Figure 4
Figure 4. Silencing of endogenous THAP1 inhibits EC proliferation, S-phase DNA synthesis, and G1/S cell-cycle progression. (A) siRNA-mediated knock-down of THAP1 mRNA in primary human ECs. Levels of THAP1 mRNA were analyzed by qPCR at different time points after transfection of siTHAP1 or siLuc control siRNAs (20 nM final concentration). (B) siRNA-mediated silencing of THAP1 protein in primary human ECs. Levels of THAP1 and αtubulin proteins were analyzed by Western blot at different time points after transfection of siTHAP1 or siLuc control siRNAs (20 nM final concentration). (C) Knock-down of THAP1 in primary human ECs inhibits S-phase DNA synthesis. The graph shows the percentage of BrdU+ cells in HUVECs transfected with siLuc, siTHAP1 (pool), siTHAP1-1, siTHAP1-2, siTHAP1-3, or siTHAP1-4 siRNAs. At least 500 cells were counted for each condition. Results are the mean of 2 independent experiments performed 48 hours after siRNA transfection. (D,E) siRNA-mediated knock-down of THAP1 inhibits proliferation of primary human ECs. Representative photos (D) of HUVECs treated with siLuc or the 4 individual THAP1 siRNAs are shown. Cell count assay (E) showing inhibition of proliferation in ECs treated with the 4 individual THAP1 siRNAs compared with untreated cells or ECs treated with siLuc control siRNA. Results are the mean of 3 independent experiments. (F,G) Knock-down of THAP1 in primary human ECs inhibits G1/S cell-cycle progression. Flow cytometry analysis of cell-cycle distribution (F) in HUVECs transfected with siLuc, siTHAP1-1, or siTHAP1-3 siRNAs. Black area represents cells in S phase, and gray area represents cells in G1 and G2/M phases. The graph shows the percentage of cells in G1, S, and G2/M phases of the cell cycle (G) in HUVECs transfected with siLuc, siTHAP1-1, or siTHAP1-3 siRNAs. Results are the mean of 3 independent experiments performed 48 hours after siRNA transfection. (H) Apoptosis levels were quantified in HUVECs transfected with siLuc, siTHAP1-1, or siTHAP1-3 siRNAs. TUNEL labeling of apoptotic nuclei was performed 48 hours after siRNA transfection, and at least 500 cells were counted for each condition. Results are the mean of 3 independent experiments. Error bars in panels C, G, H indicate SD.

Silencing of endogenous THAP1 inhibits EC proliferation, S-phase DNA synthesis, and G1/S cell-cycle progression. (A) siRNA-mediated knock-down of THAP1 mRNA in primary human ECs. Levels of THAP1 mRNA were analyzed by qPCR at different time points after transfection of siTHAP1 or siLuc control siRNAs (20 nM final concentration). (B) siRNA-mediated silencing of THAP1 protein in primary human ECs. Levels of THAP1 and αtubulin proteins were analyzed by Western blot at different time points after transfection of siTHAP1 or siLuc control siRNAs (20 nM final concentration). (C) Knock-down of THAP1 in primary human ECs inhibits S-phase DNA synthesis. The graph shows the percentage of BrdU+ cells in HUVECs transfected with siLuc, siTHAP1 (pool), siTHAP1-1, siTHAP1-2, siTHAP1-3, or siTHAP1-4 siRNAs. At least 500 cells were counted for each condition. Results are the mean of 2 independent experiments performed 48 hours after siRNA transfection. (D,E) siRNA-mediated knock-down of THAP1 inhibits proliferation of primary human ECs. Representative photos (D) of HUVECs treated with siLuc or the 4 individual THAP1 siRNAs are shown. Cell count assay (E) showing inhibition of proliferation in ECs treated with the 4 individual THAP1 siRNAs compared with untreated cells or ECs treated with siLuc control siRNA. Results are the mean of 3 independent experiments. (F,G) Knock-down of THAP1 in primary human ECs inhibits G1/S cell-cycle progression. Flow cytometry analysis of cell-cycle distribution (F) in HUVECs transfected with siLuc, siTHAP1-1, or siTHAP1-3 siRNAs. Black area represents cells in S phase, and gray area represents cells in G1 and G2/M phases. The graph shows the percentage of cells in G1, S, and G2/M phases of the cell cycle (G) in HUVECs transfected with siLuc, siTHAP1-1, or siTHAP1-3 siRNAs. Results are the mean of 3 independent experiments performed 48 hours after siRNA transfection. (H) Apoptosis levels were quantified in HUVECs transfected with siLuc, siTHAP1-1, or siTHAP1-3 siRNAs. TUNEL labeling of apoptotic nuclei was performed 48 hours after siRNA transfection, and at least 500 cells were counted for each condition. Results are the mean of 3 independent experiments. Error bars in panels C, G, H indicate SD.

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