Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Pathological Ace2-to-Ace enzyme switch in the stressed heart is transcriptionally controlled by the endothelial Brg1–FoxM1 complex

doi: 10.1073/pnas.1525078113

Figure Lengend Snippet: Endothelial FoxM1 deletion reduces cardiomyocyte mass after TAC operation. (A) Immunostaining of FoxM1 in embryonic day (E) 11.5 hearts. Original magnification: 200×. Brown, FoxM1; blue, DAPI nuclear staining. (B and C) Western blot analysis (B) and quantitation (C) of FoxM1 protein extracted from mouse hearts 7 d after sham or TAC operation. P value: Student's t test. Error bar: SEM. (D and E) Western blot analysis (D) and quantitation (E) of FoxM1 protein in cardiomyocytes isolated from mice hearts 7 d after sham or TAC operation. P value: Student's t test. Error bar: SEM. (F–J) Quantitation (J) of cardiomyocyte size by WGA immunostaining (F–I) in control and SclCreERT;FoxM1fl/fl hearts treated with tamoxifen 4 wk after sham or TAC operation. Green, WGA. P value: Student's t test. Error bar: SEM. (Scale bars, 10 μm.)

Article Snippet: The following primary antibodies were used for immunostaining: anti-Brg1 (J1) ( 24 ), anti-FoxM1 (catalog no. H00002305-M02, Abnova), anti-PECAM (catalog no. 553370, BD Pharmingen), anti-ACE (catalog no. ab75762, Abcam), and anti-ACE2 (catalog no. ab59351, Abcam).

Techniques: Immunostaining, Staining, Western Blot, Quantitation Assay, Isolation, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Pathological Ace2-to-Ace enzyme switch in the stressed heart is transcriptionally controlled by the endothelial Brg1–FoxM1 complex

doi: 10.1073/pnas.1525078113

Figure Lengend Snippet: Endothelial FoxM1 is essential for cardiac hypertrophy and pathological switch of Ace/Ace2. (A) Quantitation of FoxM1 mRNA in mouse hearts after 7 d after sham or TAC operation. (B and C) Coimmunostaining of FoxM1 (red) and Pecam (green) in mouse hearts 7 d after sham or TAC operation. Arrows, endothelial cell nuclei; arrowheads, myocardial cell nuclei. (Scale bars, 10 μm.) (D and E) Western blot analysis (D) and quantitation (E) of FoxM1 proteins in cardiac endothelial cells isolated from mouse hearts 7 d after sham or TAC operation. P value: Student's t test. Error bar: SEM. (F) Quantitation of ventricle–body weight ratio of mice treated with DMSO (Ctrl) and thiostrepton (Thio) after 4 wk of sham or TAC operation. (G and H) Trichrome staining of cardiac fibrosis in mice treated with DMSO (Ctrl) and thiostrepton (Thio) after 4 wk sham or TAC operation. Red, cardiomyocytes; blue, fibrosis. Arrow, interstitial space. (Scale bars, 20 μm.) (I) Echocardiographic measurement of FS of the LV after 4 wk (4W) of TAC. Ctrl, DMSO; Thio, thiostrepton. (J and K) Western blot analysis (J) and quantitation (K) of Ace and Ace2 proteins in the heart of DMSO-treated (Ctrl) and thiostrepton-treated (Thio) mice 2 wk (2W) after sham or TAC operation. (L and M) Coimmunostaining of FoxM1 (red) and Pecam (green) in control (Ctrl) and SclCreERT;FoxM1fl/fl mouse hearts 4 wk after TAC operation with tamoxifen treatment. Arrows, endothelial cell nuclei; arrowheads, myocardial cell nuclei. (Scale bars, 10 μm.) (N) Quantitation of ventricle–body weight ratio in control (Ctrl) and SclCreERT;FoxM1fl/fl (Mut) mice 4 wk after sham or TAC operation. P value: Student's t test. Error bar: SEM. (O) Echocardiographic measurement of FS of the left ventricle of control (Ctrl) and SclCreERT;FoxM1fl/fl (Mut) hearts after 4 wk of TAC. P value: Student's t test. Error bar: SEM. (P and Q) Trichrome staining of cardiac fibrosis in control (P) and SclCreERT;FoxM1fl/fl (Q) mice 4 wk after sham or TAC operation. Original magnification: 200×. Red, cardiomyocytes; blue, fibrosis. (R) Representative LV pressure–volume (PV) loops taken after cardiac catheterization of control (Ctrl) and SclCreERT; FoxM1fl/fl (Mut) mice 4 wk after sham or TAC operation. (S) Quantitation of Ace, Ace2, and Ace/Ace2 mRNA in control (Ctrl) and SclCreERT;FoxM1fl/fl (Mut) heart ventricles after sham or TAC operation. P value: Student's t test. Error bar: SEM.

