Fig 1.
Expression of SESN2 in the skin.
Human skin sections (A, B) or in vitro cultured primary human dermal fibroblasts (C, D) were immunolabeled with anti-SESN2 (red) either without (A, C) or with (B, D) preabsorption of the antibody with the antigen. Inhibition of labeling by antigen preabsorption is a negative control reaction to confirm the specificity of the antibody. Insets in A and B show higher magnification of dermal cells from the boxed areas of the sections. The dermo-epidermal junction and the surface of the epidermis are indicated by dotted lines. Bars: A and B, 100 μm; C and D, 50 μm.
Fig 2.
Compensatory upregulation of SESN2 upon knockdown of SESN1 in fibroblasts.
Human primary fibroblasts were cultured in triplicates and transfected with siRNAs directed against SESN1 or SESN2. 48 h after the transfection, cells were harvested, RNA was extracted, transcribed into cDNA, and subjected to quantitative PCRs for SESN1 (A) as well as SESN2 (B). Arbitrary units (a.u.) were calculated by normalizing the mRNA levels of SESN1 (A) or SESN2 (B) to B2M levels. Values relative to the expression levels in untreated cells are shown. Error bars indicate standard deviations. Student’s t-test was performed for comparisons between each treatment and the control siRNA treatment. *p < 0.05, **p < 0.01, ***p < 0.001. SESN2 protein expression was determined by Western blot analysis (C). The band intensities, normalized to the intensities of the GAPDH bands on the same blot and relative to the control siRNA-treated samples, are indicated below the blot. The results are representative for three independent experiments using cells from different donors.
Fig 3.
UVB induces SESN2 protein in isolated keratinocytes and skin explants.
Human skin explants (A) and cultured keratinocytes (KC) (B) were irradiated with the indicated doses of UVA (J/cm2) or UVB (mJ/cm2) or treated with UVA-induced oxidized phospholipids (UVA-oxPL, μg/ml). 24 h after treatment, cells were lyzed and Western blots for SESN2 and GAPDH were performed. Bands at the predicted sizes of SESN2 and GAPDH are indicated. An asterisk marks an unidentified band immunoreactive with anti-SESN2. The expression of SESN2 relative to GAPDH (lower panels) was normalized to the non-irradiated control. One of three independent experiments with similar results is shown.
Fig 4.
UVB induces upregulation of SESN1 and SESN2 via p53.
Fibroblasts (FB) were treated with p53 siRNA, 48 h after transfection fibroblasts were irradiated with UVB 20 mJ/cm2. RNA was extracted 12 h later to monitor the expression levels of the p53 target gene p21 (A), SESN1 (B) and SESN2 (C) at the mRNA level by RT-qPCR, relative to untreated cells, normalized to B2M. Error bars indicate standard deviations. For comparisons of untransfected non-irradiated versus untransfected UVB-irradiated cells and siRNA-treated versus control siRNA-treated cells (each within the non-irradiated and the irradiated groups), Student’s t-test was calculated, with p<0.05 being considered significant. *p < 0.05, **p < 0.01, ***p < 0.001. Protein lysates were prepared 8 h (D) and 24 h (E) post-irradiation to analyze the expression of p53 (D) and SESN2 (E) by Western blot. In each case, the membrane was re-probed with anti-GAPDH. The expression levels of SESN2 (relative to the non-irradiated control siRNA transfected cells, normalized to GAPDH) are indicated. One representative experiment of three independent experiments with cells of different donors is shown.