 μm. ( N ) Representative images of F4/80 and Bhlhe40 double-immunofluorescence staining of LPS-induced lungs. Scale bar = 50 μm and 20 μm. n = 6. Data are shown as the mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Bonferroni’s multiple comparisons test or unpaired two-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ns p > 0.05

Bhlhe40, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/bhlhe40/product/Novus Biologicals
Average 92 stars, based on 1 article reviews

bhlhe40 - by Bioz Stars, 2026-04

92/100 stars

Buy from Supplier

anti bhlhe40 antibodies (Novus Biologicals)

Novus Biologicals anti bhlhe40 antibodies

<t>BHLHE40</t> affected phosphorylation of AMPKα at Ser172, OCRs, and ECARs. A , gene expression profiles of BHLHE40, PPM1A, PPM1B, PPM1E, and PPM1F in EC cell lines by immunoblotting. ACTB and GAPDH were used as internal controls. B–E , BHLHE40 was knocked down in HHUA ( B ) and KLE ( C ) cells using two different shRNA constructs . BHLHE40 was overexpressed in HEC-1 ( D ) and Ishikawa ( E ) cells. B–E , values under panels indicate the relative expression levels of BHLHE40/ACTB, GAPDH/ACTB, p-AMPKα/AMPKα, and p-ACC/ACC. A , Data are representative of two technical replicates. B–E , Data are representative of at least three biological replicates. Extracellular flux analysis of HHUA ( F and G ), KLE ( H and I ), HEC-1 ( J and K ), and Ishikawa ( L and M ) cells. Real-time OCRs ( F , H , J , and L ) and ECARs ( G , I , K , and M ) were measured upon treatment with the indicated inhibitors or glucose. F–M , data are from three technical replicates. The experiments were biologically replicated twice and representative data are shown. LtCtrl, control lentiviral vector; LtE40, lentiviral vector to express BHLHE40; shCtrl, control shRNA; shE40, shRNA to knockdown BHLHE40 expression.

Anti Bhlhe40 Antibodies, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti bhlhe40 antibodies/product/Novus Biologicals
Average 92 stars, based on 1 article reviews

anti bhlhe40 antibodies - by Bioz Stars, 2026-04

92/100 stars

Buy from Supplier

Image Search Results

Bhlhe40 is upregulated in lung tissues and macrophages in LPS-induced ALI. ( A ) Representative Hematoxylin-eosin staining (H&E) of lung tissues in mice stimulated by LPS for different times. Scale bar = 100 μm. ( B ) Lung injury score based on H&E staining. ( C - I ) Relative mRNA levels of inflammatory cytokines ( Il-1β, Il-6, Tnf-α, Ifn-γ , Cxcl10, Mcp-1, Il-10 ) in the lungs of mice after LPS induction for different times. ( J - L ) qRT-PCR and Western blots and statistical analyses of Bhlhe40 in the lungs of mice after LPS induction for different times. ( M ) Representative images of immunohistochemistry for Bhlhe40 expression in the lungs of mice. Scale bar = 50 μm and 25 μm. ( N ) Representative images of F4/80 and Bhlhe40 double-immunofluorescence staining of LPS-induced lungs. Scale bar = 50 μm and 20 μm. n = 6. Data are shown as the mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Bonferroni’s multiple comparisons test or unpaired two-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ns p > 0.05

Journal: Respiratory Research

Article Title: Bhlhe40 deficiency attenuates LPS-induced acute lung injury through preventing macrophage pyroptosis

doi: 10.1186/s12931-024-02740-2

Figure Lengend Snippet: Bhlhe40 is upregulated in lung tissues and macrophages in LPS-induced ALI. ( A ) Representative Hematoxylin-eosin staining (H&E) of lung tissues in mice stimulated by LPS for different times. Scale bar = 100 μm. ( B ) Lung injury score based on H&E staining. ( C - I ) Relative mRNA levels of inflammatory cytokines ( Il-1β, Il-6, Tnf-α, Ifn-γ , Cxcl10, Mcp-1, Il-10 ) in the lungs of mice after LPS induction for different times. ( J - L ) qRT-PCR and Western blots and statistical analyses of Bhlhe40 in the lungs of mice after LPS induction for different times. ( M ) Representative images of immunohistochemistry for Bhlhe40 expression in the lungs of mice. Scale bar = 50 μm and 25 μm. ( N ) Representative images of F4/80 and Bhlhe40 double-immunofluorescence staining of LPS-induced lungs. Scale bar = 50 μm and 20 μm. n = 6. Data are shown as the mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Bonferroni’s multiple comparisons test or unpaired two-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ns p > 0.05

Article Snippet: Antibodies used for Western blot, immunohistochemistry and Immunofluorescence included Bhlhe40 (NB100-1800SS) from Novus (Centennial, CO, United States), GAPDH (ab181602), GSDMD (ab209845), Caspase-11 (ab180673) from Abcam (Cambridge, United Kingdom), F4/80 (70076T), NLRP3 (15101 S), ASC (67824T) from Cell Signal Technology (Danvers, MA, United States), GSDMD-NT (orb1495171) from biorbyt (Cambridge, United Kingdom), Caspase-1 (A0964) from Abclone (Wuhan, China), and TLR4 (66350-1-Ig), MYD88 (67969-1-Ig) from Proteintech (Rosemont, IL, United States).

