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