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bmpr2 fc  (R&D Systems)


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    R&D Systems bmpr2 fc
    Bmpr2 Fc, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+bmpr2/us11970531-718-15-18?v=R%26D+Systems
    Average 94 stars, based on 16 article reviews
    bmpr2 fc - by Bioz Stars, 2026-07
    94/100 stars

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    Fig. 6 Functional studies related to loss of <t>BMPR2</t> in human monocytes. Induced pluripotent stem cells (iPSC) derived from HPAH patients or Controls were differentiated into induced monocytes (iMono) using a stepwise differentiation protocol outlined in the Methods. iMono of HPAH patients and Controls were compared for (A) CD14 cell surface expression, assessed by FACS; (B) Number of iMono per mL. on day 19 of differentiation. (C)STAT1 gene expression by RT-qPCR. (D) Schematic demonstrating iEC and iMono co-culture approach for assessing adhesion. iMono adhesion to Control or HPAH iEC, per field of view. (E) Schematic demonstrating iEC and iMono co-culture approach for assessing transendothelial migration. Migrated of iMono across Control or HPAH iEC. (F) VE-cadherin and ICAM1 expression in co-culture of iMono and iEC with quantification based on mean fluorescent intensity (MFI) of 10 FOV for each replicate. Representative images of the iEC/iMono co-culture including phase, DAPI (blue) VE-cadherin (CDH5) (green) and ICAM1 (red). Red dotted outline on phase and DAPI images show adhered iMono. White dotted outline on merged images show corresponding iMono (pink). Bars represent mean ± SEM. For all experiments, n = 3 biological replications. + denotes the minimum achievable P-value for n = 3 was reached by the non-parametric t test
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    Calcitriol inhibits PASMC proliferation via <t>BMPR2.</t> Human control PASMC were transfected with siRNA for BMPR2 (siBMPR2) or control siRNA (scramble). ( A ) BMPR2 mRNA expression assessed by qRT-PCR after 48 h post-transfection. Data is expressed as scatter plots and bars with medians, * p < 0.05, Mann–Whitney test. ( B ) Effects of calcitriol on proliferation in silenced BMPR2 PASMC measured by MTT assay. Results are expressed as mean ± SEM. *p < 0.05, t-test vs scramble. ( C ) and ( D ) Effects of calcitriol (1–100 nmol/l) on proliferation in PASMC transfected with siBMPR2 or scramble, measured by MTT and BrdU assays, respectively. * p < 0.05, two-way ANOVA, Bonferroni post hoc test, vs scramble. ( E ) Effects of calcitriol on proliferation in PASMC in the presence or absence of DMH1 (5 µmol/l), by MTT and BrdU assay; *** p < 0.001 calcitriol vs vehicle (black column), two way-ANOVA. n = 4–5 different cultures in duplicate or triplicate.
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    ( a ) Schematic diagram of sample processing for RNA sequencing. ( b ) Under the condition of BMRP2 knockdown, the stimulation of progesterone promoted the enrichment of pro-proliferative genes in PASMCs. ( c ) Both infecting shBMPR2 lentivirus (MOI=30, 48 hr) and transfecting siBMPR2 (50 nM, 48 hr) effectively decreased the protein level of <t>BMPR2</t> in PASMCs. ( d ) Progesterone had little effect on the proliferation of normal PASMCs. ( e-h ) Under the condition of BMPR2 knockdown, progesterone (for 24 hr) significantly promoted the proliferation of PASMCs. (e) used Alarmar blue assay, (f-g) were EdU assay and its statistical graph, and (h) was the expression of PCNA. Abbreviation : PASMCs, pulmonary artery smooth muscle cells; NC, normal PASMCs; NC_C, normal PASMCs stimulated with 17β-estradiol; NC_Y, normal PASMCs stimulated with progesterone; KD, shBMRP2 lentivirus-infected PASMCs; P100, 100nM of progesterone; P1, 1μM of progesterone; PCNA, proliferating cell nuclear antigen. ns, non-significance; *, P <0.05; **, P <0.01; ****, P <0.001.
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    ( a ) Schematic diagram of sample processing for RNA sequencing. ( b ) Under the condition of BMRP2 knockdown, the stimulation of progesterone promoted the enrichment of pro-proliferative genes in PASMCs. ( c ) Both infecting shBMPR2 lentivirus (MOI=30, 48 hr) and transfecting siBMPR2 (50 nM, 48 hr) effectively decreased the protein level of <t>BMPR2</t> in PASMCs. ( d ) Progesterone had little effect on the proliferation of normal PASMCs. ( e-h ) Under the condition of BMPR2 knockdown, progesterone (for 24 hr) significantly promoted the proliferation of PASMCs. (e) used Alarmar blue assay, (f-g) were EdU assay and its statistical graph, and (h) was the expression of PCNA. Abbreviation : PASMCs, pulmonary artery smooth muscle cells; NC, normal PASMCs; NC_C, normal PASMCs stimulated with 17β-estradiol; NC_Y, normal PASMCs stimulated with progesterone; KD, shBMRP2 lentivirus-infected PASMCs; P100, 100nM of progesterone; P1, 1μM of progesterone; PCNA, proliferating cell nuclear antigen. ns, non-significance; *, P <0.05; **, P <0.01; ****, P <0.001.
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    GATA6 expression is induced via <t>BMP10-BMPR2/ALK1</t> axis. ( A–F ) HPAECs were transfected with ALK1, BMPR2, Endoglin, or control scr siRNA for 48 h, and then treated with 10 ng/ml BMP10 or vehicle for 6 h for RNA isolation and 24 h for protein isolation. ( A,C,E ): GATA6 mRNA measured by qPCR. Data are means ± SE; each experiment was repeated at least three times. ( B,D,F ): GATA6 protein levels were measured by immunoblot analysis. Data are means ± SE, each experiment was repeated at least three times. Representative blots are shown. *p < 0.05, **p < 0.01, ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s test for multiple comparisons. ( G–I ) Human PAH PASMC were treated with 10 ng/ml BMP10 or vehicle ( − ) for 48 h and immunoblot analysis to detect indicated proteins was performed. Data are means ± SE from n = 4 subjects/group. *p < 0.05 by Mann Whitney U test. ( J,K ) Equal amounts of human PAH HPAEC and PASMC were plated at 6-well plates and treated with 10 ng/ml BMP10 or vehicle ( − ). 48 h later cell counts were performed. Data are means ± SE from n = 3 subjects/group, 3 technical repetitions/subject. *p < 0.05 by Mann Whitney U test. ( L,M ) HPAECs were transfected with SMAD1 siRNA, and then treated with BMP10 for 6 h. GATA6 and SMAD1 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated at least three times. *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post-hoc correction for multiple comparisons. ( N,O ) HPAEC were treated for 30 min with diluent ( − ), 5 µM ERK1/2 inhibitor SCH772984 (ERKi), or 5 µM GSK3 inhibitor CHIR99021 (GSK3i) and then stimulated with BMP10 (10 ng/ml) or vehicle for 24 h. Representative immunoblots ( N ) and statistical analysis ( O ) are shown. ( O ): Data represent GATA6/β-actin ratio. Data are means ± SE from five independent experiments. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. ( E ) HPAECs were treated with BMP10 in the presence or absence of 10 µM ERK1/2 inhibitor SCH772984 for 24 h. GATA6 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated six times. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. The original blots are presented in Supplementary Fig. .
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    Fig. 6 Functional studies related to loss of BMPR2 in human monocytes. Induced pluripotent stem cells (iPSC) derived from HPAH patients or Controls were differentiated into induced monocytes (iMono) using a stepwise differentiation protocol outlined in the Methods. iMono of HPAH patients and Controls were compared for (A) CD14 cell surface expression, assessed by FACS; (B) Number of iMono per mL. on day 19 of differentiation. (C)STAT1 gene expression by RT-qPCR. (D) Schematic demonstrating iEC and iMono co-culture approach for assessing adhesion. iMono adhesion to Control or HPAH iEC, per field of view. (E) Schematic demonstrating iEC and iMono co-culture approach for assessing transendothelial migration. Migrated of iMono across Control or HPAH iEC. (F) VE-cadherin and ICAM1 expression in co-culture of iMono and iEC with quantification based on mean fluorescent intensity (MFI) of 10 FOV for each replicate. Representative images of the iEC/iMono co-culture including phase, DAPI (blue) VE-cadherin (CDH5) (green) and ICAM1 (red). Red dotted outline on phase and DAPI images show adhered iMono. White dotted outline on merged images show corresponding iMono (pink). Bars represent mean ± SEM. For all experiments, n = 3 biological replications. + denotes the minimum achievable P-value for n = 3 was reached by the non-parametric t test