Techniques: Staining, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Expressing, Double Immunofluorescence Staining, Two Tailed Test

Bhlhe40 deficiency increases resistance to LPS-induced ALI in mice. ( A ) Representative Hematoxylin-eosin staining (H&E) of lung tissues in mice after LPS induction. Scale bar = 100 μm. ( B ) Lung injury score based on H&E staining. ( C ) Total number of cells counted in the BALF of mice. ( D ) Total protein concentration in the BALF of mice. ( E ) IL-1β levels in BALF of mice. ( F ) IL-10 levels in BALF of mice. ( G - K ) Relative mRNA levels of inflammatory cytokines ( Il-6, Tnf-α, Ifn-γ , Mcp-1, Cxcl10 ) in the lungs of mice after LPS induction. n = 6. Data are shown as the mean ± SEM. Statistical analysis was performed by two-way ANOVA followed by Bonferroni’s multiple comparisons test. **p < 0.01, ***p < 0.001

Journal: Respiratory Research

Article Title: Bhlhe40 deficiency attenuates LPS-induced acute lung injury through preventing macrophage pyroptosis

doi: 10.1186/s12931-024-02740-2

Figure Lengend Snippet: Bhlhe40 deficiency increases resistance to LPS-induced ALI in mice. ( A ) Representative Hematoxylin-eosin staining (H&E) of lung tissues in mice after LPS induction. Scale bar = 100 μm. ( B ) Lung injury score based on H&E staining. ( C ) Total number of cells counted in the BALF of mice. ( D ) Total protein concentration in the BALF of mice. ( E ) IL-1β levels in BALF of mice. ( F ) IL-10 levels in BALF of mice. ( G - K ) Relative mRNA levels of inflammatory cytokines ( Il-6, Tnf-α, Ifn-γ , Mcp-1, Cxcl10 ) in the lungs of mice after LPS induction. n = 6. Data are shown as the mean ± SEM. Statistical analysis was performed by two-way ANOVA followed by Bonferroni’s multiple comparisons test. **p < 0.01, ***p < 0.001

Techniques: Staining, Protein Concentration

Bhlhe40 deficiency alleviates GSDMD-mediated pyroptosis and caspase-1 and caspase-11 pathways in LPS-induced ALI mice. ( A ) Representative Western blot of GSDMD FL and GSDMD NT in the lung tissues of mice. ( B ) Representative Western blot of cleaved IL-1β in the lung tissues of mice. ( C ) Representative images of F4/80 and GSDMD NT double-immunofluorescence staining of LPS-induced lungs. Scale bar = 50 μm and 20 μm. (D) Representative Western blot of pro-caspase-1, cleaved caspase-1, pro-caspase-11, cleaved caspase-11, NLRP3, ASC, TLR4 and MYD88 in the lung tissues of mice. n = 6

Journal: Respiratory Research

Article Title: Bhlhe40 deficiency attenuates LPS-induced acute lung injury through preventing macrophage pyroptosis

doi: 10.1186/s12931-024-02740-2

Figure Lengend Snippet: Bhlhe40 deficiency alleviates GSDMD-mediated pyroptosis and caspase-1 and caspase-11 pathways in LPS-induced ALI mice. ( A ) Representative Western blot of GSDMD FL and GSDMD NT in the lung tissues of mice. ( B ) Representative Western blot of cleaved IL-1β in the lung tissues of mice. ( C ) Representative images of F4/80 and GSDMD NT double-immunofluorescence staining of LPS-induced lungs. Scale bar = 50 μm and 20 μm. (D) Representative Western blot of pro-caspase-1, cleaved caspase-1, pro-caspase-11, cleaved caspase-11, NLRP3, ASC, TLR4 and MYD88 in the lung tissues of mice. n = 6

Techniques: Western Blot, Double Immunofluorescence Staining

Bhlhe40 deficiency inhibits GSDMD-mediated pyroptosis through both canonical and non-canonical pathways in BMDMs and in RAW264.7 cell line. ( A - C ) Representative Western blot of GSDMD FL , GSDMD NT , cleaved IL-1β, pro-caspase-1, cleaved caspase-1, pro-caspase-11, cleaved caspase-11, NLRP3 and ASC in BMDMs. ( D - E ) RAW264.7 cells were transfected with three Bhlhe40 siRNAs (siRNA#1, siRNA#2 and siRNA#3) or normal control siRNA (NC) for 48 h. The protein levels of Bhlhe40 were detected by western blots and quantified analysis. ( E - F ) Representative Western blot of GSDMD FL , GSDMD NT , cleaved IL-1β, pro-caspase-1, cleaved caspase-1, pro-caspase-11, cleaved caspase-11, NLRP3 and ASC in RAW246.7 cells. n = 3. Data are shown as the mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Bonferroni’s multiple comparisons test. *p < 0.05, ns p > 0.05