Article Snippet: The following primary antibodies were used for immunostaining: anti-Brg1 (J1) ( 24 ), anti-FoxM1 (catalog no. H00002305-M02, Abnova), anti-PECAM (catalog no. 553370, BD Pharmingen), anti-ACE (catalog no. ab75762, Abcam), and anti-ACE2 (catalog no. ab59351, Abcam).

Techniques: Quantitation Assay, Western Blot, Isolation, Staining, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Pathological Ace2-to-Ace enzyme switch in the stressed heart is transcriptionally controlled by the endothelial Brg1–FoxM1 complex

doi: 10.1073/pnas.1525078113

Figure Lengend Snippet: Endothelial FoxM1 is essential for stress-induced cardiac hypertrophy and dysfunction. Cardiac function PV-loop analysis of control (Ctrl) and SclCreERT; Foxm1fl/fl (Mut) mice after 4 wk of sham or TAC operation is shown. Quantitation of EF (A), SV (B), CO (C), plPwr (D), ESV (E), EDV (F), Tau (G), and EDP (H) is shown. P value: Student's t test. Error bar: SEM.

Article Snippet: The following primary antibodies were used for immunostaining: anti-Brg1 (J1) ( 24 ), anti-FoxM1 (catalog no. H00002305-M02, Abnova), anti-PECAM (catalog no. 553370, BD Pharmingen), anti-ACE (catalog no. ab75762, Abcam), and anti-ACE2 (catalog no. ab59351, Abcam).

Techniques: Control, Quantitation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Pathological Ace2-to-Ace enzyme switch in the stressed heart is transcriptionally controlled by the endothelial Brg1–FoxM1 complex

doi: 10.1073/pnas.1525078113

Figure Lengend Snippet: Brg1 cooperates with FoxM1 to control Ace and Ace2 expression. (A) Coimmunoprecipitation of Brg1 with FoxM1 in heart ventricles after 7 d of TAC. (B and C) Proximity ligation assay of Brg1–FoxM1 complex in nuclei of cultured mouse cardiac endothelial cells. Original magnification: 400×. Red, proximity ligation signal; blue, DAPI. IgG control, cells treated with IgG but not primary anti-Brg1 or -FoxM1 antibodies. (D and E) ChIP-qPCR analysis of Ace (D) and Ace2 (E) promoters using antibodies against FoxM1 7 d after sham or TAC operation. (F and G) Luciferase reporter assays of the Ace (−2,983 to +174 bp) (F) and Ace2 (−7,063 to +786 bp) (G) proximal promoters (described in Fig. 3 L and N) in mouse cardiac endothelial cells. siBrg1, siRNA-mediated knockdown of Brg1; Thio, thiostrepton. P value: Student's t test. Error bar: SEM. (H) Schematic illustration of FoxM1 repression, transactivation domains, and mutations. NRD, N-terminal repression domain; TAD, C-terminal transactivation domain. (I and J) Luciferase reporter assays of the Ace (I) and Ace2 (J) promoters with FoxM1 mutants in mouse cardiac endothelial cells. P value: Student's t test. Error bar: SEM.

Article Snippet: The following primary antibodies were used for immunostaining: anti-Brg1 (J1) ( 24 ), anti-FoxM1 (catalog no. H00002305-M02, Abnova), anti-PECAM (catalog no. 553370, BD Pharmingen), anti-ACE (catalog no. ab75762, Abcam), and anti-ACE2 (catalog no. ab59351, Abcam).