    Journal: Respiratory research

    Article Title: Altered maturation and activation state of circulating monocytes is associated with their enhanced recruitment in pulmonary arterial hypertension.

    doi: 10.1186/s12931-025-03182-0

    Figure Lengend Snippet: Fig. 6 Functional studies related to loss of BMPR2 in human monocytes. Induced pluripotent stem cells (iPSC) derived from HPAH patients or Controls were differentiated into induced monocytes (iMono) using a stepwise differentiation protocol outlined in the Methods. iMono of HPAH patients and Controls were compared for (A) CD14 cell surface expression, assessed by FACS; (B) Number of iMono per mL. on day 19 of differentiation. (C)STAT1 gene expression by RT-qPCR. (D) Schematic demonstrating iEC and iMono co-culture approach for assessing adhesion. iMono adhesion to Control or HPAH iEC, per field of view. (E) Schematic demonstrating iEC and iMono co-culture approach for assessing transendothelial migration. Migrated of iMono across Control or HPAH iEC. (F) VE-cadherin and ICAM1 expression in co-culture of iMono and iEC with quantification based on mean fluorescent intensity (MFI) of 10 FOV for each replicate. Representative images of the iEC/iMono co-culture including phase, DAPI (blue) VE-cadherin (CDH5) (green) and ICAM1 (red). Red dotted outline on phase and DAPI images show adhered iMono. White dotted outline on merged images show corresponding iMono (pink). Bars represent mean ± SEM. For all experiments, n = 3 biological replications. + denotes the minimum achievable P-value for n = 3 was reached by the non-parametric t test

    Article Snippet: SiRNA BMPR2 knockdown in THP1 cell line THP1 cells at a density of 5 × 105 cells/mL were cultured overnight in a 6-well plate, followed by siRNA transfection with siRNA BMPR2 (SMARTpool ON-TARGETplus, Origene, Rockville, MD, Cat#: L-001230-00-005) or siRNA Control (ON-TARGETplus non-targeting Control siRNA, Origene, Cat#: D-001230-01) using the TransIT-TKO reagent (Mirus, Madison, WI Cat# MIR2150) according to the manufacturer’s recommendations.

    Techniques: Functional Assay, Derivative Assay, Expressing, Gene Expression, Quantitative RT-PCR, Co-Culture Assay, Control, Migration

    Calcitriol inhibits PASMC proliferation via BMPR2. Human control PASMC were transfected with siRNA for BMPR2 (siBMPR2) or control siRNA (scramble). ( A ) BMPR2 mRNA expression assessed by qRT-PCR after 48 h post-transfection. Data is expressed as scatter plots and bars with medians, * p < 0.05, Mann–Whitney test. ( B ) Effects of calcitriol on proliferation in silenced BMPR2 PASMC measured by MTT assay. Results are expressed as mean ± SEM. *p < 0.05, t-test vs scramble. ( C ) and ( D ) Effects of calcitriol (1–100 nmol/l) on proliferation in PASMC transfected with siBMPR2 or scramble, measured by MTT and BrdU assays, respectively. * p < 0.05, two-way ANOVA, Bonferroni post hoc test, vs scramble. ( E ) Effects of calcitriol on proliferation in PASMC in the presence or absence of DMH1 (5 µmol/l), by MTT and BrdU assay; *** p < 0.001 calcitriol vs vehicle (black column), two way-ANOVA. n = 4–5 different cultures in duplicate or triplicate.

    Journal: Scientific Reports

    Article Title: Vitamin D receptor and its antiproliferative effect in human pulmonary arterial hypertension

    doi: 10.1038/s41598-024-78380-9

    Figure Lengend Snippet: Calcitriol inhibits PASMC proliferation via BMPR2. Human control PASMC were transfected with siRNA for BMPR2 (siBMPR2) or control siRNA (scramble). ( A ) BMPR2 mRNA expression assessed by qRT-PCR after 48 h post-transfection. Data is expressed as scatter plots and bars with medians, * p < 0.05, Mann–Whitney test. ( B ) Effects of calcitriol on proliferation in silenced BMPR2 PASMC measured by MTT assay. Results are expressed as mean ± SEM. *p < 0.05, t-test vs scramble. ( C ) and ( D ) Effects of calcitriol (1–100 nmol/l) on proliferation in PASMC transfected with siBMPR2 or scramble, measured by MTT and BrdU assays, respectively. * p < 0.05, two-way ANOVA, Bonferroni post hoc test, vs scramble. ( E ) Effects of calcitriol on proliferation in PASMC in the presence or absence of DMH1 (5 µmol/l), by MTT and BrdU assay; *** p < 0.001 calcitriol vs vehicle (black column), two way-ANOVA. n = 4–5 different cultures in duplicate or triplicate.

    Article Snippet: Human control PASMC were transfected with specific siRNA against BMPR2 (SR300456B, OriGene, Maryland, USA), KCNK3 (gene encoding TASK-1, SASI_Hs01_00108786, Sigma-Aldrich, St. Louis, USA), BIRC5 ( gene encoding survivin, SASI_Hs01_00052229, Sigma-Aldrich, St. Louis, USA), and control non-targeting siRNA (SIC001 scramble siRNA, Sigma-Aldrich, St. Louis, USA), using LipofectamineTM RNAiMAX (Thermo Fisher Scientific, Massachusetts, USA) according to the manufacturer’s instructions as previously reported .

    Techniques: Control, Transfection, Expressing, Quantitative RT-PCR, MANN-WHITNEY, MTT Assay, BrdU Staining

    GATA6 expression is induced via BMP10-BMPR2/ALK1 axis. ( A–F ) HPAECs were transfected with ALK1, BMPR2, Endoglin, or control scr siRNA for 48 h, and then treated with 10 ng/ml BMP10 or vehicle for 6 h for RNA isolation and 24 h for protein isolation. ( A,C,E ): GATA6 mRNA measured by qPCR. Data are means ± SE; each experiment was repeated at least three times. ( B,D,F ): GATA6 protein levels were measured by immunoblot analysis. Data are means ± SE, each experiment was repeated at least three times. Representative blots are shown. *p < 0.05, **p < 0.01, ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s test for multiple comparisons. ( G–I ) Human PAH PASMC were treated with 10 ng/ml BMP10 or vehicle ( − ) for 48 h and immunoblot analysis to detect indicated proteins was performed. Data are means ± SE from n = 4 subjects/group. *p < 0.05 by Mann Whitney U test. ( J,K ) Equal amounts of human PAH HPAEC and PASMC were plated at 6-well plates and treated with 10 ng/ml BMP10 or vehicle ( − ). 48 h later cell counts were performed. Data are means ± SE from n = 3 subjects/group, 3 technical repetitions/subject. *p < 0.05 by Mann Whitney U test. ( L,M ) HPAECs were transfected with SMAD1 siRNA, and then treated with BMP10 for 6 h. GATA6 and SMAD1 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated at least three times. *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post-hoc correction for multiple comparisons. ( N,O ) HPAEC were treated for 30 min with diluent ( − ), 5 µM ERK1/2 inhibitor SCH772984 (ERKi), or 5 µM GSK3 inhibitor CHIR99021 (GSK3i) and then stimulated with BMP10 (10 ng/ml) or vehicle for 24 h. Representative immunoblots ( N ) and statistical analysis ( O ) are shown. ( O ): Data represent GATA6/β-actin ratio. Data are means ± SE from five independent experiments. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. ( E ) HPAECs were treated with BMP10 in the presence or absence of 10 µM ERK1/2 inhibitor SCH772984 for 24 h. GATA6 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated six times. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. The original blots are presented in Supplementary Fig. .

