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

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

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

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

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

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

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

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

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

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

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

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

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

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

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