Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Docetaxel Reverses Pulmonary Vascular Remodeling by Decreasing Autophagy and Resolves Right Ventricular Fibrosis

doi: 10.1124/jpet.117.239921

Figure Lengend Snippet: Microtubule-disturbing drugs, including DTX, are effective in killing proliferating PASMCs. Proliferating/synthetic phenotype (A) and differentiated/contractile phenotype of human PASMCs (B) were treated with various antitumor drugs at 1 µM for 24 hours. Cell number was determined by counting on a hemocytometer. Equal amounts of water (for daunorubicin) and 0.1% dimethylsulfoxide (DMSO; for other drugs) were used as vehicle controls. Symbols a and b denote significantly different from DMSO and water, respectively (n = 6–9) at P < 0.05. (C) Representative photographs of control and DTX-treated PASMCs. (D–F) Proliferating/synthetic human PASMCs were treated with DTX, paclitaxel, or vincristine for 24 hours. The number of viable cells was monitored by using Cell Counting Kit-8.

Article Snippet: Human PASMCs and PAECs were purchased from ScienCell Research Laboratories (Carlsbad, CA) and Cell Applications, Inc. (San Diego, CA) and were cultured in accordance with the manufacturers’ instructions in 5% CO 2 at 37°C.

Techniques: Cell Counting

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Docetaxel Reverses Pulmonary Vascular Remodeling by Decreasing Autophagy and Resolves Right Ventricular Fibrosis

doi: 10.1124/jpet.117.239921

Figure Lengend Snippet: Inhibition of autophagy potentiates DTX-induced death of PASMCs. (A) Proliferating/synthetic human PASMCs were pretreated with dimethylsulfoxide (DMSO; 0.5%), SBI-0206965 (50 μM), or Z-VAD-FMK (50 μM) for 30 minutes and then treated with DMSO (0.1%) or DTX (50 nM) for 22 hours. The number of viable cells was monitored by using Cell Counting Kit-8 at absorbance 450 nm (A450). (B) Cells were transfected with siRNA for beclin-1 or control scrambled siRNA for 2 days. Cells were then treated with DMSO or DTX (50 nM) for 22 hours, and cell number was counted using a hemocytometer. Western blotting results demonstrate the extent of siRNA knockdown of Beclin-1 (n = 6–9). (C) Cells were transfected with siRNA for LC3B or control scrambled siRNA. Cells were then treated with DMSO or DTX, and cell number was counted. Western blotting results demonstrate the extent of siRNA knockdown of LC3B (n = 5). (D) Cells were transfected with siRNA for p62 or control scrambled siRNA. Cells were then treated with DMSO or DTX, and cell number was counted. Western blotting results demonstrate the extent of siRNA knockdown of p62 (n = 6–9). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Human PASMCs and PAECs were purchased from ScienCell Research Laboratories (Carlsbad, CA) and Cell Applications, Inc. (San Diego, CA) and were cultured in accordance with the manufacturers’ instructions in 5% CO 2 at 37°C.

Techniques: Inhibition, Cell Counting, Transfection, Western Blot

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Docetaxel Reverses Pulmonary Vascular Remodeling by Decreasing Autophagy and Resolves Right Ventricular Fibrosis

doi: 10.1124/jpet.117.239921

Figure Lengend Snippet: Effects of DTX on autophagy in PASMCs. (A and B) Human PASMCs were treated with dimethylsulfoxide (DMSO; 0.1%) or DTX (50 nM) for 22 hours, and cell lysates were subjected to Western blotting to monitor LC3B-II and p62 levels (n = 5–7). (C) Rats were treated with SU5416/hypoxia and injected with saline or DTX. Protein levels of p62 were monitored by Western blotting in isolated PA homogenates (n = 7). *Significant difference between each other at P < 0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Human PASMCs and PAECs were purchased from ScienCell Research Laboratories (Carlsbad, CA) and Cell Applications, Inc. (San Diego, CA) and were cultured in accordance with the manufacturers’ instructions in 5% CO 2 at 37°C.

Techniques: Western Blot, Injection, Isolation

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Docetaxel Reverses Pulmonary Vascular Remodeling by Decreasing Autophagy and Resolves Right Ventricular Fibrosis

doi: 10.1124/jpet.117.239921

Figure Lengend Snippet: Effects of DTX on Beclin-1. (A) Proliferating/synthetic human PASMCs were treated with dimethylsulfoxide (DMSO; 0.1%) or DTX (50 nM) for 22 hours. Beclin-1 protein expression was monitored by Western blotting (n = 6). *Values that are significantly different from each other at P < 0.05. (B) Rats with PAH and control rats were treated with saline or DTX, and Beclin-1 protein expression was monitored in the homogenates of isolated PAs (n = 7). *Values that are significantly different from each other at P < 0.05. (C) Proliferating/synthetic human PASMCs were treated with DMSO or DTX (50 nM) for 22 hours. The beclin-1 mRNA expression was monitored by reverse-transcription polymerase chain reaction (n = 6). ns, values are not significantly different from each other at P < 0.05. (D) Human PASMCs were infected with adenovirus expressing Beclin-1 for 48 hours. Cells were then treated with DMSO (0.1%) or DTX (50 nM) for 22 hours. Beclin-1 protein expression was monitored by Western blotting (n = 3). *Values that are significantly different from each other at P < 0.05. (E) Human PASMCs were pretreated with MG132 (250 nM) for 6 hours and then treated with DMSO (0.1%) or DTX (50 nM) for 22 hours. Beclin-1 protein expression was monitored by Western blotting in cell lysates (n = 6). The symbol “a” denotes values that are significantly different from the DTX value at P < 0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Human PASMCs and PAECs were purchased from ScienCell Research Laboratories (Carlsbad, CA) and Cell Applications, Inc. (San Diego, CA) and were cultured in accordance with the manufacturers’ instructions in 5% CO 2 at 37°C.