Techniques: Control, Expressing, Proximity Ligation Assay, Cell Culture, Ligation, Luciferase, Knockdown

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Pathological Ace2-to-Ace enzyme switch in the stressed heart is transcriptionally controlled by the endothelial Brg1–FoxM1 complex

doi: 10.1073/pnas.1525078113

Figure Lengend Snippet: BRG1 and FOXM1 activation in human cardiomyopathy. (A) qPCR analysis of BRG1, FOXM1, ACE, and ACE2 expression and ACE/ACE2 ratio in normal (n = 7) and LVH hearts (n = 4). (B and C) Coimmunostaining of BRG1 (red) and WGA (green) in normal and LVH hearts. Arrow, endothelial cell; arrowhead, myocardial cell. (Scale bars, 10 μm.) (D and E) Coimmunostaining of FOXM1 (red) and WGA (green) in heart of normal and LVH subjects. Arrow, endothelial cell; arrowhead, myocardial cell. (Scale bars, 10 μm.) (F) Correlation of BRG1 and FOXM1 mRNA level (x axis) with ACE/ACE2 mRNA ratio (y axis), n = 11. Red, nonlinear regression curve. e, the base of natural logarithm (∼2.718). Equations of Boltzmann sigmoidal model are listed under the graphs. (G) Working model of how cardiac endothelial Brg1–FoxM1 complex mediates stress signals to control Ace/Ace2 and angiotensin production in the heart.

Article Snippet: The following primary antibodies were used for immunostaining: anti-Brg1 (J1) ( 24 ), anti-FoxM1 (catalog no. H00002305-M02, Abnova), anti-PECAM (catalog no. 553370, BD Pharmingen), anti-ACE (catalog no. ab75762, Abcam), and anti-ACE2 (catalog no. ab59351, Abcam).

Techniques: Activation Assay, Expressing, Control

Journal: Dose-Response

Article Title: Cardamonin Promotes the Apoptosis and Chemotherapy Sensitivity to Gemcitabine of Pancreatic Cancer Through Modulating the FOXO3a-FOXM1 Axis

doi: 10.1177/15593258211042163

Figure Lengend Snippet: The effect of CAR on FOXO3a-FOXM1. PANC-1 and SW1990 cell lines were treated with CAR (0 to 20 μM) for 48 hours. (A) WB was carried out to gauge the PI3K/AKT/mTOR expression. (B) The profile of the FOXO3a/FOXM1 pathway was checked by WB. (C) The interaction between FOXO3a and FOXM1 was confirmed by IP * P < .05, ** P < .01, *** P < .001 (vs Control group), N = 3. CAR: cardamonin, WB: western blot.

Article Snippet: After incubation with the anti-FOXM1 antibody (BD Pharmingen) at 4°C, the cells were further incubated with the protein G Plus/Protein A agarose suspension (Merck) at 4°C for 4 hours.

Techniques: Expressing, Western Blot

Journal: Dose-Response

Article Title: Cardamonin Promotes the Apoptosis and Chemotherapy Sensitivity to Gemcitabine of Pancreatic Cancer Through Modulating the FOXO3a-FOXM1 Axis

doi: 10.1177/15593258211042163

Figure Lengend Snippet: Knocking down FOXO3a reduced GC cells’ chemosensitivity to GEM. (A-B) The FOXO3a knockdown cell model was constructed in PANC-1 and SW1990 cells. (A) WB was performed to monitor the expression of the FOXO3a/FOXM1 pathway after FOXO3a knockdown. (B-C) CCK8 assay was applied to examine PC cells’ viability. (D-E) PC cells with lower levels of FOXO3a were treated with 0-4 μg/mL GEM, for 24 hours. PC cells’ viability was measured by CCK8 assay. P > .05, * P < .05, ** P < .01, *** P < .001 (vs si-NC group), N = 3. WB: western blot, GEM: gemcitabine.

Article Snippet: After incubation with the anti-FOXM1 antibody (BD Pharmingen) at 4°C, the cells were further incubated with the protein G Plus/Protein A agarose suspension (Merck) at 4°C for 4 hours.

