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. 2022 Feb 8;23(3):1896. doi: 10.3390/ijms23031896

Figure 2.

Figure 2

Exposure to b-LED causes a transient block in cell proliferation. (A,B) Cell viability test of HaCaT cells exposed to b-LED. HaCaT cells were pretreated with calcein AM (2 µM) and exposed to b-LED for 4 h. Cells were then analyzed 4, 24 and 48 h after beginning of exposure to b-LED. Cells were fixed and stained with DAPI. Images were acquired by fluorescence microscopy. (A) Calcein AM/DAPI-positive cells; (B) calcein AM-positive cells. Bars represent the mean ± SEM from two independent experiments. Twelve fields containing at least 30 nuclei per field were analyzed. * p < 0.05; ** p < 0.01. (C) Western blot showing the expression of cleaved caspase 3 in HaCaT cells exposed to b-LED for 4 h. Cells were analyzed 4, 24 and 48 h after beginning of exposure to b-LED. Western blot analysis was performed on total lysates. Tubulin was detected as a loading control. Data are representative of three independent experiments. (D) Cell proliferation assay of HaCaT cells exposed to b-LED. HaCaT cells were left untreated or were exposed to b-LED for 4 h and were then evaluated for cell proliferation assays 4, 24 and 48 h after beginning of exposure to b-LED using a SpectraMax 13 microplate reader. The experiment was performed in triplicate and was repeated twice. * p < 0.05; ** p < 0.01; ns, not significant. (E–H) HaCaT cells exposed to b-LED for 4 h and then were analyzed 4 and 24 h after beginning of exposure. (E) Quantitative RT-PCR expression analysis of p21 in. (F) Western blot expression of p21 in HaCaT cells. (G) quantitative RT-PCR expression analysis of Cyclin D1 in HaCaT cells. (H) Western blot expression of Cyclin D1 in HaCaT cells. Quantitative RT-PCR was performed on total RNA. L34 mRNA levels were used for normalization. Results are expressed in terms of fold change; bars represent the mean ± SEM of duplicate determinations in four independent experiments. * p < 0.05; *** p < 0.001 and ns, not significant. Western blot analysis was performed on total lysates. Tubulin was detected as a loading control. Data are representative of three independent experiments.