Journal: Scientific Reports

Article Title: LATS kinases and SLUG regulate the transition to advanced stage in aggressive oral cancer cells

doi: 10.1038/s41598-022-16667-5

Figure Lengend Snippet: LATS1 and LATS2 phosphorylate SLUG on T208. ( A ) SAS and SAS-δ cells were co-transfected with siRNAs against LATS1 and LATS2 (siLATS1/2), and then cultured with TGF-β1 for 48 h, followed by WB with the indicated antibodies. ( B ) SAS cells transfected with GL2 (as a negative control) or LATS2 siRNA duplex were treated with cycloheximide (CHX) for the indicated periods, and then subjected to subcellular fractionation. Protein levels of SLUG and LATS2 were determined by WB. Lamin A/C and α-tubulin are nuclear and cytoplasmic fraction markers, respectively. The relative levels of cytoplasmic and nuclear SLUG normalized to the corresponding band intensities of α-tubulin and Lamin A/C, respectively, are shown below top panel. The normalized band intensities of SLUG at 0 h after CHX treatment were defined as 1.0 (lanes 1, 5, 9, and 13). ( C ) WB of parental SAS and SAS-δ, which were cultured in medium containing 10% FBS in the presence (+) or absence (–) of TGF-β1 (48 h), was performed with the indicated antibodies. The relative levels of the indicated proteins normalized to the corresponding band intensity of actin, SLUG, or SMAD2, are shown below panels. ( D ) Immunofluorescence staining with anti-SLUG-pT208 (red) and α-tubulin (green) antibodies. Cells were cultured as in ( C ). DNA was visualized by Hoechst 33258 staining (blue). The cell size were comparable between SAS and SAS-δ in the presence or absence of TGF-β1.

Article Snippet: Recombinant human LATS1 or LATS2 kinase (Carna Biosciences, Hyogo, Japan) was incubated at 30 °C for 30 min with GST-fused mouse Slug-WT, -T183A, -T209A, and all-A (S101A, S139A, S161A, T183A, S196A, T209A, S215A, S248A, and S252A) mutants in LATS1/2-kinase buffer (20 mM PIPES [pH 6.8], 4 mM MnCl 2 , 1 mM DTT, 1 mM NaF, 1 mM Na 3 VO 4 ) containing 20 μM ATP and 10 μCi [γ- 32 P] ATP.

Techniques: Transfection, Cell Culture, Negative Control, Fractionation, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Phosphorylation-dependent control of Arc protein by synaptic plasticity regulator TNIK

doi: 10.1101/2021.04.04.438383

Figure Lengend Snippet: (a) Arc is able to significantly increase TNIK activity with increased levels of ATP concentration in comparison to GST-TNIK alone, and control GST-TNIK + GST. One-way ANOVA revealed a significant dose–response difference between Arc and GST-TNIK (31.25μM, F 2,14 = 10.47, p < 0.01; 62.5μM, F 2,14 = 8.38, p < 0.01; 125 μM, F 2,14 = 8.87, p < 0.01; 250 μM, F 2,14 = 9.02, p < 0.01). Tukey’s HSD post hoc test, ** p < 0.01 in comparison to GST-TNIK (represented by the grey line), # p < 0.05; ## p < 0.01 in comparison to GST-TNIK + GST (represented by the red line). (b) Western blots showing the levels of GST to indicate Arc abundance, and phospho-serine/threonine to measure overall phosphorylation levels in Neuro2a lysate that was transfected with Arc-GST and/or TNIK and ATP (400 μM). (c) Chemical structure of TNIK inhibitor, KY-05009 (left); and inactive analog, G883-2176 (right). (d-e) KY-05009 and G883-2176 was applied in increasing concentrations (0, 0.25, 1, and 15 μM) to assess the effects on kinetic interaction between TNIK and Arc. Graphs show luminescence relative percentage difference upon addition of KY-05009 or G883-2176, respectively (±SEM). One-way ANOVA revealed a significant dose-dependent response between KY-05009 concentrations for TNIK alone (F 3,25 = 47.92, p < 0.0001) as well as TNIK + GST-Arc (F 3,35 = 37.58, p < 0.0001). Tukey’s HSD post hoc test, * p < 0.05; ** p < 0.01, *** p < 0.005; **** p < 0.0001. (f) Neuro2a cells were treated with KY-05009 (15 μM) and immunoblotted with anti-GST and anti-phospho-serine/threonine to analyze overall phosphorylation levels.

