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. 2022 Jul 25;23(15):8184. doi: 10.3390/ijms23158184

Figure 5.

Figure 5

EDEM3 depletion in prostate cancer sensitises cells to ER stress and its upregulation promotes radio-resistance. (A–D) Cellular viability of LNCaP and CWR22Rv1 cells, with knockdown of EDEM3, treated with either vehicle (DMSO), 100 nM thapsigargin or 2.5 µg/mL tunicamycin, for 24 h. Viability measured by WST-1 assay and shown as absorbance at O.D 450 nm. n = 3. Data are mean ± s.e.m. p values were calculated using two-tailed unpaired t-tests. * p < 0.05 and ** p < 0.01 (E) mRNA levels of EDEM3 in LNCaP cells 48 h after exposure to 4 Gy radiation. Data are presented as mean ± s.e.m. p value calculated using a two-tailed unpaired t-test. * p < 0.05. (F) Western blot analysis of EDEM3 expression in LNCaP cells 48 h after exposure to 4 Gy radiation. GAPDH was used as a loading control. (G,H) Cell survival determined by clonogenic assay. LNCaP and CWR22Rv1 cells transfected with either an empty vector or EDEM3 overexpression vector were irradiated with either 2 or 4 Gy and left to form colonies for 14 days. At 14 days, any colony with more than 50 cells was counted. Cell survival was calculated by comparison with relative 0 Gy controls. n = 5. Data are mean ± s.e.m. p values were calculated using a two-way ANOVA. * p < 0.05, ** p < 0.01. (I,J) Cell survival determined by clonogenic assay. Stable EDEM3 knockdown and control LNCaP and CWR22Rv1 cells were irradiated with either 2 or 4 Gy and left to form colonies for 14 days. At 14 days, any colony with more than 50 cells was counted. Cell survival was calculated by comparison with relative 0 Gy controls. n = 5. Data are mean ± s.e.m. p values were calculated using a two-way ANOVA. * p < 0.05, ** p < 0.01 and *** p < 0.001. (K) Model of the role of EDEM3 in radio-resistance. Exposure to radiation induces ER stress and an accumulation of misfolded and unfolded proteins in cells. In response, the UPR is activated by three critical sensing proteins, PERK, ATF6 and IRE1. Activation of the UPR results in the inhibition of translation, transcription of UPR-associated genes and stimulation of ERAD. EDEM3, an important ERAD-associated enzyme, begins to trim mannose from misfolded glycoproteins to signal for degradation, aiding cell survival and protecting cells from radiation.