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. 2020 May 31;53(5):284–289. doi: 10.5483/BMBRep.2020.53.5.041

Fig. 3.

Fig. 3

Sulfasalazine decreases tamoxifen-induced autophagic cell death in human RPE cells. LC3B I and II expression in the primary H-RPE cells (A, n = 3) and ARPE-19 cells (B, n = 3) was assessed by western blotting at various points after vehicle, sulfasalazine, tamoxifen,or tamoxifen plus sulfasalazine administration. Also, β-actin was used as a control for normalization. This blot is representative of the three independent experiments. Also, *P < 0.05, increased ratio of LC3 II/I protein levels after treatment with tamoxifen vs vehicle. †P < 0.05, decreased ratio of LC3 II/I protein levels after treatment with tamoxifen plus sulfasalazine vs tamoxifen only. (C) The cell viability of the primary H-RPE cells (n = 12) was analyzed at 24 hours after treatment with vehicle, tamoxifen, tamoxifen plus an autophagy inhibitor (100 nM Baf-1), or tamoxifen plus sulfasalazine in the absence or presence of Baf-1. Additionally, †P < 0.05, decreased cell viability after treatment with tamoxifen vs vehicle. *P < 0.05, increased cell viability after treatment with vs tamoxifen only. (D) The mRNA levels of LC3B in the primary H-RPE cells (n = 3) were assessed at 12 hours after treatment with vehicle, tamoxifen, or tamoxifen plus NAC. *P < 0.05, increased mRNA levels of LC3B after treatment with tamoxifen vs vehicle. †P < 0.05, decreased mRNA levels of LC3B after treatment with tamoxifen plus NAC vs tamoxifen only. (E) The LC3B I and II expression in the primary H-RPE cells (n = 3) was assessed by western blotting at 24 hours after administration of vehicle, tamoxifen, tamoxifen plus NAC. And, β-actin was used as a control for normalization. This blot is representative of the three independent experiments. And, *P < 0.05, increased ratio of LC3 II/I protein levels after treatment with tamoxifen vs vehicle. †P < 0.05, decreased ratio of LC3 II/I protein levels after treatment with tamoxifen plus NAC vs tamoxifen only. (F) The mRNA levels of LC3B were analyzed in control and the LC3B shRNA-expressing cells at 12 hours after vehicle or rapamycin (1 μM) treatment. Additionally, *P < 0.05, increased the mRNA levels of LC3B after treatment with rapamycin vs vehicle. †P < 0.05, decreased the mRNA levels of the LC3B in LC3B shRNA-expressing cells vs in control shRNA-expressing cells. (G) The cell viability of the control or LC3B shRNA-expressing cells was analyzed at 24 hours after the administration of vehicle, tamoxifen, or tamoxifen plus sulfasalazine. *P < 0.05, increased cell viability after tamoxifen administration in LC3B shRNA-expressing cells vs in control shRNA-expressing cells. Too, **P < 0.05, increased cell viability after treatment with tamoxifen plus sulfasalazine vs tamoxifen only in the control and LC3B shRNA-expressing cells. Values are presented as mean ± SD, n = 3.