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. 2020 Mar 3;10(3):392. doi: 10.3390/biom10030392

Figure 6.

Figure 6

Mutp53 knockdown abrogates the Zn (II)-curc-induced ER stress. (a) U373 cells were transfected with control pSuper (si-ctr) and pSuper-p53 (si-p53) vectors for p53 knockdown and 36 h after transfection, cells were treated with Zn (II)-curc (100 µg/mL) for 16 h, before undergoing ER-Red Fluorescence staining. Quantization of ER content in U373 cells from ER-Red Fluorescence-stained cells as evaluated by the mean fluorescence intensity (MFI) of each individual cell normalized to cell size and expressed as fold-change compared with untreated cells at the same time point. Histograms represent the mean ± SD of three independent experiments. * p ≤ 0.05. (b) Western blot analysis of BiP and p53 protein levels in U373 and HT29 cells transfected for 36 h with si-ctr and si-p53, and then treated with Zn (II)-curc (100 µg/mL) for 24 h. Densitometric analysis was performed using Image J software to calculate the ratio of BiP and p53 protein levels vs. β-actin, as indicated ns: not specific signal. (c) Total mRNA was extracted from U373 cells treated as in (b), and spliced (s) and unspliced (u) Xbp1 gene expression were assayed by the PCR of reverse-transcribed cDNA. (upper panel) Densitometric analysis was performed using Image J software to calculate the Xbp1s/28S ratio. Histograms represent the mean ± SD of three independent experiments. * p ≤ 0.05.