    Journal: Scientific Reports

    Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

    doi: 10.1038/s41598-023-33779-8

    Figure Lengend Snippet: GATA6 expression is induced via BMP10-BMPR2/ALK1 axis. ( A–F ) HPAECs were transfected with ALK1, BMPR2, Endoglin, or control scr siRNA for 48 h, and then treated with 10 ng/ml BMP10 or vehicle for 6 h for RNA isolation and 24 h for protein isolation. ( A,C,E ): GATA6 mRNA measured by qPCR. Data are means ± SE; each experiment was repeated at least three times. ( B,D,F ): GATA6 protein levels were measured by immunoblot analysis. Data are means ± SE, each experiment was repeated at least three times. Representative blots are shown. *p < 0.05, **p < 0.01, ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s test for multiple comparisons. ( G–I ) Human PAH PASMC were treated with 10 ng/ml BMP10 or vehicle ( − ) for 48 h and immunoblot analysis to detect indicated proteins was performed. Data are means ± SE from n = 4 subjects/group. *p < 0.05 by Mann Whitney U test. ( J,K ) Equal amounts of human PAH HPAEC and PASMC were plated at 6-well plates and treated with 10 ng/ml BMP10 or vehicle ( − ). 48 h later cell counts were performed. Data are means ± SE from n = 3 subjects/group, 3 technical repetitions/subject. *p < 0.05 by Mann Whitney U test. ( L,M ) HPAECs were transfected with SMAD1 siRNA, and then treated with BMP10 for 6 h. GATA6 and SMAD1 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated at least three times. *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post-hoc correction for multiple comparisons. ( N,O ) HPAEC were treated for 30 min with diluent ( − ), 5 µM ERK1/2 inhibitor SCH772984 (ERKi), or 5 µM GSK3 inhibitor CHIR99021 (GSK3i) and then stimulated with BMP10 (10 ng/ml) or vehicle for 24 h. Representative immunoblots ( N ) and statistical analysis ( O ) are shown. ( O ): Data represent GATA6/β-actin ratio. Data are means ± SE from five independent experiments. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. ( E ) HPAECs were treated with BMP10 in the presence or absence of 10 µM ERK1/2 inhibitor SCH772984 for 24 h. GATA6 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated six times. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. The original blots are presented in Supplementary Fig. .

    Article Snippet: Antibodies were as follows: Goat anti-human GATA6 (R&D; AF1700, 1:500), mouse anti-human GATA6 (Santa-Cruz, 517554, 1:500), Rabbit anti-human/mouse SOD2 (Cell signaling technology; 13141, 1:1000; 13145, 1:5000), Goat anti-human ALK1 (R&D; AF370, 1:500), Rabbit anti-mouse ALK1 (ABGENT AP7807a, 1:1000), rabbit anti-human/mouse ActR2B (LS bio; LS-B7781, 1:1000), mouse anti-human BMPR2 (Novus; NBP2-37624 Clone 3F6; 1:1000), rabbit anti-mouse BMPR2 (Proteintech; 19087–1-AP; 1:1000), mouse anti-mouse/human/rat BMPR2 (Abcam, catalog #130206 1:500), rabbit anti-human/mouse Endoglin (Proteintech; 10862–1-AP; 1:1000), rabbit anti-mouse BiP (Abcam; ab53068; 1:1000), rabbit anti-mouse CHOP (Novus; NBP2-13172; 1:1000), mouse anti-beta-actin (Sigma; A1978; 1:2000), mouse anti-human PRPF4 (Abcam, 69878, 1:1000), rabbit anti-human MYEOV (Invitrogen, PA5-48938 1:1000), rabbit anti-human STING (Invitrogen, PA5-26751, 1:1000), rabbit anti-human/mouse/rat EAF1 (Invitrogen, PA5-100493, 1:1000), rabbit anti-human/mouse/rat Histone H3 (Cell Signaling, 9715 1:1000), rabbit anti-human/mouse/rat a-b Tubulin (Cell Signaling, 2148, 1:1000).

    Techniques: Expressing, Transfection, Isolation, Western Blot, MANN-WHITNEY

    GATA6 deficiency in PAEC and PASMC results in loss of BMP receptors. ( A ) qPCR of HPAECs transfected with GATA6 or control scr siRNA ( − ) to measure indicated mRNA, each experiment was repeated at least three times. Data are means ± SE, n = 4–7. **p < 0.01, ***p < 0.001 by Mann Whitney U test. ( B ) Chromatin immune precipitation (ChiP) assay in HPAECs, n = 8–10. Representative gel images and data quantification are shown. Data are means ± SE, ***p < 0.001, ****p < 0.0001 by Mann Whitney U test. ( C,D ) Immunoblot analysis of control human PASMC transfected with siRNA GATA6 or control scr siRNA for 48 h. Data are means ± SE, 3 subjects/group, *p < 0.05 by Mann Whitney U test. Please see Fig. F,G for GATA6 immunoblots. ( E,F ) Expression of BmpR2, Alk1 , ActRIIB, and endoglin measured by qPCR in PAEC ( F ) and whole lungs ( G ) from WT and Gata6 CKO mice. Data are means ± SE; E: n = 4–5/group; F: n = 6–11 mice/group. Male and female mice responded similarly. *p < 0.05, **p < 0.01 by Mann Whitney U test (( E,F ) BmpR2, Alk1 , and ActRIIB ) and unpaired τ test (F Endoglin ). ( G,H ) Control HPAECs transfected with siGATA6 or control scr siRNA ( − ) were assayed by immunoblot analysis to detect indicated BMP receptors. Values are means ± SE of the relative protein levels by densitometry, n = 4–7.*p < 0.05, ***p < 0.001 by Mann Whitney U test. ( I,J ) Immunoblot analysis of whole lung tissue from Gata6 CKO and WT mice. Values are means ± SE of the relative protein levels by densitometry, n = 3–5/group. Male and female mice responded similarly. *p < 0.05, **p < 0.01 by Mann Whitney U test. The original blots are presented in Supplementary Fig. .

    Journal: Scientific Reports

    Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

    doi: 10.1038/s41598-023-33779-8

    Figure Lengend Snippet: GATA6 deficiency in PAEC and PASMC results in loss of BMP receptors. ( A ) qPCR of HPAECs transfected with GATA6 or control scr siRNA ( − ) to measure indicated mRNA, each experiment was repeated at least three times. Data are means ± SE, n = 4–7. **p < 0.01, ***p < 0.001 by Mann Whitney U test. ( B ) Chromatin immune precipitation (ChiP) assay in HPAECs, n = 8–10. Representative gel images and data quantification are shown. Data are means ± SE, ***p < 0.001, ****p < 0.0001 by Mann Whitney U test. ( C,D ) Immunoblot analysis of control human PASMC transfected with siRNA GATA6 or control scr siRNA for 48 h. Data are means ± SE, 3 subjects/group, *p < 0.05 by Mann Whitney U test. Please see Fig. F,G for GATA6 immunoblots. ( E,F ) Expression of BmpR2, Alk1 , ActRIIB, and endoglin measured by qPCR in PAEC ( F ) and whole lungs ( G ) from WT and Gata6 CKO mice. Data are means ± SE; E: n = 4–5/group; F: n = 6–11 mice/group. Male and female mice responded similarly. *p < 0.05, **p < 0.01 by Mann Whitney U test (( E,F ) BmpR2, Alk1 , and ActRIIB ) and unpaired τ test (F Endoglin ). ( G,H ) Control HPAECs transfected with siGATA6 or control scr siRNA ( − ) were assayed by immunoblot analysis to detect indicated BMP receptors. Values are means ± SE of the relative protein levels by densitometry, n = 4–7.*p < 0.05, ***p < 0.001 by Mann Whitney U test. ( I,J ) Immunoblot analysis of whole lung tissue from Gata6 CKO and WT mice. Values are means ± SE of the relative protein levels by densitometry, n = 3–5/group. Male and female mice responded similarly. *p < 0.05, **p < 0.01 by Mann Whitney U test. The original blots are presented in Supplementary Fig. .

