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. Author manuscript; available in PMC: 2021 Apr 19.
Published in final edited form as: Nat Cell Biol. 2020 Oct 19;22(11):1382–1395. doi: 10.1038/s41556-020-00592-8

Extended Data Fig. 4. PLCγ1 suppression decreases mitochondrial respiration and enhances cancer cell glycolytic capacity in human NSCLC cells.

Extended Data Fig. 4

(a) Immunoblot analysis of the indicated targets in A549 cells transduced with either an empty vector control (Tet-pLKO-puro, shControl) or a doxycycline-inducible shRNA against PLCγ1, incubated without/with doxycycline for 48h and further incubated for 48h in normoxia or hypoxia (3% O2).

(b-e) Graphs showing oxygen consumption rate (OCR, b and c) and extracellular acidification rate (ECAR, d and e) of A549 cells transduced and treated as in (a); n replicates/group: (b) = 8, (c) = 7 normoxia/8 hypoxia, (d) = 9 normoxia/ 8 hypoxia, (e) = 8. Panels (c and e) are treated without/with doxycycline and serve as a control for the doxycycline effect on cells.

(f) Bar graph showing ECAR parameters in A549 cells transduced as in (a). Cells were transfected with either pcDNA3.1 empty vector or pcDNA3.1-rPLCγ1 (shRNA-resistant PLCγ1) to rescue the shPLCγ1 #2 and incubated for 48h in normoxia or hypoxia; n = 8.

(g-h) Oxygen consumption rate (g) and extracellular acidification rate (h) parameters of A549 cells transfected with either pcDNA3.1 empty vector or pcDNA3.1-PLCγ1 and incubated for 48h in normoxia or hypoxia; (g) n = 8 for normoxia and 6 for hypoxia groups, (h) n = 7.

OCR was determined during sequential treatments with oligomycin, FCCP and rotenone/antimycin (AA). ECAR was determined during sequential treatments with glucose (Glc), oligomycin and 2 deoxyglucose (2DG).

Graphical data are mean ± SD. Statistical analyses were done using one-way ANOVA; n, number of biologically independent samples. **** p < 0.0001. Statistical source data and unprocessed immunoblots are provided in Source Data Extended Data Fig. 4.