Journal: Respiratory Research

Article Title: Bhlhe40 deficiency attenuates LPS-induced acute lung injury through preventing macrophage pyroptosis

doi: 10.1186/s12931-024-02740-2

Figure Lengend Snippet: Bhlhe40 deficiency inhibits GSDMD-mediated pyroptosis through both canonical and non-canonical pathways in BMDMs and in RAW264.7 cell line. ( A - C ) Representative Western blot of GSDMD FL , GSDMD NT , cleaved IL-1β, pro-caspase-1, cleaved caspase-1, pro-caspase-11, cleaved caspase-11, NLRP3 and ASC in BMDMs. ( D - E ) RAW264.7 cells were transfected with three Bhlhe40 siRNAs (siRNA#1, siRNA#2 and siRNA#3) or normal control siRNA (NC) for 48 h. The protein levels of Bhlhe40 were detected by western blots and quantified analysis. ( E - F ) Representative Western blot of GSDMD FL , GSDMD NT , cleaved IL-1β, pro-caspase-1, cleaved caspase-1, pro-caspase-11, cleaved caspase-11, NLRP3 and ASC in RAW246.7 cells. n = 3. Data are shown as the mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Bonferroni’s multiple comparisons test. *p < 0.05, ns p > 0.05

Techniques: Western Blot, Transfection, Control

Bhlhe40 deficiency suppresses LPS-induced inflammatory cytokine production in BMDMs and in RAW264.7 cell line. ( A ) Il-1β levels in the culture supernatant of BMDMs. ( B ) Il-1β levels in the culture supernatant of RAW264.7 cells. ( C ) Relative mRNA levels of Il-6, Tnf-α, Cxcl10 and iNOS in BMDMs were assessed by qRT-PCR. ( D ) Relative mRNA levels of Il-6, Tnf-α, Cxcl10 and iNOS in RAW264.7 cells were assessed by qRT-PCR. n = 3. Data are shown as the mean ± SEM. Statistical analysis was performed by two-way ANOVA followed by Bonferroni’s multiple comparisons test or unpaired two-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001

Journal: Respiratory Research

Article Title: Bhlhe40 deficiency attenuates LPS-induced acute lung injury through preventing macrophage pyroptosis

doi: 10.1186/s12931-024-02740-2

Figure Lengend Snippet: Bhlhe40 deficiency suppresses LPS-induced inflammatory cytokine production in BMDMs and in RAW264.7 cell line. ( A ) Il-1β levels in the culture supernatant of BMDMs. ( B ) Il-1β levels in the culture supernatant of RAW264.7 cells. ( C ) Relative mRNA levels of Il-6, Tnf-α, Cxcl10 and iNOS in BMDMs were assessed by qRT-PCR. ( D ) Relative mRNA levels of Il-6, Tnf-α, Cxcl10 and iNOS in RAW264.7 cells were assessed by qRT-PCR. n = 3. Data are shown as the mean ± SEM. Statistical analysis was performed by two-way ANOVA followed by Bonferroni’s multiple comparisons test or unpaired two-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001

Techniques: Quantitative RT-PCR, Two Tailed Test

BHLHE40 affected phosphorylation of AMPKα at Ser172, OCRs, and ECARs. A , gene expression profiles of BHLHE40, PPM1A, PPM1B, PPM1E, and PPM1F in EC cell lines by immunoblotting. ACTB and GAPDH were used as internal controls. B–E , BHLHE40 was knocked down in HHUA ( B ) and KLE ( C ) cells using two different shRNA constructs . BHLHE40 was overexpressed in HEC-1 ( D ) and Ishikawa ( E ) cells. B–E , values under panels indicate the relative expression levels of BHLHE40/ACTB, GAPDH/ACTB, p-AMPKα/AMPKα, and p-ACC/ACC. A , Data are representative of two technical replicates. B–E , Data are representative of at least three biological replicates. Extracellular flux analysis of HHUA ( F and G ), KLE ( H and I ), HEC-1 ( J and K ), and Ishikawa ( L and M ) cells. Real-time OCRs ( F , H , J , and L ) and ECARs ( G , I , K , and M ) were measured upon treatment with the indicated inhibitors or glucose. F–M , data are from three technical replicates. The experiments were biologically replicated twice and representative data are shown. LtCtrl, control lentiviral vector; LtE40, lentiviral vector to express BHLHE40; shCtrl, control shRNA; shE40, shRNA to knockdown BHLHE40 expression.

Journal: The Journal of Biological Chemistry

Article Title: The BHLHE40‒PPM1F‒AMPK pathway regulates energy metabolism and is associated with the aggressiveness of endometrial cancer

doi: 10.1016/j.jbc.2024.105695

Figure Lengend Snippet: BHLHE40 affected phosphorylation of AMPKα at Ser172, OCRs, and ECARs. A , gene expression profiles of BHLHE40, PPM1A, PPM1B, PPM1E, and PPM1F in EC cell lines by immunoblotting. ACTB and GAPDH were used as internal controls. B–E , BHLHE40 was knocked down in HHUA ( B ) and KLE ( C ) cells using two different shRNA constructs . BHLHE40 was overexpressed in HEC-1 ( D ) and Ishikawa ( E ) cells. B–E , values under panels indicate the relative expression levels of BHLHE40/ACTB, GAPDH/ACTB, p-AMPKα/AMPKα, and p-ACC/ACC. A , Data are representative of two technical replicates. B–E , Data are representative of at least three biological replicates. Extracellular flux analysis of HHUA ( F and G ), KLE ( H and I ), HEC-1 ( J and K ), and Ishikawa ( L and M ) cells. Real-time OCRs ( F , H , J , and L ) and ECARs ( G , I , K , and M ) were measured upon treatment with the indicated inhibitors or glucose. F–M , data are from three technical replicates. The experiments were biologically replicated twice and representative data are shown. LtCtrl, control lentiviral vector; LtE40, lentiviral vector to express BHLHE40; shCtrl, control shRNA; shE40, shRNA to knockdown BHLHE40 expression.