    Article Snippet: Antibodies were as follows: Goat anti-human GATA6 (R&D; AF1700, 1:500), mouse anti-human GATA6 (Santa-Cruz, 517554, 1:500), Rabbit anti-human/mouse SOD2 (Cell signaling technology; 13141, 1:1000; 13145, 1:5000), Goat anti-human ALK1 (R&D; AF370, 1:500), Rabbit anti-mouse ALK1 (ABGENT AP7807a, 1:1000), rabbit anti-human/mouse ActR2B (LS bio; LS-B7781, 1:1000), mouse anti-human BMPR2 (Novus; NBP2-37624 Clone 3F6; 1:1000), rabbit anti-mouse BMPR2 (Proteintech; 19087–1-AP; 1:1000), mouse anti-mouse/human/rat BMPR2 (Abcam, catalog #130206 1:500), rabbit anti-human/mouse Endoglin (Proteintech; 10862–1-AP; 1:1000), rabbit anti-mouse BiP (Abcam; ab53068; 1:1000), rabbit anti-mouse CHOP (Novus; NBP2-13172; 1:1000), mouse anti-beta-actin (Sigma; A1978; 1:2000), mouse anti-human PRPF4 (Abcam, 69878, 1:1000), rabbit anti-human MYEOV (Invitrogen, PA5-48938 1:1000), rabbit anti-human STING (Invitrogen, PA5-26751, 1:1000), rabbit anti-human/mouse/rat EAF1 (Invitrogen, PA5-100493, 1:1000), rabbit anti-human/mouse/rat Histone H3 (Cell Signaling, 9715 1:1000), rabbit anti-human/mouse/rat a-b Tubulin (Cell Signaling, 2148, 1:1000).

    Techniques: Transfection, MANN-WHITNEY, Western Blot, Expressing

    Treatment with DMF restores expression of the BMP receptors, reverses oxidative stress and pulmonary hypertension in Gata6 CKO mice. (DMF or vehicle were administered daily via i.p. injection for 3 weeks. ( A ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of indicated BMP receptors. Data are means ± SE, n = 6–12, *p < 0.05. **p < 0.01 by Kruskal–Wallis test followed by Dunn’s multiple comparisons test ( BmpR2, ActRIIB, Alk1 ) and one-way ANOVA followed by post hoc Tukey’s multiple comparison ( Endoglin ). ( B ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of the antioxidant enzymes and eNOS . Data are means ± SE, n = 6–17, *p < 0.05., **p < 0.01, ***p < 0.001 by one-way ANOVA followed by post hoc Tukey’s multiple comparisons test ( SOD2, GPX1, CAT ) and Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test ( eNOS ). ( C,D ) mRNA levels of indicated BMP receptors and antioxidant enzymes measured by qPCR in PAEC from WT and Gata6 CKO mice treated with DMF or vehicle. Data are means ± SE, n = 3–6 mice/group, *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test. ( E–G ) RVSP, pulmonary acceleration time as a fraction of ejection time (PAT/ET) and Fulton index (RV/[LV + S]) were evaluated in WT and Gata6 CKO mice in the presence or absence of DMF. Data are means ± SE. n = 5–11 mice/group. *p < 0.05, **p < 0.01. ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test (RVSP) and one-way ANOVA followed by post hoc Tukey’s multiple comparisons test (PAT/ET and RV/(LV + S).

    Journal: Scientific Reports

    Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

    doi: 10.1038/s41598-023-33779-8

    Figure Lengend Snippet: Treatment with DMF restores expression of the BMP receptors, reverses oxidative stress and pulmonary hypertension in Gata6 CKO mice. (DMF or vehicle were administered daily via i.p. injection for 3 weeks. ( A ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of indicated BMP receptors. Data are means ± SE, n = 6–12, *p < 0.05. **p < 0.01 by Kruskal–Wallis test followed by Dunn’s multiple comparisons test ( BmpR2, ActRIIB, Alk1 ) and one-way ANOVA followed by post hoc Tukey’s multiple comparison ( Endoglin ). ( B ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of the antioxidant enzymes and eNOS . Data are means ± SE, n = 6–17, *p < 0.05., **p < 0.01, ***p < 0.001 by one-way ANOVA followed by post hoc Tukey’s multiple comparisons test ( SOD2, GPX1, CAT ) and Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test ( eNOS ). ( C,D ) mRNA levels of indicated BMP receptors and antioxidant enzymes measured by qPCR in PAEC from WT and Gata6 CKO mice treated with DMF or vehicle. Data are means ± SE, n = 3–6 mice/group, *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test. ( E–G ) RVSP, pulmonary acceleration time as a fraction of ejection time (PAT/ET) and Fulton index (RV/[LV + S]) were evaluated in WT and Gata6 CKO mice in the presence or absence of DMF. Data are means ± SE. n = 5–11 mice/group. *p < 0.05, **p < 0.01. ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test (RVSP) and one-way ANOVA followed by post hoc Tukey’s multiple comparisons test (PAT/ET and RV/(LV + S).

    Article Snippet: Antibodies were as follows: Goat anti-human GATA6 (R&D; AF1700, 1:500), mouse anti-human GATA6 (Santa-Cruz, 517554, 1:500), Rabbit anti-human/mouse SOD2 (Cell signaling technology; 13141, 1:1000; 13145, 1:5000), Goat anti-human ALK1 (R&D; AF370, 1:500), Rabbit anti-mouse ALK1 (ABGENT AP7807a, 1:1000), rabbit anti-human/mouse ActR2B (LS bio; LS-B7781, 1:1000), mouse anti-human BMPR2 (Novus; NBP2-37624 Clone 3F6; 1:1000), rabbit anti-mouse BMPR2 (Proteintech; 19087–1-AP; 1:1000), mouse anti-mouse/human/rat BMPR2 (Abcam, catalog #130206 1:500), rabbit anti-human/mouse Endoglin (Proteintech; 10862–1-AP; 1:1000), rabbit anti-mouse BiP (Abcam; ab53068; 1:1000), rabbit anti-mouse CHOP (Novus; NBP2-13172; 1:1000), mouse anti-beta-actin (Sigma; A1978; 1:2000), mouse anti-human PRPF4 (Abcam, 69878, 1:1000), rabbit anti-human MYEOV (Invitrogen, PA5-48938 1:1000), rabbit anti-human STING (Invitrogen, PA5-26751, 1:1000), rabbit anti-human/mouse/rat EAF1 (Invitrogen, PA5-100493, 1:1000), rabbit anti-human/mouse/rat Histone H3 (Cell Signaling, 9715 1:1000), rabbit anti-human/mouse/rat a-b Tubulin (Cell Signaling, 2148, 1:1000).

