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Fig. 6 | Journal of Experimental & Clinical Cancer Research

Fig. 6

From: The non-canonical mechanism of ER stress-mediated progression of prostate cancer

Fig. 6

Alcohol-induced ER stress in PCa cells. (A) Representative IF images of giantin (green) and ATF6 (red) in LNCaP cells: control and treated with 50 mM EtOH for 96 h; bars, 10 μm. (B) Quantification of the ratio of nuclear/cytoplasmic ATF6 IF in cells presented in A (N = 3; **P < 0.001, t test). (C) ATF6 W-B of the nuclear fraction from cells presented in A; lamin B1 is a loading control. (D) GCC185, GRP78, and ATF6 W-B of the lysates from the cells presented in A; β-actin is a loading control. (E) Calreticulin and HSP90 W-B of the lysates from the cells presented in A; β-actin is a loading control. (F and G) S1P (F) and S2P (G) W-B of the ER fraction isolated from the cells presented in A; HSP70 is a loading control. (H and I) IF staining of LNCaP cells to evaluate colocalization of S1P (red) and S2P (red) with GCC185 (green); bars, 10 μm. White boxes indicate area magnified at the right. (J) Quantification of the Pearson coefficient of colocalization for the cells presented in H and I (N = 90 cells from three repeats; **P < 0.001, t test). (K) AR W-B of the nuclear fraction from LNCaP cells: control, treated with ETOH, and ATF6 siRNA followed by EtOH. (L) HDAC6-P and HSP90-Ac W-B of the lysate of cells from K. (M) Anchorage-independent colony formation was measured by the soft agar assay for LNCaP cells: control, treated with ETOH, and ATF6 siRNA followed by EtOH; bars, 200 μm. (N) Quantification of the number of colonies in 20 randomly selected areas for cells from M (N = 3; **P < 0.001, t test). (O) Representative LNCaP xenograft tumors from control and EtOH-fed mice. (P) The tumor growth is presented as a ratio of tumor weight (g)/number of days after injection; medians (min – max); N = 9 mice from each group; ***P < 0.05, unpaired t-test). (Q) ATF6 immunostaining (red) of the tissue sections from xenograft tumors presented in O. (R) Quantification of the ratio of nuclear/cytoplasmic ATF6 IF in cells presented in Q (means ± SD; N = 3; **P < 0.001, t test). All images were acquired with the same imaging parameters

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