Article Snippet: Reactions were performed according to manufacturer’s protocol with 1 µM full-length GST-tagged mouse Arc and 10 nM of human TNIK catalytic domain (Carna Biosciences, Japan, #07-138) in reaction buffer (50 mM Tris [pH 7.5], 5 mM MgCl 2 , 0.01% Brij-35).

Techniques: Activity Assay, Concentration Assay, Comparison, Western Blot, Transfection

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 loss induces SOD2 and other anti-oxidant enzymes deficiency in human and mouse PAECs. ( A ) An expression of the antioxidant enzymes measured by qPCR in human PAECs (HPAECs) transfected with siGATA6 or control scr siRNA. mRNA levels in scr siRNA transfected cells were set as one. Data are means ± SE, *p < 0.05 vs scr siRNA by Kruskal–Wallis test with post hoc Dunn’s test for multiple comparisons; n = 3–10 (see also Supplemental Table ). ( B ) Chromatin immune precipitation (ChiP) assay in HPAECs, n = 8 for SOD2 ; n = 5 for GPX1 . The experiments were repeated three times. Representative gel images and data quantification are shown. Values are means ± SE, **p < 0.01, ***p < 0.001 by Mann Whitney U test. ( C,D ) Control HPAECs transfected with siGATA6 or scr siRNA (siContr) were assayed by Western blot analysis to detect GATA6 and SOD2. Representative blots are shown. Values are means ± SE of the relative protein levels by densitometry, n = 5, **p < 0.01 by Mann Whitney U test. ( E,F ) Western blot analysis of whole lung tissue from Gata6 CKO and WT mice. Representative blots are shown. Values are means ± SE of the relative protein levels by densitometry, n = 4, *p < 0.05 by Mann Whitney U test. ( G ) SOD2, GPX, and Catalase activity were measured in Human HPAECs transfected with siGATA6 or scr siRNA, Values are means ± SE, n = 3, *p < 0.05 by Mann Whitney U test. ( H ) Mouse Sod2 (n = 5), Gpx (n = 10), and Cat (n = 9) activity were measured in mouse lung tissue in Gata6 CKO and WT mice. *p < 0.05, **p < 0.01 by Mann Whitney U test (Sod activity) and unpaired τ test (Gpx and Cat activities). ( I,J ) mRNA levels of the antioxidant enzymes measured by qPCR in whole lung tissues ( I ) and in PAEC from Gata6 CKO mice (J). Data are means ± SE. I: n = 12–13 for Sod , n = 10/group for Gpx1 , n = 9/group for Cat . J: n = 4 mice/group. *p < 0.05, **p < 0.01 by unpaired τ test (I) or Mann Whitney U test ( J ). 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, MANN-WHITNEY, Western Blot, Activity Assay

