Figure 5

ATF-4 protects cells from cellular stress.

HaCaT cells were transfected with ATF-4 siRNA and were treated with different laser doses. Cellular viability (a) (n = 3) was assessed at 24 hrs. (b) Conversely, over expressing ATF-4 stable HaCaT cells was treated with increasing doses of laser and cellular viability was assessed. Statistical significance was determined using two-way ANOVA among different treatments using the Bonferroni’s multiple comparison test (n = 3). (c) Outline shows HaCaT cells treated with phototoxic dose of laser that generates two populations, surviving cells that remain adherent (A) and dying or dead cells that float (F) in media. These were collected separately, lysed and immunoblotting (d) was performed to assess ER stress pathway. Histological assessment of mice skin showing damaged area in laser-treated skin was performed by immunohistochemistry for ATF-4 (e,f), HSP70 (g,h) and (i,j) TUNEL positivity. (Scale bar = 70 μm). Quantitation of high power images from mice sections are plotted (n = 5). Insets show high power magnifications. Images in a gel are cropped (horizontally) and placed together for better clarity of results and were run in the same experimental conditions. Significance was assessed with paired Student’s t-test. Statistical significance are indicated as P < 0.05 (*), <0.001 (**), <0.0001 (***) and <0.00001(****).