Fig 2: Knocking down OXR1 selectively kills IR‐SCs by downregulating the expression of antioxidant enzymes to sensitize the cells to oxidative stress. (a) The levels of OXR1 mRNA (top) and OXR1 protein (bottom) in NCs and IR‐SCs transfected with either scramble control shRNA (shCtrl) or two different shRNA constructs targeting OXR1 (shOXR1‐1 or shOXR1‐2). (b) Expression of glutathione peroxidase 2 (GPX2), heme oxygenase 1 (HO‐1), catalase (CAT), superoxide dismutase 1 (SOD1), and SOD2 mRNA in NCs and IR‐SCs transfected with shCtrl or shOXR1. Data in (a) and (b) are the mean ± SE of fold changes in mRNA expression compared with shCtrl‐transfected cells from two to three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. shCtrl by unpaired t test. (c) Percent viability of NCs and IR‐SCs transfected with shCtrl or shOXR1 presented as the mean ± SE of two independent experiments. *p < 0.05 vs. IR‐SCs transfected with shCtrl by unpaired t test. (d) Levels of intracellular ROS in NCs and IR‐SCs transfected with shCtrl or shOXR1 in the presence or absence of 2 mM N‐acetyl‐l‐cysteine (NAC). Data are presented as the mean ± SE of mean fluorescent intensity (MFI) of dihydrorhodamine 123 (DHR) from two independent experiments. *p < 0.05 vs. IR‐SCs transfected with shOXR1 without NAC by unpaired t test. (e) Percent apoptotic cells in NCs and IR‐SCs transfected with shCtrl or shOXR1 in the presence or absence of 2 mM NAC are presented as the mean ± SE of two independent experiments. *p < 0.05 vs. IR‐SCs transfected with shOXR1 without NAC by unpaired t test. (f) Percent viable cells in NCs and IR‐SCs transfected with either shCtrl or shOXR1 after incubation with 100 μM H2O2 for 3 day. Data are presented as the mean ± SE of percent viable cells compared with their respective controls without H2O2 treatment from two independent experiments. *p < 0.05 vs. IR‐SCs transfected with shCtrl by unpaired t test. (g) Percent apoptotic cells in NS and IR‐SCs transfected with shCtrl or shOXR1 after incubation with 100 μM H2O2 in the presence or absence of 2 mM NAC. Data are presented as the mean ± SE of two to three independent experiments. *p < 0.05 vs. IR‐SCs transfected with shOXR1 with H2O2 alone by unpaired t test

Fig 3: Knocking down OXR1 also induces apoptosis in RE‐SCs via downregulating the expression of antioxidant enzymes and increasing ROS production. (a) The levels of (top) OXR1 mRNA and (bottom) OXR1 protein in NCs, IR‐SCs, and RE‐SCs. (b) Expression of GPX2,HO‐1,CAT,SOD1, and SOD2 mRNA in NCs and RE‐SCs. Data in (a) and (b) are the means ± SE of fold changes in mRNA expression compared with NCs from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. NCs by unpaired t test. (c) The levels of (top) OXR1 mRNA and (bottom) OXR1 protein in RE‐SCs transfected with shCtrl or shOXR1. (d) The levels of GPX2,HO‐1,CAT,SOD1, and SOD2 mRNA in IR‐SCs transfected with shCtrl or shOXR1. Data in (c) and (d) are the means ± SE of fold changes in mRNA expression compared with shCtrl‐transfected cells from two independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. shCtrl by unpaired t test. (e) The levels of intracellular ROS in RE‐SCs transfected with shCtrl or shOXR1. The data are presented as the mean ± SE of fold changes compared with shCtrl‐transfected RE‐SCs from two independent experiments. *p < 0.05 vs. shCtrl by unpaired t test. (f) Percent apoptotic cells in RE‐SCs transfected with shCtrl or shOXR1 are presented as the mean ± SE of two independent experiments. *p < 0.05 vs. shCtrl by unpaired t test

Fig 4: PL selectively decreases the level of OXR1 in SCs, in part by inducing OXR1 proteasomal degradation. (a) Percentage of SA‐β‐gal–positive cells at different days after IR. (b) The levels of OXR1 mRNA in WI‐38 cells at different days after IR are presented as a fold change from un‐irradiated control cells. (c) The levels of OXR1 protein in WI‐38 cells at different days after IR. Left panel, representative immunoblot of OXR1 and β‐actin; right panel, fold changes in OXR1 expression in irradiated cells relative to un‐irradiated cells after normalization to β‐actin. (d) The levels of OXR1 mRNA in NCs and IR‐SCs after incubation with vehicle (Veh) or PL (5 μM) for 6 hr. (e) The levels of OXR1 protein in NCs and IR‐SCs after incubation with vehicle (Veh) or PL (5 μM) for 6 hr. Left panel, representative immunoblot of OXR1 and β‐actin; right panel, fold changes in OXR1 expression in cells relative to vehicle‐treated NCs after normalization to β‐actin. (f) The levels of OXR1 were determined in NCs and IR‐SCs after incubation with vehicle (Veh) or PL (5 μM) for 6 hr in the presence or absence of 5 μM MG‐132 (MG). (g) The levels of poly‐ubiquitylated (Poly‐Ub) proteins in NCs and IR‐SCs after incubation with vehicle (Veh) or PL (5 μM) for 6 hr; β‐actin was included as a loading control. (h) The levels of poly‐ubiquitylated (Poly‐Ub) OXR1 in IR‐SCs after incubation with vehicle (Veh) or PL (5 μM) for 6 hr; the cell lysates were incubated with OXR1 or IgG control and resin for overnight. After extensive washing, ubiquitinated OXR1 was eluted by SDS sample buffer and immunoblotted with ubiquitin and OXR1 antibodies. The input cell lysates were also immunoblotted with antibodies to OXR1 or β‐actin as indicated. ns indicates the nonspecific band. Data in bar graphs are the mean ± SE of two to three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. un‐irradiated cells by one‐way ANOVA. a p < 0.05 vs. NCs treated with Veh; b p < 0.05 vs. IR‐SCs treated with Veh by two‐way ANOVA