Article Snippet: We tested three anti-BHLHE40 antibodies (sc-101023 from Santa Cruz Biotechnology; HPA028921 from Atlas Antibodies; NB100 to 1800 from Novus Biologicals) in the ChIP assay.

Techniques: Phospho-proteomics, Gene Expression, Western Blot, shRNA, Construct, Expressing, Control, Plasmid Preparation, Knockdown

Comprehensive analysis of BHLHE40-knocked down HHUA cells by microarray and iMPAQT analyses. A , Gene Set Enrichment Analysis (GSEA) of upregulated genes in wild-type mouse embryonic fibroblasts (MEFs) and AMPKα1 and AMPKα2 double knockout MEFs ( https://www.ncbi.nlm.nih.gov/geo/ : accession number GSE97735 ) were compared with upregulated genes in shCtrl-transfected HHUA cells relative to those transfected with shBHLHE40. B , GSEA of upregulated genes in control cardiac fibroblasts transfected with scrambled siRNA and those transfected with siAMPKα1 ( https://www.ncbi.nlm.nih.gov/geo/ : accession number GSE147470 ) were compared with upregulated genes in HHUA cells transfected with shCtrl relative to those transfected with shBHLHE40. C , an annotated gene set of 187 genes upregulated in AKT-transgenic murine prostate (M2666, MSigDB, https://www.gsea-msigdb.org/gsea/msigdb/ ) was compared with upregulated genes in HHUA cells transfected with shCtrl relative to those transfected with shBHLHE40. D , an annotated gene set of 62 genes from the glycolysis/gluconeogenesis pathway by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (M11521, MSigDB, https://www.gsea-msigdb.org/gsea/msigdb/ ) was compared with upregulated genes in HHUA cells transfected with shCtrl relative to those transfected with shBHLHE40. E , a clustered heat map analysis of iMPAQT data. F , Volcano plotting analysis of iMPAQT data. Both microarray and iMPAQT data were from three biological replicates. shCtrl, shControl; shE40, shBHLHE40.

Journal: The Journal of Biological Chemistry

Article Title: The BHLHE40‒PPM1F‒AMPK pathway regulates energy metabolism and is associated with the aggressiveness of endometrial cancer

doi: 10.1016/j.jbc.2024.105695

Figure Lengend Snippet: Comprehensive analysis of BHLHE40-knocked down HHUA cells by microarray and iMPAQT analyses. A , Gene Set Enrichment Analysis (GSEA) of upregulated genes in wild-type mouse embryonic fibroblasts (MEFs) and AMPKα1 and AMPKα2 double knockout MEFs ( https://www.ncbi.nlm.nih.gov/geo/ : accession number GSE97735 ) were compared with upregulated genes in shCtrl-transfected HHUA cells relative to those transfected with shBHLHE40. B , GSEA of upregulated genes in control cardiac fibroblasts transfected with scrambled siRNA and those transfected with siAMPKα1 ( https://www.ncbi.nlm.nih.gov/geo/ : accession number GSE147470 ) were compared with upregulated genes in HHUA cells transfected with shCtrl relative to those transfected with shBHLHE40. C , an annotated gene set of 187 genes upregulated in AKT-transgenic murine prostate (M2666, MSigDB, https://www.gsea-msigdb.org/gsea/msigdb/ ) was compared with upregulated genes in HHUA cells transfected with shCtrl relative to those transfected with shBHLHE40. D , an annotated gene set of 62 genes from the glycolysis/gluconeogenesis pathway by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (M11521, MSigDB, https://www.gsea-msigdb.org/gsea/msigdb/ ) was compared with upregulated genes in HHUA cells transfected with shCtrl relative to those transfected with shBHLHE40. E , a clustered heat map analysis of iMPAQT data. F , Volcano plotting analysis of iMPAQT data. Both microarray and iMPAQT data were from three biological replicates. shCtrl, shControl; shE40, shBHLHE40.

Article Snippet: We tested three anti-BHLHE40 antibodies (sc-101023 from Santa Cruz Biotechnology; HPA028921 from Atlas Antibodies; NB100 to 1800 from Novus Biologicals) in the ChIP assay.

