Journal: Experimental & Molecular Medicine

Article Title: USP15 promotes pulmonary vascular remodeling in pulmonary hypertension in a YAP1/TAZ-dependent manner

doi: 10.1038/s12276-022-00920-y

Figure Lengend Snippet: a Identification of hPASMCs by immunofluorescence staining. Scale bar: 100 μm. b , c The mRNA and protein levels of USP15 in the hypoxia-treated hPASMCs. d The protein levels of t-YAP1 and t-TAZ in the hypoxia-treated hPASMCs. e The protein levels of n-YAP1 and n-TAZ in the hypoxia-treated hPASMCs. Data are represented as the mean ± SD, n = 4 in each group. Compared with values at 0 h, * P < 0.05, ** P < 0.01.

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were purchased from iCell Bioscience, Inc., Shanghai. hPASMCs were cultured in specific medium (PriMed-iCell-004-LS, iCell Bioscience, China) at 37 °C in a humidified incubator with 5% CO 2 . hPASMCs at passages 2-7 were used for further study.

Techniques: Immunofluorescence, Staining

Journal: Experimental & Molecular Medicine

Article Title: USP15 promotes pulmonary vascular remodeling in pulmonary hypertension in a YAP1/TAZ-dependent manner

doi: 10.1038/s12276-022-00920-y

Figure Lengend Snippet: Adenovirus delivering shRNA against hUSP15 was used to downregulate USP15 expression in hPASMCs. After 48 h of infection, cells were cultured under hypoxia for another 24 h. The protein level ( a ) and mRNA level ( b ) of USP15 in the treated cells were measured by western blotting and real-time PCR, respectively. c The abundance of PCNA protein was detected by western blot analysis. d A CCK-8 assay was performed to determine cell viability after treatments. e , f The protein levels of t-YAP1, t-TAZ, n-YAP1, and n-TAZ in the treated cells were measured by western blotting. g , h Cell migration in each group was determined by wound healing assay. Scale bar: 200 μm. Data are represented as the mean ± SD, n = 4 in each group.

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were purchased from iCell Bioscience, Inc., Shanghai. hPASMCs were cultured in specific medium (PriMed-iCell-004-LS, iCell Bioscience, China) at 37 °C in a humidified incubator with 5% CO 2 . hPASMCs at passages 2-7 were used for further study.

Techniques: shRNA, Expressing, Infection, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, CCK-8 Assay, Migration, Wound Healing Assay

Journal: Experimental & Molecular Medicine

Article Title: USP15 promotes pulmonary vascular remodeling in pulmonary hypertension in a YAP1/TAZ-dependent manner

doi: 10.1038/s12276-022-00920-y

Figure Lengend Snippet: Adenovirus delivering overexpressed hUSP15 was used to upregulate USP15 expression in hPASMCs under normoxia. Western blot analysis ( a ) and real-time PCR ( b ) were conducted to determine the infectivity of Ad-hUSP15-OV after 72 h of infection. Afterward, hPASMCs were coinfected with adenovirus delivering overexpressed hUSP15 and hYAP1-sh or hTAZ-sh. c The expression level of PCNA in hPASMCs was detected by immunoblot analysis after coinfection. d A CCK-8 assay was performed to detect the viability of hPASMCs after coinfection. e , f The expression levels of t-YAP1, t-TAZ, n-YAP1, and n-TAZ in hPASMCs were detected by western blotting after coinfection. g , h Cell migration in hPASMCs was determined by wound healing assays after coinfection. Scale bar: 200 μm. Data are represented as the mean ± SD, n = 4 in each group. Compared to the Vector+NCsh-2 group, # P < 0.05, ## P < 0.01; compared to the hUSP15-OV + NCsh-2 group, * P < 0.05, ** P < 0.01.

