Figure 3.

In the presence of ER-stress, IRAK2 is upregulated, and its downregulation affects the ERN1 pathway. (A) Expression of IRAK2, ERN1, XBP1s and CHOP at the mRNA level in control and IRAK2 knockdown BCSC1 and BCSC3. The transcription of the genes was evaluated in control cells (CV), control cells treated with thapsigargin (CV + TG), IRAK2 knockdown cells (shIRAK2) and IRAK2 knockdown cells treated with thapsigargin (shIRAK2 + TG). All cells (thapsigargin-treated and untreated, control and shIRAK2 cells) were cultured in the presence of doxycycline. Cells were harvested, and RNA was isolated from their pellet. The gene of interest (IRAK2, ERN1, XBP1s and CHOP) expression was normalised to the HKG β-actin. The relative expression was calculated using the 2^−ΔΔCT method and represented as the normalised quantity of mRNA in shIRAK2 cells, CV + TG cells and shIRAK2 + TG cells relative to the normalised quantity of mRNA in CV cells, which was set as 1. (B–E) The expression of ERN1 (B) and CHOP (D) at the protein level and their relative quantification ((C) for ERN1 and (E) for CHOP) in the CV and shIRAK2 BCSC1, BCSC3 and MDA-MB-468. Cells were cultured for 5 days in a medium containing 100-ng/mL doxycycline, harvested, and the proteins were subsequently isolated from the cell pellet. The expression of the gene of interest (ERN1 or CHOP) was normalised to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Subsequently, the expression evaluated in shIRAK2 cells was compared with that calculated in CV cells, which was set as 1. Data (n = 3) are presented as means ± SEM using unpaired Student’s t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.