    Techniques: Expressing, Injection

    ( a ) Schematic diagram of sample processing for RNA sequencing. ( b ) Under the condition of BMRP2 knockdown, the stimulation of progesterone promoted the enrichment of pro-proliferative genes in PASMCs. ( c ) Both infecting shBMPR2 lentivirus (MOI=30, 48 hr) and transfecting siBMPR2 (50 nM, 48 hr) effectively decreased the protein level of BMPR2 in PASMCs. ( d ) Progesterone had little effect on the proliferation of normal PASMCs. ( e-h ) Under the condition of BMPR2 knockdown, progesterone (for 24 hr) significantly promoted the proliferation of PASMCs. (e) used Alarmar blue assay, (f-g) were EdU assay and its statistical graph, and (h) was the expression of PCNA. Abbreviation : PASMCs, pulmonary artery smooth muscle cells; NC, normal PASMCs; NC_C, normal PASMCs stimulated with 17β-estradiol; NC_Y, normal PASMCs stimulated with progesterone; KD, shBMRP2 lentivirus-infected PASMCs; P100, 100nM of progesterone; P1, 1μM of progesterone; PCNA, proliferating cell nuclear antigen. ns, non-significance; *, P <0.05; **, P <0.01; ****, P <0.001.

    Journal: bioRxiv

    Article Title: Progesterone is an Inducement of Heritable Pulmonary Arterial Hypertension with BMPR2 Mutation

    doi: 10.1101/2023.04.21.537897

    Figure Lengend Snippet: ( a ) Schematic diagram of sample processing for RNA sequencing. ( b ) Under the condition of BMRP2 knockdown, the stimulation of progesterone promoted the enrichment of pro-proliferative genes in PASMCs. ( c ) Both infecting shBMPR2 lentivirus (MOI=30, 48 hr) and transfecting siBMPR2 (50 nM, 48 hr) effectively decreased the protein level of BMPR2 in PASMCs. ( d ) Progesterone had little effect on the proliferation of normal PASMCs. ( e-h ) Under the condition of BMPR2 knockdown, progesterone (for 24 hr) significantly promoted the proliferation of PASMCs. (e) used Alarmar blue assay, (f-g) were EdU assay and its statistical graph, and (h) was the expression of PCNA. Abbreviation : PASMCs, pulmonary artery smooth muscle cells; NC, normal PASMCs; NC_C, normal PASMCs stimulated with 17β-estradiol; NC_Y, normal PASMCs stimulated with progesterone; KD, shBMRP2 lentivirus-infected PASMCs; P100, 100nM of progesterone; P1, 1μM of progesterone; PCNA, proliferating cell nuclear antigen. ns, non-significance; *, P <0.05; **, P <0.01; ****, P <0.001.

    Article Snippet: Human BMPR2 ELISA Kit (orb406355, Biorbyt, United Kingdom) was used to detect the level of soluble BMPR2 in serum.

    Techniques: RNA Sequencing, Knockdown, EdU Assay, Expressing, Infection

    ( a ) Progesterone (24 hr) enriched the genes of “MAPK pathways” and “Pathways in cancer” in BMRP2-knockdown PASMCs. ( b ) Progesterone (for 2 hr) activated the ERK pathway in BMPR2-knockdown PASMCs. ( c ) Venn graph, which brought into all the differentially-expressed genes with the fold change > 1.4, showed that MYC and EDN1 were upregulated by progesterone (24 hr) only under the condition of BMPR2 knockdown. ( d-e ) Progesterone upregulated the mRNA and protein of c-MYC and EDN1 in BMPR2-knockdown PASMCs. Abbreviation : ERK, extracellular regulated protein kinases; EDN1, endothelin1; MAPK, mitogen-activated protein kinase. **, P <0.01; ****, P <0.001.

    Journal: bioRxiv

    Article Title: Progesterone is an Inducement of Heritable Pulmonary Arterial Hypertension with BMPR2 Mutation

    doi: 10.1101/2023.04.21.537897

    Figure Lengend Snippet: ( a ) Progesterone (24 hr) enriched the genes of “MAPK pathways” and “Pathways in cancer” in BMRP2-knockdown PASMCs. ( b ) Progesterone (for 2 hr) activated the ERK pathway in BMPR2-knockdown PASMCs. ( c ) Venn graph, which brought into all the differentially-expressed genes with the fold change > 1.4, showed that MYC and EDN1 were upregulated by progesterone (24 hr) only under the condition of BMPR2 knockdown. ( d-e ) Progesterone upregulated the mRNA and protein of c-MYC and EDN1 in BMPR2-knockdown PASMCs. Abbreviation : ERK, extracellular regulated protein kinases; EDN1, endothelin1; MAPK, mitogen-activated protein kinase. **, P <0.01; ****, P <0.001.

    Article Snippet: Human BMPR2 ELISA Kit (orb406355, Biorbyt, United Kingdom) was used to detect the level of soluble BMPR2 in serum.

    Techniques: Knockdown

    ( a ) Alarmar blue assay showed that PD0325901 and Bosentan (for 24 hr) reversed the pro-proliferative effects of progesterone in BMPR2-knockdown PASMCs . (b-c ) EdU assay showed that PD0325901 and Bosentan (for 24 hr) also reversed the effects of progesterone. ( d ) Inhibiting ERK pathway by PD0325901 reversed the upregulation of these proteins induced by progesterone in BMPR2-knockdown PASMCs. Abbreviation : ECE1, endothelin converting enzyme 1; PD, PD0325901; Bos, Bosentan. ns, non-significance; *, P <0.05; **, P <0.01; ***, P <0.001; ****, P <0.001.

    Journal: bioRxiv

    Article Title: Progesterone is an Inducement of Heritable Pulmonary Arterial Hypertension with BMPR2 Mutation

    doi: 10.1101/2023.04.21.537897

    Figure Lengend Snippet: ( a ) Alarmar blue assay showed that PD0325901 and Bosentan (for 24 hr) reversed the pro-proliferative effects of progesterone in BMPR2-knockdown PASMCs . (b-c ) EdU assay showed that PD0325901 and Bosentan (for 24 hr) also reversed the effects of progesterone. ( d ) Inhibiting ERK pathway by PD0325901 reversed the upregulation of these proteins induced by progesterone in BMPR2-knockdown PASMCs. Abbreviation : ECE1, endothelin converting enzyme 1; PD, PD0325901; Bos, Bosentan. ns, non-significance; *, P <0.05; **, P <0.01; ***, P <0.001; ****, P <0.001.

    Article Snippet: Human BMPR2 ELISA Kit (orb406355, Biorbyt, United Kingdom) was used to detect the level of soluble BMPR2 in serum.

    Techniques: Knockdown, EdU Assay

    ( a-b ) Transfecting siPGR (200nM, 72 hr) effectively downregulated the mRNA and protein of PGR. ( c-e ) Knockdown of PGR reversed the pro-proliferative effects of progesterone (for 24 hr) in BMPR2-knockdown PASMCs. (c) used Alarmar blue assay, and (d-e) were EdU assay and its statistical graph. ( f ) Knockdown of PGR reversed the upregulation of these proteins induced by progesterone (for 2 hr) in BMPR2-knockdown PASMCs. Abbreviation: PGR, progesterone receptor. *, P <0.05; **, P <0.01; ***, P <0.001.

    Journal: bioRxiv

    Article Title: Progesterone is an Inducement of Heritable Pulmonary Arterial Hypertension with BMPR2 Mutation

    doi: 10.1101/2023.04.21.537897

    Figure Lengend Snippet: ( a-b ) Transfecting siPGR (200nM, 72 hr) effectively downregulated the mRNA and protein of PGR. ( c-e ) Knockdown of PGR reversed the pro-proliferative effects of progesterone (for 24 hr) in BMPR2-knockdown PASMCs. (c) used Alarmar blue assay, and (d-e) were EdU assay and its statistical graph. ( f ) Knockdown of PGR reversed the upregulation of these proteins induced by progesterone (for 2 hr) in BMPR2-knockdown PASMCs. Abbreviation: PGR, progesterone receptor. *, P <0.05; **, P <0.01; ***, P <0.001.

    Article Snippet: Human BMPR2 ELISA Kit (orb406355, Biorbyt, United Kingdom) was used to detect the level of soluble BMPR2 in serum.