Techniques: Microarray, Double Knockout, Transfection, Control, Transgenic Assay

PDH and LDH activity and PDHA1 and LDHA expression in BHLHE40-modulated EC cells. PDH activity ( A , C , E , and G ) and LDH activity ( B , D , F , and H ) were measured in HHUA ( A and B ), KLE ( C and D ), HEC-1 ( E and F ), and Ishikawa ( G and H ) cells after culturing for 24 h in DMEM or DMEM:F12 with 10% FBS with 1 mM sodium pyruvate without glucose. A–H , data are from three technical replicates. The experiments were biologically replicated three times and representative data are shown. I–L , immunoblotting analysis of EC cells cultured for 24 h in DMEM or DMEM:F12 with the indicated concentrations of glucose with 1 mM sodium pyruvate and 10% FBS. HHUA ( I ), KLE ( J ), HEC-1 ( K ), and Ishikawa ( L ) cells. I–L , values under panels indicate the relative expression levels of p-PDHA1/PDHA1, p-LDHA/LDHA, p-AMPKα/AMPKα, p-ACC/ACC, and LDHA/ACTB. Data are representative of at least three biological replicates. shCtrl, shControl; shE40, shBHLHE40; LtCtrl, LtControl; LtE40, LtBHLHE40. A–H , unpaired two-sided Student’s t test or the Mann–Whitney U test was used. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Journal: The Journal of Biological Chemistry

Article Title: The BHLHE40‒PPM1F‒AMPK pathway regulates energy metabolism and is associated with the aggressiveness of endometrial cancer

doi: 10.1016/j.jbc.2024.105695

Figure Lengend Snippet: PDH and LDH activity and PDHA1 and LDHA expression in BHLHE40-modulated EC cells. PDH activity ( A , C , E , and G ) and LDH activity ( B , D , F , and H ) were measured in HHUA ( A and B ), KLE ( C and D ), HEC-1 ( E and F ), and Ishikawa ( G and H ) cells after culturing for 24 h in DMEM or DMEM:F12 with 10% FBS with 1 mM sodium pyruvate without glucose. A–H , data are from three technical replicates. The experiments were biologically replicated three times and representative data are shown. I–L , immunoblotting analysis of EC cells cultured for 24 h in DMEM or DMEM:F12 with the indicated concentrations of glucose with 1 mM sodium pyruvate and 10% FBS. HHUA ( I ), KLE ( J ), HEC-1 ( K ), and Ishikawa ( L ) cells. I–L , values under panels indicate the relative expression levels of p-PDHA1/PDHA1, p-LDHA/LDHA, p-AMPKα/AMPKα, p-ACC/ACC, and LDHA/ACTB. Data are representative of at least three biological replicates. shCtrl, shControl; shE40, shBHLHE40; LtCtrl, LtControl; LtE40, LtBHLHE40. A–H , unpaired two-sided Student’s t test or the Mann–Whitney U test was used. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: We tested three anti-BHLHE40 antibodies (sc-101023 from Santa Cruz Biotechnology; HPA028921 from Atlas Antibodies; NB100 to 1800 from Novus Biologicals) in the ChIP assay.

Techniques: Activity Assay, Expressing, Western Blot, Cell Culture, MANN-WHITNEY

Impact of AMPKα expression on BHLHE40-expressing EC cells. Immunoblotting analysis of EC cells transfected with an siRNA against AMPKα1/2 (siAMPKα) and cultured for 24 h in DMEM with 10% FBS and 1 mM sodium pyruvate without glucose. HEC1 ( A ) and Ishikawa ( B ) cells. A and B , values under panels indicate relative expression levels of p- AMPKα/AMPKα, p-ACC/ACC, p-PDHA1/PDHA1, p-LDHA/LDHA, AMPKα/ACTB and LDHA/ACTB. A and B , data are representative of at least three biological replicates. PDH activity ( C and E ) and LDH activity ( D and F ) were measured in HEC1 ( C and D ) and Ishikawa ( E and F ) cells after culturing for 24 h in DMEM with 10% FBS and 1 mM sodium pyruvate without glucose. ( C – F ) Data are from three technical replicates. The experiments were biologically replicated three times and representative data are shown. Extracellular flux analysis of HEC-1 ( G and H ) and Ishikawa ( I and J ) cells. Real-time OCRs ( G and I ) and ECARs ( H and J ) were measured upon treatment with the indicated inhibitors or glucose. G–J , data are from three technical replicates. The experiments were biologically replicated twice and representative data are shown. C–F , unpaired two-sided Student’s t test or the Mann–Whitney U test was used. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. LtCtrl, LtControl; LtE40, LtBHLHE40; shCtrl, shControl; shE40, shBHLHE40.