Techniques: Construct, Expressing, CCK-8 Assay, Western Blot

Journal: Dose-Response

Article Title: Cardamonin Promotes the Apoptosis and Chemotherapy Sensitivity to Gemcitabine of Pancreatic Cancer Through Modulating the FOXO3a-FOXM1 Axis

doi: 10.1177/15593258211042163

Figure Lengend Snippet: Down-regulating FOXO3a reversed CAR-mediated anti-tumor effects in PC cells. PANC-1 cells transfected with si-NC or si-FOXO3a were treated with 20 μM CAR for 48 hours respectively. (A) CCK8 assay was adopted to measure cell viability. (B) The colony formation experiment was conducted to test the colony-forming ability of PANC-1 cells. (C) PC cells with lower levels of FOXO3a were treated with 20 μM CAR and 0-4 μg/mL GEM. Cell viability was testified by CCK8 assay. (D) WB was conducted to monitor FOXO3a-FOXM1 profiles in PANC-1 cells. *** P < .001 (vs control group); ## P < .01, ### P < .001 (vsCAR+si-NC group). CAR: cardamonin, GEM: gemcitabine, WB: western blot, CCK8: cell counting kit-8.

Article Snippet: After incubation with the anti-FOXM1 antibody (BD Pharmingen) at 4°C, the cells were further incubated with the protein G Plus/Protein A agarose suspension (Merck) at 4°C for 4 hours.

Techniques: Transfection, CCK-8 Assay, Western Blot, Cell Counting

Journal: Dose-Response

Article Title: Cardamonin Promotes the Apoptosis and Chemotherapy Sensitivity to Gemcitabine of Pancreatic Cancer Through Modulating the FOXO3a-FOXM1 Axis

doi: 10.1177/15593258211042163

Figure Lengend Snippet: CAR curbed PC cells’ growth in vivo . PANC-1 and SW1990 cells were subjected to xenograft tumor experiment in nude mice, which were then treated with CAR (5 μg/kg body weight) or the same volume of saline by intraperitoneal injection. (A-F) The tumor images formed in the nude mice. The weight and volume of the tumors were calculated. (G-H) WB was conducted to measure the FOXO3a-FOXM1 and PI3K/AKT/mTOR pathways in the formed tumor tissues. I. Immunohistochemistry was used for detecting FOXO3a-FOXM1 in the tumor tissues. ** P < .01, *** P < .001. N = 5. CAR: cardamonin, WB: western blot.

Article Snippet: After incubation with the anti-FOXM1 antibody (BD Pharmingen) at 4°C, the cells were further incubated with the protein G Plus/Protein A agarose suspension (Merck) at 4°C for 4 hours.

Techniques: In Vivo, Injection, Immunohistochemistry, Western Blot

Journal: Dose-Response

Article Title: Cardamonin Promotes the Apoptosis and Chemotherapy Sensitivity to Gemcitabine of Pancreatic Cancer Through Modulating the FOXO3a-FOXM1 Axis

doi: 10.1177/15593258211042163

Figure Lengend Snippet: The Specific Primer Sequence.

Article Snippet: After incubation with the anti-FOXM1 antibody (BD Pharmingen) at 4°C, the cells were further incubated with the protein G Plus/Protein A agarose suspension (Merck) at 4°C for 4 hours.

Techniques: Sequencing

Journal: Journal of Cancer

Article Title: Lasalocid inhibits melanoma by down-regulating FOXM1 through PI3K/AKT and JNK/P38 MAPK pathways

doi: 10.7150/jca.101798

Figure Lengend Snippet: FOXM1 is a key gene in the inhibition of melanoma by lasalocid (A) Volcano plot of differential genes. (B) KEGG pathway analysis of differentially expressed genes. (C) GO functional enrichment analysis of differentially expressed genes. (D) Venn diagram was used to screen the key genes of lasalocid inhibiting melanoma. (E) GEPIA2 online analysis platform was used to verify the candidate genes.

Article Snippet: Afterwards, the melanoma cells were blocked with BSA blocking solution (Solarbio, China) for 30 min and then incubated overnight at 4°C with anti-FOXM1 antibody (Sanying, China).

Techniques: Inhibition, Functional Assay

Journal: Journal of Cancer

Article Title: Lasalocid inhibits melanoma by down-regulating FOXM1 through PI3K/AKT and JNK/P38 MAPK pathways

doi: 10.7150/jca.101798

Figure Lengend Snippet: Lasalocid inhibits the proliferation, migration and invasion of melanoma cells by down-regulating FOXM1. (A,B) The inhibition of FOXM1 mRNA and protein expression by lasalocid in human melanoma cells was assessed using real-time PCR and Western blot techniques. (C,D) Immunofluorescence assay was used to detect the inhibition of FOXM1 protein expression by lasalocid. (E,F) The mRNA expression of FOXM1-regulated genes in human melanoma cells treated with lasalocid for 24 h was quantitatively detected using real-time quantitative PCR. (G,H) Western blot was used to detect the expression of FOXM1-regulated proteins in human melanoma cells treated with lasalocid for 24 h. (Data are mean±SD * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001).

