Extended Data Fig. 6. PLCγ1 suppression depletes mitochondrial ROS through impairment of Ca²⁺ entry into the mitochondria.

(a) Flow cytometry histograms (left) and quantification of DHR (green, right) of A549 cells transduced with an empty vector (Tet-pLKO-puro, shControl) and incubated without/with doxycycline (DOX) for 48h, incubated for additional 48h in normoxia or hypoxia and stained with DHR for flow-cytometry. H₂O₂: positive control. DHR: Dihydrorhodamine. Normo: normoxia. Hypo: hypoxia. n = 3.

(b) MCU mRNA levels in A549 cells transduced with an empty vector (Tet-pLKO-puro, shControl) or 2 doxycycline-inducible shRNAs against MCU and incubated with doxycycline for 72h; n = 3.

(c) Immunoblot analysis of the indicated targets in A549 cells transduced with an empty vector (Tet-pLKO-puro, shControl) or 2 doxycycline-inducible shRNA against PLCγ1, transfected with either pcDNA3.1 empty vector or pcDNA3.1-MCU-flag plasmid and incubated in normoxia or hypoxia for 48h.

(d) Representative flow cytometry histograms of DHR (green) mean fluorescent intensity of A549 cells from experiment reported in main Fig. 4f. Sh#2: shPLCγ1 #2.

(e) Representative flow cytometry histograms (left) and quantification of DHR (green) of A549 cells transduced with an empty vector (Tet-pLKO-puro, shControl) or a doxycycline-inducible shRNA against PLCγ1. Cells were then treated with doxycycline for 24h, transfected with either pcDNA3.1 empty vector or pcDNA3.1-MCU-flag plasmid and moved to hypoxia for additional 48h before staining cells with DHR for analysis. DHR: Dihydrorhodamine. Sh#1: shPLCγ1 #1; n = 3.

(f) Cytosolic (left) and mitochondrial (right) Ca²⁺ response of A549 cells transduced as in (c), transfected with appropriate targeted-aequorin and histamine-induced Ca²⁺ was measured 48h after incubation in normoxia or hypoxia; n/group = cytosolic normoxia 7, 8, 7, 7, 8, 7/cytosolic hypoxia 8, 6, 7, 8, 7, 8. n/group = mitochondrial normoxia 11, 7, 9, 10, 8, 6/mitochondrial hypoxia 7, 10, 9, 9, 9, 11.

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. 6.