Journal: The Journal of Biological Chemistry

Article Title: The BHLHE40‒PPM1F‒AMPK pathway regulates energy metabolism and is associated with the aggressiveness of endometrial cancer

doi: 10.1016/j.jbc.2024.105695

Figure Lengend Snippet: Impact of AMPKα expression on BHLHE40-expressing EC cells. Immunoblotting analysis of EC cells transfected with an siRNA against AMPKα1/2 (siAMPKα) and cultured for 24 h in DMEM with 10% FBS and 1 mM sodium pyruvate without glucose. HEC1 ( A ) and Ishikawa ( B ) cells. A and B , values under panels indicate relative expression levels of p- AMPKα/AMPKα, p-ACC/ACC, p-PDHA1/PDHA1, p-LDHA/LDHA, AMPKα/ACTB and LDHA/ACTB. A and B , data are representative of at least three biological replicates. PDH activity ( C and E ) and LDH activity ( D and F ) were measured in HEC1 ( C and D ) and Ishikawa ( E and F ) cells after culturing for 24 h in DMEM with 10% FBS and 1 mM sodium pyruvate without glucose. ( C – F ) Data are from three technical replicates. The experiments were biologically replicated three times and representative data are shown. Extracellular flux analysis of HEC-1 ( G and H ) and Ishikawa ( I and J ) cells. Real-time OCRs ( G and I ) and ECARs ( H and J ) were measured upon treatment with the indicated inhibitors or glucose. G–J , data are from three technical replicates. The experiments were biologically replicated twice and representative data are shown. C–F , unpaired two-sided Student’s t test or the Mann–Whitney U test was used. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. LtCtrl, LtControl; LtE40, LtBHLHE40; shCtrl, shControl; shE40, shBHLHE40.

Article Snippet: We tested three anti-BHLHE40 antibodies (sc-101023 from Santa Cruz Biotechnology; HPA028921 from Atlas Antibodies; NB100 to 1800 from Novus Biologicals) in the ChIP assay.

Techniques: Expressing, Western Blot, Transfection, Cell Culture, Activity Assay, MANN-WHITNEY

Expression of PPM1s in BHLHE40-modulated EC cells. A–D , immunoblotting analysis of EC cells cultured for 24 h in DMEM or DMEM:F12 with the indicated concentrations of glucose with 1 mM sodium pyruvate and 10% FBS. HHUA ( A ), KLE ( B ). HEC-1 ( C ), and Ishikawa ( D ) cells. A–D , values under panels indicate relative expression levels of PPM1A/ACTB, PPM1F/ACTB, and PPM1E/ACTB. A–D , data are representative of three biological replicates. E–L , real-time RT-PCR analysis of EC cells cultured for 24 h in DMEM or DMEM:F12 with 1.0 g/L glucose, 1 mM sodium pyruvate, and 10% FBS. HHUA ( E and F ), KLE ( G and H ). HEC-1 ( I and J ), and Ishikawa ( K and L ) cells. PPM1A ( E , G , I , and K ), PPM1F ( F , H , and J ), and PPM1E ( L ). E–L , data are from three biological replicates. Unpaired two-sided Student’s t test was used. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. LtCtrl, LtControl; LtE40, LtBHLHE40; shCtrl, shControl; shE40, shBHLHE40.

Journal: The Journal of Biological Chemistry

Article Title: The BHLHE40‒PPM1F‒AMPK pathway regulates energy metabolism and is associated with the aggressiveness of endometrial cancer

doi: 10.1016/j.jbc.2024.105695

Figure Lengend Snippet: Expression of PPM1s in BHLHE40-modulated EC cells. A–D , immunoblotting analysis of EC cells cultured for 24 h in DMEM or DMEM:F12 with the indicated concentrations of glucose with 1 mM sodium pyruvate and 10% FBS. HHUA ( A ), KLE ( B ). HEC-1 ( C ), and Ishikawa ( D ) cells. A–D , values under panels indicate relative expression levels of PPM1A/ACTB, PPM1F/ACTB, and PPM1E/ACTB. A–D , data are representative of three biological replicates. E–L , real-time RT-PCR analysis of EC cells cultured for 24 h in DMEM or DMEM:F12 with 1.0 g/L glucose, 1 mM sodium pyruvate, and 10% FBS. HHUA ( E and F ), KLE ( G and H ). HEC-1 ( I and J ), and Ishikawa ( K and L ) cells. PPM1A ( E , G , I , and K ), PPM1F ( F , H , and J ), and PPM1E ( L ). E–L , data are from three biological replicates. Unpaired two-sided Student’s t test was used. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. LtCtrl, LtControl; LtE40, LtBHLHE40; shCtrl, shControl; shE40, shBHLHE40.

Article Snippet: We tested three anti-BHLHE40 antibodies (sc-101023 from Santa Cruz Biotechnology; HPA028921 from Atlas Antibodies; NB100 to 1800 from Novus Biologicals) in the ChIP assay.

Techniques: Expressing, Western Blot, Cell Culture, Quantitative RT-PCR

BHLHE40 t ranscriptionally suppresses PPM1F expression. A, top , schematic presentation of four E-boxes in the promoter of PPM1F . A, bottom , Gel shift assay using nuclear extracts from 293T cells transfected with FLAG-BHLHE40 was incubated with labeled E-box1, 2, 3, and 4 probes ( <xref ref-type=