    Techniques: Knockdown, EdU Assay

    ( a-b ) Progesterone (for 2 hr) promoted the nuclear translocation of c-JUN in BMPR2– knockdown PASMCs, which could be reversed by knockdown of PGR. ( c ) Nucleus and cytoplasm ratio of mean fluorescence intensity of c-JUN. ( d ) Compared with RLP30 (the positive control) and IgG (the negative control), the %input of cJUN was over 4%, demonstrating its binding to the promoter region of EDN1. ( e ) JASPAR database predicted that AP-1 (the dimer of c-JUN and c-FOS) could combine onto the promoter region of EDN1. Abbreviation : ChIP, chromatin immunoprecipitation; AP-1, activator protein-1. **, P <0.01; ***, P <0.001.

    Journal: bioRxiv

    Article Title: Progesterone is an Inducement of Heritable Pulmonary Arterial Hypertension with BMPR2 Mutation

    doi: 10.1101/2023.04.21.537897

    Figure Lengend Snippet: ( a-b ) Progesterone (for 2 hr) promoted the nuclear translocation of c-JUN in BMPR2– knockdown PASMCs, which could be reversed by knockdown of PGR. ( c ) Nucleus and cytoplasm ratio of mean fluorescence intensity of c-JUN. ( d ) Compared with RLP30 (the positive control) and IgG (the negative control), the %input of cJUN was over 4%, demonstrating its binding to the promoter region of EDN1. ( e ) JASPAR database predicted that AP-1 (the dimer of c-JUN and c-FOS) could combine onto the promoter region of EDN1. Abbreviation : ChIP, chromatin immunoprecipitation; AP-1, activator protein-1. **, P <0.01; ***, P <0.001.

    Article Snippet: Human BMPR2 ELISA Kit (orb406355, Biorbyt, United Kingdom) was used to detect the level of soluble BMPR2 in serum.

    Techniques: Translocation Assay, Knockdown, Fluorescence, Positive Control, Negative Control, Binding Assay, Chromatin Immunoprecipitation

    ( a ) Alarmar blue assay showed that progesterone (24 hr) upregulated the proliferation in iPSCs-VSMCs of a HPAH patient, but not influenced iPSCs-VSMCs of a normal person. ( b-c ) Transwell assay showed that progesterone upregulated the migration (8 hr) in iPSCs-VSMCs of a HPAH patient, but not influenced iPSCs-VSMCs of a normal person. ( d ) Progesterone (2 hr) induced the phosphorylation of ERK in iPSCs-VSMCs. ( e ) In comparison with normal iPSCs-VSMCs, EDN1 was significantly increased in PAH iPSCs-VSMCs that could be further upregulated by progesterone (24 hr). Abbreviation: iPSCs, induced pluripotent stem cells; VSMCs, vascular smooth muscle cells; HPAH, hereditary pulmonary artery hypertension; BMPR2 mut, BMRP2-mutation carrier; WT, wild type. ns, non-significance; *, P <0.05.

    Journal: bioRxiv

    Article Title: Progesterone is an Inducement of Heritable Pulmonary Arterial Hypertension with BMPR2 Mutation

    doi: 10.1101/2023.04.21.537897

    Figure Lengend Snippet: ( a ) Alarmar blue assay showed that progesterone (24 hr) upregulated the proliferation in iPSCs-VSMCs of a HPAH patient, but not influenced iPSCs-VSMCs of a normal person. ( b-c ) Transwell assay showed that progesterone upregulated the migration (8 hr) in iPSCs-VSMCs of a HPAH patient, but not influenced iPSCs-VSMCs of a normal person. ( d ) Progesterone (2 hr) induced the phosphorylation of ERK in iPSCs-VSMCs. ( e ) In comparison with normal iPSCs-VSMCs, EDN1 was significantly increased in PAH iPSCs-VSMCs that could be further upregulated by progesterone (24 hr). Abbreviation: iPSCs, induced pluripotent stem cells; VSMCs, vascular smooth muscle cells; HPAH, hereditary pulmonary artery hypertension; BMPR2 mut, BMRP2-mutation carrier; WT, wild type. ns, non-significance; *, P <0.05.

    Article Snippet: Human BMPR2 ELISA Kit (orb406355, Biorbyt, United Kingdom) was used to detect the level of soluble BMPR2 in serum.

    Techniques: Transwell Assay, Migration, Phospho-proteomics, Comparison, Mutagenesis

    ( a ) Time flowchart of animal intervention and phenotype detection. ( b-c ) Right heart catheterization experiments showed the increase of RVSP in CKO and CKO+P group. (n=4 per group) ( d-e ) H&E stain showed the vascular remodeling of pulmonary arterioles (<50μM and 50-100μM) in CKO and CKO+P group and the statistical analyses (4 mice per group, 3 fields per mouse). ( f ) IF stain of vWF (green) and αSMA (red) showed the proliferation and hypertrophy of PASMCs in CKO and CKO+P group. Abbreviation: ♀, female; RVSP, right ventricle systolic pressure; CKO, SM22-cre BMPR2 flox +/- mice; P, progesterone; IF, immunofluorescence; vWF, von Willebrand factor; αSMA, smooth muscle actin. *, P <0.05; **, P <0.01; ****, P <0.0001.

    Journal: bioRxiv

    Article Title: Progesterone is an Inducement of Heritable Pulmonary Arterial Hypertension with BMPR2 Mutation

    doi: 10.1101/2023.04.21.537897

    Figure Lengend Snippet: ( a ) Time flowchart of animal intervention and phenotype detection. ( b-c ) Right heart catheterization experiments showed the increase of RVSP in CKO and CKO+P group. (n=4 per group) ( d-e ) H&E stain showed the vascular remodeling of pulmonary arterioles (<50μM and 50-100μM) in CKO and CKO+P group and the statistical analyses (4 mice per group, 3 fields per mouse). ( f ) IF stain of vWF (green) and αSMA (red) showed the proliferation and hypertrophy of PASMCs in CKO and CKO+P group. Abbreviation: ♀, female; RVSP, right ventricle systolic pressure; CKO, SM22-cre BMPR2 flox +/- mice; P, progesterone; IF, immunofluorescence; vWF, von Willebrand factor; αSMA, smooth muscle actin. *, P <0.05; **, P <0.01; ****, P <0.0001.

    Article Snippet: Human BMPR2 ELISA Kit (orb406355, Biorbyt, United Kingdom) was used to detect the level of soluble BMPR2 in serum.

    Techniques: Staining, Immunofluorescence

    GATA6 expression is induced via BMP10-BMPR2/ALK1 axis. ( A–F ) HPAECs were transfected with ALK1, BMPR2, Endoglin, or control scr siRNA for 48 h, and then treated with 10 ng/ml BMP10 or vehicle for 6 h for RNA isolation and 24 h for protein isolation. ( A,C,E ): GATA6 mRNA measured by qPCR. Data are means ± SE; each experiment was repeated at least three times. ( B,D,F ): GATA6 protein levels were measured by immunoblot analysis. Data are means ± SE, each experiment was repeated at least three times. Representative blots are shown. *p < 0.05, **p < 0.01, ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s test for multiple comparisons. ( G–I ) Human PAH PASMC were treated with 10 ng/ml BMP10 or vehicle ( − ) for 48 h and immunoblot analysis to detect indicated proteins was performed. Data are means ± SE from n = 4 subjects/group. *p < 0.05 by Mann Whitney U test. ( J,K ) Equal amounts of human PAH HPAEC and PASMC were plated at 6-well plates and treated with 10 ng/ml BMP10 or vehicle ( − ). 48 h later cell counts were performed. Data are means ± SE from n = 3 subjects/group, 3 technical repetitions/subject. *p < 0.05 by Mann Whitney U test. ( L,M ) HPAECs were transfected with SMAD1 siRNA, and then treated with BMP10 for 6 h. GATA6 and SMAD1 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated at least three times. *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post-hoc correction for multiple comparisons. ( N,O ) HPAEC were treated for 30 min with diluent ( − ), 5 µM ERK1/2 inhibitor SCH772984 (ERKi), or 5 µM GSK3 inhibitor CHIR99021 (GSK3i) and then stimulated with BMP10 (10 ng/ml) or vehicle for 24 h. Representative immunoblots ( N ) and statistical analysis ( O ) are shown. ( O ): Data represent GATA6/β-actin ratio. Data are means ± SE from five independent experiments. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. ( E ) HPAECs were treated with BMP10 in the presence or absence of 10 µM ERK1/2 inhibitor SCH772984 for 24 h. GATA6 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated six times. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. The original blots are presented in Supplementary Fig. .