Article Snippet: Afterwards, the melanoma cells were blocked with BSA blocking solution (Solarbio, China) for 30 min and then incubated overnight at 4°C with anti-FOXM1 antibody (Sanying, China).

Techniques: Migration, Inhibition, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence

Journal: Journal of Cancer

Article Title: Lasalocid inhibits melanoma by down-regulating FOXM1 through PI3K/AKT and JNK/P38 MAPK pathways

doi: 10.7150/jca.101798

Figure Lengend Snippet: FOXM1 was down-regulated by lasalocid through inhibition of PI3K/AKT pathway. (A,B) The protein expression levels of PI3K, AKT, P-PI3K, and P-AKT in melanoma cells were assessed after 24 h of lasalocid treatment using Western blot analysis. (C,D) Western blot analysis was employed to assess the expression levels of PI3K, AKT, P-PI3K, P-AKT and FOXM1 following activation of the PI3K/AKT pathway by IGF1 (100 ng/mL, 24 h). (E,F) Statistical diagram of the proliferation ability of melanoma cells significantly increased by IGF1 detected by CCK-8. (Data are mean±SD * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001).

Article Snippet: Afterwards, the melanoma cells were blocked with BSA blocking solution (Solarbio, China) for 30 min and then incubated overnight at 4°C with anti-FOXM1 antibody (Sanying, China).

Techniques: Inhibition, Expressing, Western Blot, Activation Assay, CCK-8 Assay

Journal: Journal of Cancer

Article Title: Lasalocid inhibits melanoma by down-regulating FOXM1 through PI3K/AKT and JNK/P38 MAPK pathways

doi: 10.7150/jca.101798

Figure Lengend Snippet: Lasalocid inhibits the proliferation of melanoma cells by activating JNK/P38 MAPK pathway and down-regulating FOXM1. (A,B) The protein expression levels of JNK, P-JNK, P38 and P-P38 in melanoma cells treated with lasalocid for 24 h were detected by Western blot. (C,D) The expression of P38, P-P38 and FOXM1 proteins and the proliferation ability of melanoma cells after inhibiting the P38 MAPK pathway by SB202190 (20 μM, 24 h). (E,F) The expression of JNK, P-JNK and FOXM1 proteins and the proliferation ability of melanoma cells after inhibiting JNK MAPK pathway by SP600125 (20 μM, 24 h). (Data are mean±SD * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: Afterwards, the melanoma cells were blocked with BSA blocking solution (Solarbio, China) for 30 min and then incubated overnight at 4°C with anti-FOXM1 antibody (Sanying, China).

Techniques: Expressing, Western Blot

Journal: Journal of Cancer

Article Title: Lasalocid inhibits melanoma by down-regulating FOXM1 through PI3K/AKT and JNK/P38 MAPK pathways

doi: 10.7150/jca.101798

Figure Lengend Snippet: Lasalocid inhibits melanoma cell proliferation in vivo (A) Schematic representation of in vivo experiments. (B) Photos of nude mice after tumor formation. (C) Images of the stripped tumor. (D) Statistical plot of tumor mass. (E) Statistical plot of mouse weight. (F) Statistical plot of mouse weight. (G) Immunohistochemistry was used to detect the expression of FOXM1, MPP2, MMP9 and Ki67 protein in the tumors. TUNEL staining was used to detect the apoptosis of tumors in vivo . (H) Heart, liver, spleen, lung and kidney tissues were examined by HE to assess the systemic toxicity of lasalocid. (Data are mean±SD * P <0.05, ** P <0.01).

Article Snippet: Afterwards, the melanoma cells were blocked with BSA blocking solution (Solarbio, China) for 30 min and then incubated overnight at 4°C with anti-FOXM1 antibody (Sanying, China).

Techniques: In Vivo, Immunohistochemistry, Expressing, TUNEL Assay, Staining