Table S4 ). A canonical E-box probe from the BHLHE41 promoter was used as a positive control . B , anti-FLAG antibody was used to form supershifted bands. An anti-SRF antibody was used as a negative control. A and B , data are representative of two biological replicates. C and D, top , reporter analysis of the wild type and mutant PPM1F promoter in HEC-6 cells transfected with FLAG-BHLHE40 ( Table S3 ). C and D, bottom , Reporter analysis of the wild type and mutant PPM1F promoter in HHUA cells transfected with siBHLHE40 at a concentration of 50 nM. See also Fig. S6 . C and D , data are from four technical replicates. The experiments were biologically replicated three times and representative data are shown. E and F , ChIP assay using 293T cells transfected with empty vector (pCDNA3) or HA-BHLHE40 (pCDNA3-HA-BHLHE40). Protein–DNA complexes immunoprecipitated with anti-HA, anti-HDAC1, anti-acetylated-histone H3 (Ac-H3), or anti-PCAF antibodies were used to amplify indicated promoter regions by PCR ( Table S1 ). The −6602 to −6544 and −4166 to −4089 regions contain E-Box2 and E-Box4, respectively. The −375 to −252 region represents a negative control. The occupancy ratios (%) were calculated using 10% input samples as standards. αHA1, anti-HA (HA-7; Sigma–Aldrich) antibody; αHA2, anti-HA (ab9110; Abcam) antibody. E and F , data are from three biological replicates. ( C–F ) Unpaired two-sided Student’s t test or the Mann–Whitney U test was used. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. " width="100%" height="100%">

Journal: The Journal of Biological Chemistry

Article Title: The BHLHE40‒PPM1F‒AMPK pathway regulates energy metabolism and is associated with the aggressiveness of endometrial cancer

doi: 10.1016/j.jbc.2024.105695

Figure Lengend Snippet: BHLHE40 t ranscriptionally suppresses PPM1F expression. A, top , schematic presentation of four E-boxes in the promoter of PPM1F . A, bottom , Gel shift assay using nuclear extracts from 293T cells transfected with FLAG-BHLHE40 was incubated with labeled E-box1, 2, 3, and 4 probes ( Table S4 ). A canonical E-box probe from the BHLHE41 promoter was used as a positive control . B , anti-FLAG antibody was used to form supershifted bands. An anti-SRF antibody was used as a negative control. A and B , data are representative of two biological replicates. C and D, top , reporter analysis of the wild type and mutant PPM1F promoter in HEC-6 cells transfected with FLAG-BHLHE40 ( Table S3 ). C and D, bottom , Reporter analysis of the wild type and mutant PPM1F promoter in HHUA cells transfected with siBHLHE40 at a concentration of 50 nM. See also Fig. S6 . C and D , data are from four technical replicates. The experiments were biologically replicated three times and representative data are shown. E and F , ChIP assay using 293T cells transfected with empty vector (pCDNA3) or HA-BHLHE40 (pCDNA3-HA-BHLHE40). Protein–DNA complexes immunoprecipitated with anti-HA, anti-HDAC1, anti-acetylated-histone H3 (Ac-H3), or anti-PCAF antibodies were used to amplify indicated promoter regions by PCR ( Table S1 ). The −6602 to −6544 and −4166 to −4089 regions contain E-Box2 and E-Box4, respectively. The −375 to −252 region represents a negative control. The occupancy ratios (%) were calculated using 10% input samples as standards. αHA1, anti-HA (HA-7; Sigma–Aldrich) antibody; αHA2, anti-HA (ab9110; Abcam) antibody. E and F , data are from three biological replicates. ( C–F ) Unpaired two-sided Student’s t test or the Mann–Whitney U test was used. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: We tested three anti-BHLHE40 antibodies (sc-101023 from Santa Cruz Biotechnology; HPA028921 from Atlas Antibodies; NB100 to 1800 from Novus Biologicals) in the ChIP assay.

Techniques: Expressing, Gel Shift, Transfection, Incubation, Labeling, Positive Control, Negative Control, Mutagenesis, Concentration Assay, Plasmid Preparation, Immunoprecipitation, MANN-WHITNEY

Phosphatase activity of PPM1F on phospho-AMPKα Ser172. A , immunoblotting of HHUA cells transfected with FLAG-tagged PPM1A-, PPM1B-, PPM1E-, or PPM1F-expressing vector. B , immunoblotting of HHUA cells transfected with wild type and phosphatase inactive mutant (R326A-I328A) PPM1F. C , in vitro phosphatase assay reconstituted with FLAG-PPM1F and activated AMPK complex. D and E , immunoblotting analysis of HHUA ( D ) and HEC-1 ( E ) cells transfected with indicated siRNAs. Values under panels indicate relative expression levels of p-AMPKα/AMPKα, p-ACC/ACC, p-PDHA1/PDHA1, p-LDHA/LDHA, LDHA/ACTB, BHLHE40/ACTB, PPM1A/ACTB, and PPM1F/ACTB. Data are representative of at least two technical replicates from three biological replicates.

Journal: The Journal of Biological Chemistry

Article Title: The BHLHE40‒PPM1F‒AMPK pathway regulates energy metabolism and is associated with the aggressiveness of endometrial cancer

doi: 10.1016/j.jbc.2024.105695

Figure Lengend Snippet: Phosphatase activity of PPM1F on phospho-AMPKα Ser172. A , immunoblotting of HHUA cells transfected with FLAG-tagged PPM1A-, PPM1B-, PPM1E-, or PPM1F-expressing vector. B , immunoblotting of HHUA cells transfected with wild type and phosphatase inactive mutant (R326A-I328A) PPM1F. C , in vitro phosphatase assay reconstituted with FLAG-PPM1F and activated AMPK complex. D and E , immunoblotting analysis of HHUA ( D ) and HEC-1 ( E ) cells transfected with indicated siRNAs. Values under panels indicate relative expression levels of p-AMPKα/AMPKα, p-ACC/ACC, p-PDHA1/PDHA1, p-LDHA/LDHA, LDHA/ACTB, BHLHE40/ACTB, PPM1A/ACTB, and PPM1F/ACTB. Data are representative of at least two technical replicates from three biological replicates.

