HSC70 Overexpression and Knockdown in Cell Lines

HEK 293 T cells were transiently transfected with plasmid pLX307 encoding for HSC70 (HSPA8) using the calcium phosphate method [24 (link)]. pLKO.1-puro lentiviral vectors expressing shRNAs targeting HSC70 (shHSC70_1: NM_006597.3-976s1c1, shHSC70_2: NM_006597.3-335s21c1, shHSC70_3: NM_006597.3-2040s21c1) were purchased from the Sigma-Aldrich. These vectors contain a puromycin antibiotic resistance gene for selection of transduced mammalian cells. Sequences of shRNAs to target LAMP2A were (1) shRNA: CTGCAACCTGATTGATTA and (2) shRNA: GGCAGGAGTACTTATTCTAGT. These shRNA sequences were cloned into a U6-EF1a-IRES-hygro lentiviral vector backbone. Lentivirus production and transduction were done as described [25 (link), 26 (link)]. Transduced NB4 and SKBR3 cell populations were selected with 1.5 μg/mL puromycin for 4 days, and knockdown efficiency was assessed by western blot analysis (Figure 2).