Fig. 4

The direct addition of DEIX inhibits LPS-mediated oxidative stress in hPDLCs and stimulates in vitro wound healing of the cells. (A) CCK-8 assay results showing the proliferation rate (%) of hPDLCs depending on the indicated concentrations (0–50 µM) of DEIX 24 h after incubation (n = 7). (B) Proliferation rate (%) of hPDLCs exposed to LPS (2 µg/mL) and/or DEIX (20 µM) or not to them for 24 h (n = 7). (C) IF assay-derived CLSM images showing the expression of Ki-67 in hPDLCs in relation to the presence and absence of LPS, DEIX, or both 24 h after the incubation (Bar = 10 μm). (D) Ki-67-positive cells (%)/100 counted cells in the hPDLCs untreated or exposed to LPS, DEIX, or both (n = 5). (E) Flow cytometric histograms showing the DCF-specific signals of hPDLCs 24 h after the incubation with and without LPS, DEIX, or both. (F) LPS-mediated increase of DCF-positive hPDLCs (%) and its inhibition by adding DEIX are shown (n = 4). (G) Western blot image of γ-H₂AX that is expressed in the hPDLCs, untreated or exposed to LPS, DEIX, or both for 24 h, along with its expression ratio to that of the loading control, β-actin (n = 4). Original blots are presented in Figure S4. (H) In vitro wound healing images 24 h after the incubation (Bar = 50 μm). (I)The areas (%) of migrated hPDLCs in the scratched regions (n = 4). (J) The RT-qPCR results showing the expression patterns of vimentin, scleraxis, and periostin in the hPDLCs exposed or not to LPS, DEIX, or both for 24 h (n = 4). The letters in panel A indicate significant differences at p < 0.05 among the cells by one-way ANOVA followed by Tukey’s multiple comparisons test. ^*p < 0.05 and ^**p < 0.01 in panel B were determined by an unpaired Student’s t-test. ^*p < 0.05 and ^**p < 0.01 in panels D, F, G, I, and J were calculated using an unpaired non-parametric test (Kolmogorov-Smirnov test). ns, not significant.