Techniques: Expressing, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Infection

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Docetaxel Reverses Pulmonary Vascular Remodeling by Decreasing Autophagy and Resolves Right Ventricular Fibrosis

doi: 10.1124/jpet.117.239921

Figure Lengend Snippet: The identification of a protein that interacts with Beclin-1 in response to DTX. (A) Proliferating/synthetic human PASMCs were treated with dimethylsulfoxide (DMSO; 0.1%) or DTX (50 nM) for 24 hours. Cell lysates were subjected to immunoprecipitation with rabbit Beclin-1 IgG or normal rabbit IgG, SDS-PAGE, and Coomassie Blue staining. The arrow indicates a band that is consistently upregulated by DTX. (B) Immunoprecipitated (IP) samples with the Beclin-1 IgG were Western blotted (WB) with goat MYH9 IgG (n = 6). *Values that are significantly different from each other at P < 0.05. (C) PA homogenates from rats with PAH treated with saline or DTX were immunoprecipitated with goat MYH9 IgG and subjected to Western blotting with rabbit Beclin-1 IgG (n = 4). *Values that are significantly different from each other at P < 0.05. (D) Human PASMCs were transfected with siRNA for MYH9. The extent of the MYH9 knockdown was determined by Western blotting. (E) Human PASMCs with MYH9 knocked down were treated with DMSO or DTX. Cell number was counted on a hemocytometer (N = 6). The symbol “a” denotes values that are significantly different from the control siRNA + DTX value at P < 0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Human PASMCs and PAECs were purchased from ScienCell Research Laboratories (Carlsbad, CA) and Cell Applications, Inc. (San Diego, CA) and were cultured in accordance with the manufacturers’ instructions in 5% CO 2 at 37°C.

Techniques: Immunoprecipitation, SDS Page, Staining, Western Blot, Transfection

Journal: International Journal of Molecular Medicine

Article Title: Reversal of the Warburg effect with DCA in PDGF-treated human PASMC is potentiated by pyruvate dehydrogenase kinase-1 inhibition mediated through blocking Akt/GSK-3β signalling

doi: 10.3892/ijmm.2018.3745

Figure Lengend Snippet: Effect of DCA, LY294002 or combination of DCA and LY294002 on the growth of human PASMCs. The human PASMCs were seeded in 96-well plates in RPMI-1640 medium supplemented with 10% FBS followed by 48 h serum starving prior to exposure to 20 ng/ml PDGF or with increased concentrations of (A) DCA or (B) LY294002 in fresh culture medium with 0.5% foetal bovine serum for 72 h. * P<0.05, ** P<0.01 and *** P<0.001 vs. PDGF-treated cells; #P<0.05 and ## P<0.01 vs. cells treated with 5 mM DCA or 5 µ M LY294002. (C) Cells were exposed to PDGF or 5 µ M LY294002, 10 or 20 mM DCA and combination of 5 µ M LY294002 and DCA (10 mM) for 24 h. * P<0.05, ** P<0.01 as compared with PDGF cells. The data are presented as the mean ± standard deviation of 6 duplicated wells in three separate experiments. PASMCs, pulmonary arterial smooth muscle cells; DCA, dichloroacetate; PDGF, platelet-derived growth factor; D5, D10, D20 and D50, cells treated with dichloroacetate at 5, 10, 20, 50 mM, respectively, following PDGF exposure. L5, 10 and 20, cells treated with LY294002 at 5, 10, 20 µ M, respectively, following PDGF exposure. L5D10, cells treated in a combination of 5 µ M LY294002 and 10 mM dichloroacetate following PDGF exposure.

Article Snippet: Human PASMCs were purchased from Wuxi BioHermes Biomedical Technology Co., Ltd (Wuxi, China).

Techniques: Standard Deviation, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: Reversal of the Warburg effect with DCA in PDGF-treated human PASMC is potentiated by pyruvate dehydrogenase kinase-1 inhibition mediated through blocking Akt/GSK-3β signalling

doi: 10.3892/ijmm.2018.3745

Figure Lengend Snippet: Effect of DCA, LY294002 or combination of DCA and LY294002 on the apoptosis and mitochondria membrane potential of human PASMCs. The PASMCs were seeded into 25 cm 2 tissue culture flask at a density of 5×10 5 cell/flask and cultured in RPMI-1640 complete culture medium for 16 h followed by serum starvation for 24 h. The cells were then exposed to PDGF alone or 5 µ M LY294002, DCA at 10 mM or a combination of 5 µ M LY294002 and 10 mM DCA for 48 h prior to (A) apoptosis or (B) JC-1 assay. (C) The expression levels of caspase-3 and cleaved caspase-3 were analyzed with western blot analysis. The representative change of one of the three experiments is presented, as all assays exhibited identical results. (D) Results were pooled from three separate experiments and are presented as mean ± standard deviation. * P<0.05, ** P<0.01 and *** P<0.001 vs. control cells. ## P<0.01 and ### P<0.001 vs. cells treated with PDGF. C, control; PASMCs, pulmonary arterial smooth muscle cells; DCA, dichloroacetate; PDGF, platelet-derived growth factor; D5 and D10, cells treated with DCA at 5 and 10 mM, respectively, following PDGF exposure; L5, cells treated with LY294002 at 5 µ M following PDGF exposure; L5D10, cells treated with a combination of 5 µ M LY294002 and 10 mM DCA following PDGF exposure.

Article Snippet: Human PASMCs were purchased from Wuxi BioHermes Biomedical Technology Co., Ltd (Wuxi, China).

Techniques: Membrane, Cell Culture, Expressing, Western Blot, Standard Deviation, Control, Derivative Assay