Fig 1: Knockdown of FOXM1 alleviated UV irradiation-mediated dysfunction of keratinocytes. (a) si-NC and si-FOXM1 were transfected into HaCaT cells and transfection efficiency was measured by RT-PCR after 24 hours. The transfected HaCaT cells were processed by UV irradiation and then treated with curcumin (5 µM) for 24 hours. (b) CCK-8 was employed to examine the proliferation of UV-irradiated HaCaT cells. (c)–(e) The contents of SOD, GSH-PX, and MDA in UV-irradiated HaCaT cells were evaluated using the Oxidative Stress Assay Kit. (f) Cytofluorimetry was applied to assay the level of ROS in UV-irradiated HaCaT cells. (g) The expression of inflammatory cytokines in UV-irradiated HaCaT cells was monitored by RT-PCR. (h) WB was adopted to verify the expression of apoptosis-related proteins in UV-irradiated HaCaT cells. (i) WB gauged the expression of oxidative stress-related proteins in UV-irradiated HaCaT cells. (j) The profiles of inflammatory response proteins in UV-irradiated HaCaT cells were compared by WB. (k) WB assessed the SPAG5/FOXM1 pathway expression in UV-irradiated HaCaT cells. N = 3. *p < 0.05 (vs. si-NC or UV group), **p < 0.01, ***p < 0.001; #p < 0.05 (vs. si-FOXM1+UV group), ##p < 0.01, ###p < 0.001.
Fig 2: Overexpression of FOXM1 stimulated UV irradiation-mediated dysfunction of keratinocytes. (a) Vectors and FOXM1 overexpression plasmids were transfected into HaCaT cells, and the transfection efficiency was inspected by RT-PCR after 24 hours. The transfected HaCaT cells were then subjected to UV irradiation and treated with 5 μM curcumin for 24 hours. (b) UV-irradiated HaCaT cell proliferation was assayed using CCK-8. (c)–(e) Levels of SOD, GSH-PX, and MDA in UV-irradiated HaCaT cells were compared using the Oxidative Stress Assay Kit. (f) The amount of ROS in UV-irradiated HaCaT cells was determined by cytofluorimetry. (g) ELISA was implemented to verify the profiles of inflammatory factors (IL-1β, IL-6, IL-18, TNFα) in UV-irradiated HaCaT cells. (h)–(k) Expression of Bax, Bcl-xL, Caspase3, Caspase8, Caspase9, Keap1, Nrf2, HO-1, COX2, iNOS, NF-κB, MMP1, MMP9, SPAG5, and FOXM1 in UV-irradiated HaCaT cells was examined with WB. N = 3. ∗p < 0.05 (vs. vector or UV group), ∗∗p < 0.01, ∗∗∗p < 0.001; #p < 0.05 (vs. FOXM1+UV group), ##p < 0.01, ###p < 0.001.
Fig 3: Knockdown of SPAG5 ameliorated UV irradiation-mediated dysfunction of keratinocytes. (a) The si-NC and si-SPAG5 were transfected into HaCaT cells and the expression of SPAG5 was evaluated by RT-PCR 24 hours later. We then exposed the transfected HaCaT cells to UV irradiation and treated them with 5 µM curcumin for 24 hours. (b) UV-irradiated HaCaT cell proliferation was assayed with CCK-8. (c)–(f) The levels of SOD, GSH-PX, MDA, and ROS in UV-irradiated HaCaT cells were tested using an oxidative stress assay kit and cytofluorimetry. (g) Expression of inflammatory factors (IL-1ß, IL-6, IL-18, and TNFa) in UV-irradiated HaCaT cells was checked by RT-PCR. (h) Expression of apoptosis-related proteins (Bax, Bcl-xL, Caspase3, Caspase8, Caspase9) in UV-irradiated HaCaT cells was detected by WB. (i) WB was implemented to testify the profiles of oxidative stress-related proteins (Keap1, Nrf2, HO-1, COX2, and iNOS) in UV-irradiated HaCaT cells. (j) The expression of inflammatory response proteins (NF-?B, MMP1, and MMP9) in UV-irradiated HaCaT cells was evaluated by WB. (k) The SPAG5/FOXM1 pathway expression in UV-irradiated HaCaT cells was testified by WB. N = 3. *p < 0.05 (vs. si-NC or UV group), **p < 0.01, ***p < 0.001; #p < 0.05 (vs. si-SPAG5+UV group), ##p < 0.01, ###p < 0.001.
Fig 4: Overexpression of SPAG5 potentiated UV irradiation-mediated dysfunction of keratinocytes. (a) Vectors and SPAG5 overexpression plasmids were transfected into HaCaT cells, and the SPAG5 expression in HaCaT cells was assayed by RT-PCR 24 hours later. The transfected HaCaT cells were irradiated with UV and then treated with curcumin (5 µM) for 24 hours. (b) CCK-8 was applied to check the proliferation of UV-irradiated HaCaT cells. (c)–(f) Levels of SOD, GSH-PX, MDA, and ROS in UV-irradiated HaCaT cells were assessed using an oxidative stress assay kit and cytofluorimetry. (g) ELISA tested the expression of inflammatory factors IL-1ß, IL-6, IL-18, and TNFa in UV-irradiated HaCaT cells. (h)–(j) WB was conducted to investigate the profiles of apoptosis-associated proteins (Bax, Bcl-xL, Caspase3, Caspase8, Caspase9), oxidative stress-associated proteins (Keap1, Nrf2, HO-1, COX2, iNOS), and inflammatory response proteins (NF-?B, MMP1, MMP). (k) The expression of the SPAG5/FOXM1 pathway in UV-irradiated HaCaT cells was estimated by WB. N = 3. *p < 0.05 (vs. vector or UV group), **p < 0.01, ***p < 0.001; #p < 0.05 (vs. SPAG5+UV group), ##p < 0.01, ###p < 0.001.
Fig 5: Curcumin choked the SPAG5/FOXM1 pathway. (a) Expression of SPAG5 and FOXM1 in HaCaT cells was probed by WB. (b) RT-PCR was implemented to verify the profiles of SPAG5 and FOXM1 mRNA in HaCaT cells. N = 3. *p < 0.05 (vs. CON group), **p < 0.01, ***p < 0.001; #p < 0.05 (vs. UV group), ##p < 0.01, ###p < 0.001.
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