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were purchased from iCell Bioscience, Inc., Shanghai. hPASMCs were cultured in specific medium (PriMed-iCell-004-LS, iCell Bioscience, China) at 37 °C in a humidified incubator with 5% CO 2 . hPASMCs at passages 2-7 were used for further study.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Infection, CCK-8 Assay, Migration, Plasmid Preparation

Journal: Experimental & Molecular Medicine

Article Title: USP15 promotes pulmonary vascular remodeling in pulmonary hypertension in a YAP1/TAZ-dependent manner

doi: 10.1038/s12276-022-00920-y

Figure Lengend Snippet: a Anti-USP15 immunoprecipitation was used to determine the interaction between USP15 and YAP1 or TAZ in the control and hypoxic hPASMCs. b Anti-YAP1 immunoprecipitation to determine the interaction between YAP1 and USP15 in the control or hypoxic hPASMCs. c Anti-TAZ immunoprecipitation to determine the interaction between TAZ and USP15 in the control or hypoxic hPASMCs. d hPASMCs were infected with Ad-hUSP15-sh or Ad-NCsh-2 for 48 h. After 3 h of starvation, cells were exposed to hypoxia for 24 h and treated with CHX (25 μg/ml) or MG132 (5 μM) for 0, 1, 4, or 8 h (at the last 8 h of hypoxic time). Cell samples were harvested for immunoblot analysis to detect YAP1 expression. The percentage of YAP1/actin is shown in the right panel. e hPASMCs infected with Ad-hUSP15-OV or Ad-Vector were treated with MG132 (5 μM) under normoxia for 8 h, and cells were harvested for immunoprecipitation using anti-YAP1 antibody. f Under normoxia, infected hPASMCs were subjected to a CHX pulse-chase assay. Cell samples were harvested for immunoblot analysis to detect YAP1 expression. The percentage of YAP1/Actin is shown in the right panel. g His-tagged USP15 and Myc-tagged YAP1 were cotransfected into HEK293T cells. At 48 h post-transfection, cell samples were harvested for immunoprecipitation using an anti-His antibody. h HEK293T cells were cotransfected with Myc-tagged YAP1 and His-tagged USP15/vector. At 48 h post-transfection, cells were treated with CHX (25 μg/ml) for 0, 1, 4, and 8 h. YAP1 expression in cell samples was determined by western blot analysis using an anti-Myc antibody. The percentage of YAP1/actin is shown in the right panel. i His-tagged USP15, HA-tagged Ubi and Myc-tagged YAP1 were cotransfected into HEK293T cells. At 48 h post-transfection, cells were treated with MG132 (5 μM) for 5 h. Cell samples were then harvested for immunoprecipitation using anti-Myc antibody. Data are represented as the mean ± SD, n = 4 in each group.

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were purchased from iCell Bioscience, Inc., Shanghai. hPASMCs were cultured in specific medium (PriMed-iCell-004-LS, iCell Bioscience, China) at 37 °C in a humidified incubator with 5% CO 2 . hPASMCs at passages 2-7 were used for further study.

Techniques: Immunoprecipitation, Control, Infection, Western Blot, Expressing, Plasmid Preparation, Pulse Chase, Transfection

Journal: The European Respiratory Journal

Article Title: Targeting peptidyl-prolyl isomerase 1 in experimental pulmonary arterial hypertension