    Journal: Scientific Reports

    Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

    doi: 10.1038/s41598-023-33779-8

    Figure Lengend Snippet: GATA6 expression is induced via BMP10-BMPR2/ALK1 axis. ( A–F ) HPAECs were transfected with ALK1, BMPR2, Endoglin, or control scr siRNA for 48 h, and then treated with 10 ng/ml BMP10 or vehicle for 6 h for RNA isolation and 24 h for protein isolation. ( A,C,E ): GATA6 mRNA measured by qPCR. Data are means ± SE; each experiment was repeated at least three times. ( B,D,F ): GATA6 protein levels were measured by immunoblot analysis. Data are means ± SE, each experiment was repeated at least three times. Representative blots are shown. *p < 0.05, **p < 0.01, ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s test for multiple comparisons. ( G–I ) Human PAH PASMC were treated with 10 ng/ml BMP10 or vehicle ( − ) for 48 h and immunoblot analysis to detect indicated proteins was performed. Data are means ± SE from n = 4 subjects/group. *p < 0.05 by Mann Whitney U test. ( J,K ) Equal amounts of human PAH HPAEC and PASMC were plated at 6-well plates and treated with 10 ng/ml BMP10 or vehicle ( − ). 48 h later cell counts were performed. Data are means ± SE from n = 3 subjects/group, 3 technical repetitions/subject. *p < 0.05 by Mann Whitney U test. ( L,M ) HPAECs were transfected with SMAD1 siRNA, and then treated with BMP10 for 6 h. GATA6 and SMAD1 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated at least three times. *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post-hoc correction for multiple comparisons. ( N,O ) HPAEC were treated for 30 min with diluent ( − ), 5 µM ERK1/2 inhibitor SCH772984 (ERKi), or 5 µM GSK3 inhibitor CHIR99021 (GSK3i) and then stimulated with BMP10 (10 ng/ml) or vehicle for 24 h. Representative immunoblots ( N ) and statistical analysis ( O ) are shown. ( O ): Data represent GATA6/β-actin ratio. Data are means ± SE from five independent experiments. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. ( E ) HPAECs were treated with BMP10 in the presence or absence of 10 µM ERK1/2 inhibitor SCH772984 for 24 h. GATA6 mRNA levels were measured by qPCR. Data shown as means ± SE. Each experiment was repeated six times. *p < 0.05 by Kruskal–Wallis test with post-hoc Dunn’s correction for multiple comparisons. The original blots are presented in Supplementary Fig. .

    Article Snippet: Antibodies were as follows: Goat anti-human GATA6 (R&D; AF1700, 1:500), mouse anti-human GATA6 (Santa-Cruz, 517554, 1:500), Rabbit anti-human/mouse SOD2 (Cell signaling technology; 13141, 1:1000; 13145, 1:5000), Goat anti-human ALK1 (R&D; AF370, 1:500), Rabbit anti-mouse ALK1 (ABGENT AP7807a, 1:1000), rabbit anti-human/mouse ActR2B (LS bio; LS-B7781, 1:1000), mouse anti-human BMPR2 (Novus; NBP2-37624 Clone 3F6; 1:1000), rabbit anti-mouse BMPR2 (Proteintech; 19087–1-AP; 1:1000), mouse anti-mouse/human/rat BMPR2 (Abcam, catalog #130206 1:500), rabbit anti-human/mouse Endoglin (Proteintech; 10862–1-AP; 1:1000), rabbit anti-mouse BiP (Abcam; ab53068; 1:1000), rabbit anti-mouse CHOP (Novus; NBP2-13172; 1:1000), mouse anti-beta-actin (Sigma; A1978; 1:2000), mouse anti-human PRPF4 (Abcam, 69878, 1:1000), rabbit anti-human MYEOV (Invitrogen, PA5-48938 1:1000), rabbit anti-human STING (Invitrogen, PA5-26751, 1:1000), rabbit anti-human/mouse/rat EAF1 (Invitrogen, PA5-100493, 1:1000), rabbit anti-human/mouse/rat Histone H3 (Cell Signaling, 9715 1:1000), rabbit anti-human/mouse/rat a-b Tubulin (Cell Signaling, 2148, 1:1000).

    Techniques: Expressing, Transfection, Control, Isolation, Western Blot, MANN-WHITNEY

    GATA6 deficiency in PAEC and PASMC results in loss of BMP receptors. ( A ) qPCR of HPAECs transfected with GATA6 or control scr siRNA ( − ) to measure indicated mRNA, each experiment was repeated at least three times. Data are means ± SE, n = 4–7. **p < 0.01, ***p < 0.001 by Mann Whitney U test. ( B ) Chromatin immune precipitation (ChiP) assay in HPAECs, n = 8–10. Representative gel images and data quantification are shown. Data are means ± SE, ***p < 0.001, ****p < 0.0001 by Mann Whitney U test. ( C,D ) Immunoblot analysis of control human PASMC transfected with siRNA GATA6 or control scr siRNA for 48 h. Data are means ± SE, 3 subjects/group, *p < 0.05 by Mann Whitney U test. Please see Fig. F,G for GATA6 immunoblots. ( E,F ) Expression of BmpR2, Alk1 , ActRIIB, and endoglin measured by qPCR in PAEC ( F ) and whole lungs ( G ) from WT and Gata6 CKO mice. Data are means ± SE; E: n = 4–5/group; F: n = 6–11 mice/group. Male and female mice responded similarly. *p < 0.05, **p < 0.01 by Mann Whitney U test (( E,F ) BmpR2, Alk1 , and ActRIIB ) and unpaired τ test (F Endoglin ). ( G,H ) Control HPAECs transfected with siGATA6 or control scr siRNA ( − ) were assayed by immunoblot analysis to detect indicated BMP receptors. Values are means ± SE of the relative protein levels by densitometry, n = 4–7.*p < 0.05, ***p < 0.001 by Mann Whitney U test. ( I,J ) Immunoblot analysis of whole lung tissue from Gata6 CKO and WT mice. Values are means ± SE of the relative protein levels by densitometry, n = 3–5/group. Male and female mice responded similarly. *p < 0.05, **p < 0.01 by Mann Whitney U test. The original blots are presented in Supplementary Fig. .

