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. 2022 Nov 3;12:18636. doi: 10.1038/s41598-022-19850-w

Figure 4.

Figure 4

PF4 is internalized and degraded by hGFs. (A) Serum-starved cells were cultured in the absence or presence of recombinant PF4 at the indicated concentrations. Whole cell lysates were resolved by SDS-PAGE and the immunoblots were probed with an antibody against PF4. GAPDH is shown as a loading control. (B) Confocal micrographs illustrate the intracellular distribution of PF4 (green), DAPI (blue), and actin (red) in cells treated with recombinant PF4 (1 µg/mL) for 24 h prior to fixation. Cells were imaged with a Leica SP5 laser scanning confocal microscope. Bar = 20 µm. (C) Cells were treated with PF4 (10 µg/mL) for the indicated time periods. Cell lysates were resolved by SDS-PAGE and the immunoblots were probed with an antibody against PF4. β-actin is shown as a loading control. (D) Confocal micrographs illustrate the intracellular distribution of PF4 (green, top panels) and PF4/actin (green/red, bottom panels) in cells cultured in the absence (−) or presence (+) of recombinant PF4 for the indicated times prior to fixation and processing for microscopy. Slides were imaged with Zeiss spinning disk confocal microscope. Bar = 20 µm. (E) Serum-starved cells were cultured in the presence of recombinant PF4 (10 µg/mL) for 30 min (0.5 h) prior to replacement with PF4-free medium. Cells were harvested at the indicated time points. Cell lysates were resolved by SDS-PAGE and immunoblots probed with an anti-PF4 antibody. β-actin is shown as a loading control. (F) Top: Schematic illustration of the experimental strategy (medium change vs. no medium change): Scenario (i)—Medium change: cells were cultured in the absence (−) or presence (+) of recombinant PF4 for 30 min (0.5 h) prior to medium replacement with PF4-free medium. Scenario (ii)—No medium change: cells were cultured either in the absence (−) or presence (+) of recombinant PF4 for the duration of the 24 h time course. Cell culture supernatants were harvested, and cell lysates prepared, after 24 h. Bottom: Cell lysates were resolved by SDS-PAGE and immunoblots probed with anti-PF4 antibody. β-actin is shown as a loading control. (G) Top: Bar graph depicts the intracellular MMP-2 (expressed as the MMP-2:β-actin ratio) in cells cultured in the absence (-PF4, black bars) or presence (+ PF4, white bars) of recombinant PF4, both following withdrawal of PF4-containing medium (Medium change) or with the continued presence of PF4 (No change). The MMP-2:β-actin ratio of the untreated controls (-PF4, Medium change) was set at 1. Data are mean ± SD and represent three independent experiments. **, p < 0.01, ***, p < 0.001, based on two-way ANOVA and Tukey’s post-hoc multiple comparison tests. Bottom: Cell lysates were resolved by SDS-PAGE and immunoblots probed with anti-MMP-2 antibody. β-actin is shown as a loading control.