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 is deficient in both PAEC and PASMC in human PAH lungs. ( A ) IHC analyses were performed to detect GATA6 (green), CD31 (red) and α-SMA (red) in small PAs from healthy controls (HC) and patients with PAH (SSc-PAH and IPAH) and analysis of nuclear GATA6 in CD31- and SMA-positive cells in small PAs was performed. Left: Images are representative from 3–4 subjects/control, SSc-PAH, and IPAH. Bar equals 50 μm. White arrowheads indicate GATA6-positive cells. Right: Data are in optical density units (OD); Data are means ± SE from 3–4 human subjects per control, 7–8 for PAH (SSc-PAH + IPAH) groups. *p < 0.05 by Mann Whitney U test. ( B,C ) Immunoblot analysis of early-passage PAEC and PASMC from healthy controls (HC) and subjects with IPAH to detect indicated proteins. Data are means ± SE from n = 5 subjects/group. **p < 0.01 by Mann Whitney U test. ( D ) Immunoblot analysis of nuclear fractions of PASMC from healthy control (HC) and IPAH subjects. Data are means ± SE from n = 4 subjects/group. *p < 0.05 by Mann Whitney U test. ( E ) Immunocytochemical analysis of PASMC from healthy control (HC) and IPAH subjects to detect GATA6 (red) and DAPI (blue). Bar equals 50 µm. Representative images from two subjects/group by Mann Whitney U test. ( F–J ) Human non-diseased PASMC were transfected with siRNA GATA6 or control scrambled siRNA ( − ). 48 h post-transfection, immunoblot (F, G) and cell count analysis (H) were performed. Data are means ± SE from n = 3 subjects/group. *p < 0.05 by Mann Whitney U test. ( I ) qPCR analysis of PASMC from healthy controls (HC) and patients with PAH to detect SOD2 expression. Data are means ± SE, n = 5 subjects/group. *p < 0.05 by Mann Whitney U test. ( K,L ) Immunoblot analysis of healthy control (HC) and IPAH PASMC to detect indicated proteins was performed on the same membrane using strip- re-probe approach. Correlation between GATA6 and SOD was examined (R2). Data are means ± SE, n = 5 subjects/group. **p < 0.01 by Mann Whitney U test ( K ), R2 = 0.81 by Spearman analysis with Holm–Sidak adjusted p values ( L ). 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: Control, MANN-WHITNEY, Western Blot, Transfection, Cell Counting, Expressing, Membrane, Stripping Membranes

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

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

Journal: PLoS ONE

Article Title: BMP4 Increases the Expression of TRPC and Basal [Ca 2+ ] i via the p38MAPK and ERK1/2 Pathways Independent of BMPRII in PASMCs

doi: 10.1371/journal.pone.0112695

Figure Lengend Snippet: (A) represents the blots of P-Smad1/5/8, P-p38MAPK, P-ERK1/2, t-Smad1/5/8, t-p38MAPK and t-ERK1/2 upon BMP4 treatment (50 ng/ml) in dose and time dependent manner. (B) Bar values are means ± SEM (n = 3 for each group). (C) represents the blots of BMPRII upon BMP4 treatment for 5–30 min. (D) Bar values are means ± SEM (n = 3 for each group).* P <0.01 vs. respective vehicle control.

Article Snippet: All antibodies were obtained as follow: TRPC1, 6 antibodies (abcam), TRPC4 antibody (Alomone Laboratories), phospho-Smad1/5/8, phospho-ERK1/2, phospho-p38MAPK, ERK1/2, Smad5 and p38MAPK antibodies (Cell Signaling Technology), BMPRIa antibody (Proteintech Group), BMPRIb antibody (Invitrogen), ALK2 antibody (Novus), BMPRII antibody (BD), ActR2a and ActR2b antibodies (Abbiotec), α-actin antibody (Sigma-Aldrich).

Techniques: Control

Journal: PLoS ONE

Article Title: BMP4 Increases the Expression of TRPC and Basal [Ca 2+ ] i via the p38MAPK and ERK1/2 Pathways Independent of BMPRII in PASMCs

doi: 10.1371/journal.pone.0112695

Figure Lengend Snippet: (A), (B) and (C) show blots (left) and normalized bar graphs (right) of P-Smad1/5/8, P-p38MAPK, P-ERK1/2, t-Smad1/5/8, t- p38MAPK and t-ERK1/2, respectively. Bar values were means ± SEM (n = 4 for each group). * P <0.01 vs. respective siNT control.

Article Snippet: All antibodies were obtained as follow: TRPC1, 6 antibodies (abcam), TRPC4 antibody (Alomone Laboratories), phospho-Smad1/5/8, phospho-ERK1/2, phospho-p38MAPK, ERK1/2, Smad5 and p38MAPK antibodies (Cell Signaling Technology), BMPRIa antibody (Proteintech Group), BMPRIb antibody (Invitrogen), ALK2 antibody (Novus), BMPRII antibody (BD), ActR2a and ActR2b antibodies (Abbiotec), α-actin antibody (Sigma-Aldrich).

Techniques: Control