SRP54 knockdown induces a defect in proliferation and increased ER stress and autophagy. (A-D) HL-60 cell lines stably expressing either shRNA targeting SRP54 (1 or 2) or a SCR were generated after transduction with lentiviral particles and after sorting on GFP⁺ after 72 hours. Nontransduced (NT, not transduced) HL-60 cell line was used as control. (A) SRP54 mRNA expression level was checked by qRT-PCR related to PPIA (n = 4). Mean ± SEM; unpaired t test, 2-tailed; *P < .05; **P < .01. (B) Fold proliferation was assessed after Trypan blue exclusion for 3 days (n = 4). Mean ± SEM; unpaired t test, 2-tailed; *P < .05. (C) Spliced XBP1 mRNA expression level was checked by qRT-PCR related to PPIA, CFL1, and H2AZ (n = 4). Mean of SCR or shRNA_1 and 2 ± SEM; Unpaired t test, 2-tailed; *P < .05. (D) P-eIF2a, P-PERK, P-ULK1 were evaluated by western blot analysis using specific antibodies. β-Actin was used as loading control. (E-G) Normal CD34⁺ progenitors were transduced with 2 lentiviruses expressing either shRNA targeting SRP54 (ShSRP54_2) or SCR. CD34⁺GFP⁺ cells were sorted 72 hours later and were cultured for 10 days in serum-free medium with SCF, IL-3, and G-CSF. (E) Granulocytic cells were counted by Trypan blue exclusion (n = 3) or sorted the same day on the CD36⁻CD15⁺CD11b⁺ phenotype for analysis of (F) SRP54 gene expression level (n = 4) and (G) spliced XBP1 (n = 3) mRNA expression level by qRT-PCR related to PPIA and HPRT. Mean ± SEM; unpaired t test, 2-tailed; **P < .01; ***P < .001.