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. 2021 Jun 7;9:675486. doi: 10.3389/fcell.2021.675486

FIGURE 2.

FIGURE 2

Silencing of ATF4 suppressed Ihh expression and human NP cell degeneration. NP cells were treated with low dose (10 ng/ml) or high dose (20 ng/ml) of TNF-α for 3 days to induce degeneration and transfected with siATF4 to silence the ATF4 gene expression. (A) eIF2α, p-eIF2α, ATF4, Ihh, and MMP13 protein levels were assessed by WB, (B) measured by densitometric analyses, and expressed as folds relative to control. (C) Representative images of IF staining with Col-II and Col-X and measured by densitometric analyses and expressed as folds relative to control (scale bar = 100 μm). Data are presented as means ± SD (n = 7). *p < 0.05; **p < 0.01; ***p < 0.001; the orange column indicates control NP cells without any treatments, the green column indicates NP cells treated with 10 ng/ml of TNF-α, the blue column indicates NP cells treated with 20 ng/ml of TNF-α, and the gray column indicates siATF4 transfected NP cells treated with 20 ng/ml of TNF-α. ATF4, activating transcription factor 4; Ihh, Indian hedgehog; NP, nucleus pulposus; TNF, tumor necrosis factor; p-eIF2α, phosphorylated eukaryotic initiation factor-2α; eIF2α, eukaryotic initiation factor-2α; MMP13, matrix metalloproteinase-13; WB, western blot; IF, immunofluorescence; Col-II, collagen II; Col-X, collagen X.