    Journal: Scientific Reports

    Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

    doi: 10.1038/s41598-023-33779-8

    Figure Lengend Snippet: GATA6 deficiency in PAEC and PASMC results in loss of BMP receptors. ( A ) qPCR of HPAECs transfected with GATA6 or control scr siRNA ( − ) to measure indicated mRNA, each experiment was repeated at least three times. Data are means ± SE, n = 4–7. **p < 0.01, ***p < 0.001 by Mann Whitney U test. ( B ) Chromatin immune precipitation (ChiP) assay in HPAECs, n = 8–10. Representative gel images and data quantification are shown. Data are means ± SE, ***p < 0.001, ****p < 0.0001 by Mann Whitney U test. ( C,D ) Immunoblot analysis of control human PASMC transfected with siRNA GATA6 or control scr siRNA for 48 h. Data are means ± SE, 3 subjects/group, *p < 0.05 by Mann Whitney U test. Please see Fig. F,G for GATA6 immunoblots. ( E,F ) Expression of BmpR2, Alk1 , ActRIIB, and endoglin measured by qPCR in PAEC ( F ) and whole lungs ( G ) from WT and Gata6 CKO mice. Data are means ± SE; E: n = 4–5/group; F: n = 6–11 mice/group. Male and female mice responded similarly. *p < 0.05, **p < 0.01 by Mann Whitney U test (( E,F ) BmpR2, Alk1 , and ActRIIB ) and unpaired τ test (F Endoglin ). ( G,H ) Control HPAECs transfected with siGATA6 or control scr siRNA ( − ) were assayed by immunoblot analysis to detect indicated BMP receptors. Values are means ± SE of the relative protein levels by densitometry, n = 4–7.*p < 0.05, ***p < 0.001 by Mann Whitney U test. ( I,J ) Immunoblot analysis of whole lung tissue from Gata6 CKO and WT mice. Values are means ± SE of the relative protein levels by densitometry, n = 3–5/group. Male and female mice responded similarly. *p < 0.05, **p < 0.01 by Mann Whitney U test. The original blots are presented in Supplementary Fig. .

    Article Snippet: Antibodies were as follows: Goat anti-human GATA6 (R&D; AF1700, 1:500), mouse anti-human GATA6 (Santa-Cruz, 517554, 1:500), Rabbit anti-human/mouse SOD2 (Cell signaling technology; 13141, 1:1000; 13145, 1:5000), Goat anti-human ALK1 (R&D; AF370, 1:500), Rabbit anti-mouse ALK1 (ABGENT AP7807a, 1:1000), rabbit anti-human/mouse ActR2B (LS bio; LS-B7781, 1:1000), mouse anti-human BMPR2 (Novus; NBP2-37624 Clone 3F6; 1:1000), rabbit anti-mouse BMPR2 (Proteintech; 19087–1-AP; 1:1000), mouse anti-mouse/human/rat BMPR2 (Abcam, catalog #130206 1:500), rabbit anti-human/mouse Endoglin (Proteintech; 10862–1-AP; 1:1000), rabbit anti-mouse BiP (Abcam; ab53068; 1:1000), rabbit anti-mouse CHOP (Novus; NBP2-13172; 1:1000), mouse anti-beta-actin (Sigma; A1978; 1:2000), mouse anti-human PRPF4 (Abcam, 69878, 1:1000), rabbit anti-human MYEOV (Invitrogen, PA5-48938 1:1000), rabbit anti-human STING (Invitrogen, PA5-26751, 1:1000), rabbit anti-human/mouse/rat EAF1 (Invitrogen, PA5-100493, 1:1000), rabbit anti-human/mouse/rat Histone H3 (Cell Signaling, 9715 1:1000), rabbit anti-human/mouse/rat a-b Tubulin (Cell Signaling, 2148, 1:1000).

    Techniques: Transfection, Control, MANN-WHITNEY, Western Blot, Expressing

    Treatment with DMF restores expression of the BMP receptors, reverses oxidative stress and pulmonary hypertension in Gata6 CKO mice. (DMF or vehicle were administered daily via i.p. injection for 3 weeks. ( A ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of indicated BMP receptors. Data are means ± SE, n = 6–12, *p < 0.05. **p < 0.01 by Kruskal–Wallis test followed by Dunn’s multiple comparisons test ( BmpR2, ActRIIB, Alk1 ) and one-way ANOVA followed by post hoc Tukey’s multiple comparison ( Endoglin ). ( B ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of the antioxidant enzymes and eNOS . Data are means ± SE, n = 6–17, *p < 0.05., **p < 0.01, ***p < 0.001 by one-way ANOVA followed by post hoc Tukey’s multiple comparisons test ( SOD2, GPX1, CAT ) and Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test ( eNOS ). ( C,D ) mRNA levels of indicated BMP receptors and antioxidant enzymes measured by qPCR in PAEC from WT and Gata6 CKO mice treated with DMF or vehicle. Data are means ± SE, n = 3–6 mice/group, *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test. ( E–G ) RVSP, pulmonary acceleration time as a fraction of ejection time (PAT/ET) and Fulton index (RV/[LV + S]) were evaluated in WT and Gata6 CKO mice in the presence or absence of DMF. Data are means ± SE. n = 5–11 mice/group. *p < 0.05, **p < 0.01. ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test (RVSP) and one-way ANOVA followed by post hoc Tukey’s multiple comparisons test (PAT/ET and RV/(LV + S).

    Journal: Scientific Reports

    Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

    doi: 10.1038/s41598-023-33779-8

    Figure Lengend Snippet: Treatment with DMF restores expression of the BMP receptors, reverses oxidative stress and pulmonary hypertension in Gata6 CKO mice. (DMF or vehicle were administered daily via i.p. injection for 3 weeks. ( A ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of indicated BMP receptors. Data are means ± SE, n = 6–12, *p < 0.05. **p < 0.01 by Kruskal–Wallis test followed by Dunn’s multiple comparisons test ( BmpR2, ActRIIB, Alk1 ) and one-way ANOVA followed by post hoc Tukey’s multiple comparison ( Endoglin ). ( B ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of the antioxidant enzymes and eNOS . Data are means ± SE, n = 6–17, *p < 0.05., **p < 0.01, ***p < 0.001 by one-way ANOVA followed by post hoc Tukey’s multiple comparisons test ( SOD2, GPX1, CAT ) and Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test ( eNOS ). ( C,D ) mRNA levels of indicated BMP receptors and antioxidant enzymes measured by qPCR in PAEC from WT and Gata6 CKO mice treated with DMF or vehicle. Data are means ± SE, n = 3–6 mice/group, *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test. ( E–G ) RVSP, pulmonary acceleration time as a fraction of ejection time (PAT/ET) and Fulton index (RV/[LV + S]) were evaluated in WT and Gata6 CKO mice in the presence or absence of DMF. Data are means ± SE. n = 5–11 mice/group. *p < 0.05, **p < 0.01. ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test (RVSP) and one-way ANOVA followed by post hoc Tukey’s multiple comparisons test (PAT/ET and RV/(LV + S).

    Article Snippet: Antibodies were as follows: Goat anti-human GATA6 (R&D; AF1700, 1:500), mouse anti-human GATA6 (Santa-Cruz, 517554, 1:500), Rabbit anti-human/mouse SOD2 (Cell signaling technology; 13141, 1:1000; 13145, 1:5000), Goat anti-human ALK1 (R&D; AF370, 1:500), Rabbit anti-mouse ALK1 (ABGENT AP7807a, 1:1000), rabbit anti-human/mouse ActR2B (LS bio; LS-B7781, 1:1000), mouse anti-human BMPR2 (Novus; NBP2-37624 Clone 3F6; 1:1000), rabbit anti-mouse BMPR2 (Proteintech; 19087–1-AP; 1:1000), mouse anti-mouse/human/rat BMPR2 (Abcam, catalog #130206 1:500), rabbit anti-human/mouse Endoglin (Proteintech; 10862–1-AP; 1:1000), rabbit anti-mouse BiP (Abcam; ab53068; 1:1000), rabbit anti-mouse CHOP (Novus; NBP2-13172; 1:1000), mouse anti-beta-actin (Sigma; A1978; 1:2000), mouse anti-human PRPF4 (Abcam, 69878, 1:1000), rabbit anti-human MYEOV (Invitrogen, PA5-48938 1:1000), rabbit anti-human STING (Invitrogen, PA5-26751, 1:1000), rabbit anti-human/mouse/rat EAF1 (Invitrogen, PA5-100493, 1:1000), rabbit anti-human/mouse/rat Histone H3 (Cell Signaling, 9715 1:1000), rabbit anti-human/mouse/rat a-b Tubulin (Cell Signaling, 2148, 1:1000).

    Techniques: Expressing, Injection, Comparison