doi: 10.1183/13993003.01698-2021

Figure Lengend Snippet: Peptidyl-prolyl cis/trans isomerase, NIMA interacting 1 (Pin1) activation in experimental and human pulmonary arterial hypertension (PAH). a) Representative immunofluorescence micrograph of human lung sections from control and idiopathic PAH (IPAH) patients. Staining was undertaken for Pin1 (green) and vessel identity was visualised using α-smooth muscle actin (SMA) (red). Scale bar=50 µm. b, d) Protein expression of Pin1 in smooth muscle cells (control n=4, IPAH n=9) and endothelial cells (control n=4, IPAH n=5) isolated from pulmonary arteries of control and IPAH patients. Regulation at protein level was analysed using Western blot analysis followed by c, e) densitometric analysis. f–i) Correlation of Pin1 with clinical characteristics of IPAH patients, such as mean pulmonary arterial pressure (mPAP) (n=4, r=0.8177, p=0.0468), pulmonary capillary wedge pressure (n=6, r= −8825, p=0.1175), cardiac index (n=4, r= −0.8276, p=01724) and systolic pulmonary artery pressure (n=7, r= −0.6285, p=0.1306), respectively. j, l) Western blot analysis of Pin1 in lung homogenates exposed to Sugen5416/hypoxia (SuHx) (normoxia (NOX) n=4, SuHx n=4) and chronic hypoxia (HOX), respectively (NOX n=6, HOX n=7) followed by k, m) densitometric analysis. Pan-actin is taken as loading control. DAPI: 4′,6-diamidino-2-phenylindole; hPASMCs: human pulmonary artery smooth muscle cells; hPAECs: human pulmonary artery endothelial cells; ns : nonsignificant; A.U.: arbitrary unit. *: p<0.05, ***: p<0.001 (t-test).

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were either obtained from the Universities of Giessen and Marburg Lung Center Giessen Biobank, member of DZL Platform Biobanking or purchased from Lonza (Basel, Switzerland).

Techniques: Activation Assay, Immunofluorescence, Control, Staining, Expressing, Isolation, Western Blot

Journal: The European Respiratory Journal

Article Title: Targeting peptidyl-prolyl isomerase 1 in experimental pulmonary arterial hypertension

doi: 10.1183/13993003.01698-2021

Figure Lengend Snippet: Peptidyl-prolyl cis/trans isomerase, NIMA interacting 1 (Pin1) blockage results in the suppression of vascular cell proliferation in vitro . a) Human pulmonary artery smooth muscle cells (hPASMCs) from controls and idiopathic pulmonary arterial hypertension (IPAH) patients cultured in SmGM-2 were serum-starved and treated with Juglone or dimethyl sulfoxide (DMSO) (vehicle) in the presence of platelet-derived growth factor (PDGF)-BB for 24 h. b) Representative Western blots of Pin1 and proliferating cell nuclear antigen (PCNA) expression in control and IPAH hPASMCs followed by c) densitometric analysis 24 h after Pin1 mRNA knockdown. Immunofluorescence staining for Ki-67 + cells in d) Pin1-silenced (si) and f) Juglone-exposed hPASMCs. g) Human pulmonary artery endothelial cells (hPAECs) were serum-starved (0.2% fetal bovine serum (FBS) in M200) and stimulated with 10% FBS with or without Juglone for 24 h. Proliferation of Pin1-silenced e) hPASMCs and h) hPAECs of donor control and IPAH patients in presence or absence of e) PDGF-BB and h) 10% FBS determined by 5-bromo-2-deoxyuridine (BrdU) incorporation. The rate of DNA synthesis for a, e, g and h) was examined by measuring of BrdU incorporation [ A 370 nm]. Scr: scrambled; ns : nonsignificant. Statistical analysis was performed using one-way ANOVA with Newman–Keuls post hoc test for multiple comparisons. **: p<0.01, ***: p<0.001, ****: p<0.0001 versus PDGF-BB or 10% FBS treated cells; # : p<0.05, ## : p<0.01, ### : p<0.001, #### : p<0.0001 versus si scrambled or dimethyl sulfoxide (DMSO)-treated cells; § : p<0.05, §§ : p<0.01, §§§ : p<0.001, §§§§ : p<0.0001 versus si scrambled treated or IPAH cells. Data from three independent experiments are presented as mean± sem .

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were either obtained from the Universities of Giessen and Marburg Lung Center Giessen Biobank, member of DZL Platform Biobanking or purchased from Lonza (Basel, Switzerland).

