A pool of four shRNA targeting PIEZO1 cloned into a lentiviral vector (PLKO3.1‐CMV‐tGFP, MISSION tool, Sigma Aldrich) were used as previously described.
16 (link) In experiments performed with separated shRNA#1 and #2, clone used was described in TableS1 . Lentiviral particle production was performed in HEK293T cells as previously described.
16 (link) Lentiviral supernatant was ultracentrifugated for 1.5 h at 100,000 g at 4°C; THP1 cells were transduced using a multiplicity of infection (MOI) of 10 in the presence of 8 μg/mL polybrene (Sigma Aldrich). Cells were washed twice in 1X PBS (Eurobio scientific) 24 h after transduction. At D4 post‐infection, GFP cells were sorted on a FACS Aria II instrument (Becton Dickinson) and cultured for 3 additional days. Cell counts, flow cytometry, gene and proteins analysis were performed at D4, D7, and D9.
16 (link) In experiments performed with separated shRNA#1 and #2, clone used was described in Table
16 (link) Lentiviral supernatant was ultracentrifugated for 1.5 h at 100,000 g at 4°C; THP1 cells were transduced using a multiplicity of infection (MOI) of 10 in the presence of 8 μg/mL polybrene (Sigma Aldrich). Cells were washed twice in 1X PBS (Eurobio scientific) 24 h after transduction. At D4 post‐infection, GFP cells were sorted on a FACS Aria II instrument (Becton Dickinson) and cultured for 3 additional days. Cell counts, flow cytometry, gene and proteins analysis were performed at D4, D7, and D9.
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