Article Snippet: We tested three anti-BHLHE40 antibodies (sc-101023 from Santa Cruz Biotechnology; HPA028921 from Atlas Antibodies; NB100 to 1800 from Novus Biologicals) in the ChIP assay.

Techniques: Activity Assay, Western Blot, Transfection, Expressing, Plasmid Preparation, Mutagenesis, In Vitro, Phosphatase Assay

Protein expression of BHLHE40, phospho-AMPKα, and PPM1F correlates with prognosis of endometrial cancer patients. A–D , Immunohistochemical analysis of primary sites from surgically removed uteri. Stage IA, endometrioid carcinoma, grade 1 ( A ); stage IB, endometrioid carcinoma grade 1 ( B ); stage IVB, endometrioid carcinoma, grade 3 ( C ); stage IIIC2, serous carcinoma ( D ). The scale bars indicate 200 μm. Total staining score of BHLHE40 was evaluated between groups of endometrioid carcinoma grade 1 and 2 and that of grade 3, serous carcinoma ( E ). Comparison of groups at stage I and II and that of stage III and IV ( F ). E and F , Welch’s t test was applied. G , total staining scores of BHLHE40 and phospho-AMPKα were analyzed using Pearson’s product-moment correlation coefficient. Also see <xref ref-type=

Fig. S7 A . H , Total staining scores of BHLHE40 and PPM1F were analyzed using Pearson’s product-moment correlation coefficient. Also see Fig. S7 B . r-values show correlation coefficients. I , correlation between BHLHE40 mRNA levels and recurrence-free survival of endometrial cancer cases (n = 543 from Gene Expression Omnibus, European Genome-Phenome Archive, and TCGA databases) was analyzed by KM potter ( http://kmplotter.com/analysis/ ). J , correlation between PPM1F mRNA and progression-free survival from TCGA (n = 505) was visualized by the Kaplan–Meier curve and evaluated by the log-rank test. Correlation between total staining score of BHLHE40 ( K ), phospho-AMPKα ( L ), PPM1F ( M ), and prognosis ( left panels , recurrence-free survival; right panels , overall survival) of patients ( n = 39) was visualized by a Kaplan–Meier curve and evaluated by the log-rank test. " width="100%" height="100%">

Journal: The Journal of Biological Chemistry

Article Title: The BHLHE40‒PPM1F‒AMPK pathway regulates energy metabolism and is associated with the aggressiveness of endometrial cancer

doi: 10.1016/j.jbc.2024.105695

Figure Lengend Snippet: Protein expression of BHLHE40, phospho-AMPKα, and PPM1F correlates with prognosis of endometrial cancer patients. A–D , Immunohistochemical analysis of primary sites from surgically removed uteri. Stage IA, endometrioid carcinoma, grade 1 ( A ); stage IB, endometrioid carcinoma grade 1 ( B ); stage IVB, endometrioid carcinoma, grade 3 ( C ); stage IIIC2, serous carcinoma ( D ). The scale bars indicate 200 μm. Total staining score of BHLHE40 was evaluated between groups of endometrioid carcinoma grade 1 and 2 and that of grade 3, serous carcinoma ( E ). Comparison of groups at stage I and II and that of stage III and IV ( F ). E and F , Welch’s t test was applied. G , total staining scores of BHLHE40 and phospho-AMPKα were analyzed using Pearson’s product-moment correlation coefficient. Also see Fig. S7 A . H , Total staining scores of BHLHE40 and PPM1F were analyzed using Pearson’s product-moment correlation coefficient. Also see Fig. S7 B . r-values show correlation coefficients. I , correlation between BHLHE40 mRNA levels and recurrence-free survival of endometrial cancer cases (n = 543 from Gene Expression Omnibus, European Genome-Phenome Archive, and TCGA databases) was analyzed by KM potter ( http://kmplotter.com/analysis/ ). J , correlation between PPM1F mRNA and progression-free survival from TCGA (n = 505) was visualized by the Kaplan–Meier curve and evaluated by the log-rank test. Correlation between total staining score of BHLHE40 ( K ), phospho-AMPKα ( L ), PPM1F ( M ), and prognosis ( left panels , recurrence-free survival; right panels , overall survival) of patients ( n = 39) was visualized by a Kaplan–Meier curve and evaluated by the log-rank test.

Article Snippet: We tested three anti-BHLHE40 antibodies (sc-101023 from Santa Cruz Biotechnology; HPA028921 from Atlas Antibodies; NB100 to 1800 from Novus Biologicals) in the ChIP assay.

Techniques: Expressing, Immunohistochemical staining, Staining, Comparison, Gene Expression

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