Fig 2: Pemafibrate attenuates VSMC proliferation in vivo. Endothelial denudation injuries were induced in the left femoral artery of mice maintained on normal chow (A, B) or a HFD (C, D). (A, C) Sections were subjected to immunohistochemistry for PCNA (red) and counterstained with DAPI to visualize nuclei (blue). Magnification, 630×. (B, D) PCNA-positive cells were quantified by analyzing the fraction of stained cells to the total number of nuclei (n = 5). Data are the mean ± SEM. An unpaired t-test was performed to calculate statistical significance (*P < 0.05 vs control).

Fig 3: The HTR-8/SVneo cell cycle was not changed by CNTF treatment. Graphs representing untreated (a) and CNTF-treated (a’) HTR-8/SVneo cells. The area under the green curve represents the G0/G1 phases, the red curve the S phase and the blue curve the G2/M phases of the cell cycle. a’’ Histograms represent the percentage of the cells in each cell cycle phase and show that there were no significant differences in the cell cycle phases between treated and untreated cells. b PCNA Western blots of HTR-8/SVneo cells, untreated and CNTF-treated. Right, b’ histograms showing band quantification. There were no significant differences between untreated and CNTF-treated cells. Bands were normalized using ß-actin. Results are expressed in arbitrary units (A.U.) and reported as histogram bars. Data are mean ± SD (n = 3)

Fig 4: Proposed mechanism of action of palbociclib and dinaciclib in PAH. Multiple growth factors, cytokines, and mitogens induce the activation of cyclin-dependent kinases (CDKs), e.g., by increasing the expression of cyclin D1. Palbociclib specifically targets active complexes consisting of cyclin D1 and CDK4 or CDK6, whereas dinaciclib blocks the activity of the cyclin E-CDK2 complex. This leads to reduced levels of phosphorylated Rb protein and, as a consequence, transcriptional repression of E2F downstream target genes, e.g., Cyclin A2, CDK1, and PCNA. Thus, palbociclib (and dinaciclib) interfere with cell cycle progression from G1 into S phase, causing a reduction of proliferation of pulmonary vascular cells

Fig 5: Loss of replisome proteins from chromatin in ATR-deficient cells. (A) Western blot detection of total cellular and chromatin-bound proteins as indicated. (B) Western blot detection of SUMO2/3 in chromatin lysates and quantification by optical density. APH was added to the cells as indicated, and chromatin-containing fractions were Western-blotted and detected for SUMO2/3. Note that Atr?/- cells consistently exhibited reduced SUMO2/3-conjugated proteins in chromatin lysates after 6 h of APH treatment. (C) Western blot detection of SUMO2/3 in chromatin lysates from RNF4-suppressed cells. This experiment was performed similarly to that described in B, with the exception that RNF4 levels were suppressed by shRNA targeting. Note that while the amplitude of SUMO2/3 presence was variable using crude nuclear-insoluble preparations, the changes shown represent the average effects of the experiments (n = 14). RNF4 suppression prevents the loss of SUMO2/3 conjugates on chromatin in Atr?/- cells. (D) Western blot detection of PCNA in chromatin lysates from the indicated cells treated with MG132 or left untreated. Loss of PCNA in Atr?/- cells was consistently mitigated by the addition of MG132.