Techniques: In Vitro, Cell Culture, Derivative Assay, Western Blot, Expressing, Control, Knockdown, Immunofluorescence, Staining, BrdU Incorporation Assay, DNA Synthesis

Journal: The European Respiratory Journal

Article Title: Targeting peptidyl-prolyl isomerase 1 in experimental pulmonary arterial hypertension

doi: 10.1183/13993003.01698-2021

Figure Lengend Snippet: Peptidyl-prolyl cis/trans isomerase, NIMA interacting 1 (Pin1) blockage results in initiation of cell apoptosis in vitro . Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay after 24 h treatment with increasing concentration of Juglone of a) control and i) idiopathic pulmonary arterial hypertension (IPAH) human pulmonary artery smooth muscle cells (hPASMCs), and of e) control and o) IPAH human pulmonary artery endothelial cells (hPAECs). b, j, m) Representative Western blots and c, d, k, l, n) subsequent densitometric analysis of control and IPAH hPASMCs after Juglone treatment. f, p, s) Representative Western blots and g, h, q, r, t) subsequent densitometric analysis of control and IPAH hPAECs. PARP: poly (ADP-ribose) polymerase; PCNA: proliferating cell nuclear antigen. *: p<0.05; **: p<0.01; ***: p<0.001 versus dimethyl sulfoxide (DMSO)-treated control cells. Statistical analysis was performed using one-way ANOVA with Newman–Keuls post hoc test for multiple comparisons. Data from three independent experiments are presented as mean± sem .

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were either obtained from the Universities of Giessen and Marburg Lung Center Giessen Biobank, member of DZL Platform Biobanking or purchased from Lonza (Basel, Switzerland).

Techniques: In Vitro, TUNEL Assay, Concentration Assay, Control, Western Blot

Journal: The European Respiratory Journal

Article Title: Targeting peptidyl-prolyl isomerase 1 in experimental pulmonary arterial hypertension

doi: 10.1183/13993003.01698-2021

Figure Lengend Snippet: Peptidyl-prolyl cis/trans isomerase, NIMA interacting 1 (Pin1) controls the activity of multitude of transcription factors. a) Control and idiopathic pulmonary arterial hypertension (IPAH) human pulmonary artery smooth muscle cells (hPASMCs) after 24 h of serum starvation were subjected to platelet-derived growth factor (PDGF)-BB (50 ng·mL −1 ), epidermal growth factor (EGF) (5 ng·mL −1 ) and growth medium (GM) with 5% fetal bovine serum (FBS). Intracellular Pin1 levels were monitored by ELISA. *: p<0.05, ****: p<0.0001 versus control PASMCs; ## : p<0.01, #### : p<0.0001 versus IPAH hPASMCs; §§ : p<0.01 IPAH hPASMCs versus control hPASMCs. Statistical analysis was performed using one-way ANOVA with Newman–Keuls post hoc test for multiple comparisons. Data from three independent experiments are presented as mean± sem . b) Pin1-silenced and Juglone-treated hPASMCs were stimulated with GM for 24 h and nuclear protein extracts were used for transcription factor activation profile array, presented as log-transformed signals in a volcano plot. c) Log-transformed scatter plot of combined transcription factor activation/inactivation in Pin1-silenced and Juglone-treated hPASMCs. Data from two independent experiments are presented. d, f) Western blots and e, g) subsequent densitometry analyses of hypoxia-inducible factor (HIF)-1α and C/EBPα transcription factors in Pin1-silenced control and IPAH hPASMCs subjected to hypoxia for 24 h. h) Hypoxia-responsive element (HRE) luciferase activity in Pin1-silenced hPASMCs after 24 h of hypoxia. Scr: scrambled; ns : nonsignificant. *: p<0.05; ****: p<0.0001 for normoxia (NOX) si Scr versus hypoxia (HOX) si Scr; § : p<0.05; §§§§ : p<0.0001 for HOX si Scr versus HOX si Pin1. Data from three independent experiments are presented as mean± sem .

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were either obtained from the Universities of Giessen and Marburg Lung Center Giessen Biobank, member of DZL Platform Biobanking or purchased from Lonza (Basel, Switzerland).

Techniques: Activity Assay, Control, Derivative Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Transformation Assay, Western Blot, Luciferase