Sac1 decreases cellular PIP and PIP₂ and makes it easier for PLC to hydrolyze PI(4,5)P₂. (A, left) Mass spectrometry analysis for PIP. Histogram of the most abundant PIP species in control (black bars) and Sac1-overexpressing (green bars) tsA201 cells (means ± SEM). (right) Summary of the total PIP signal in control and Sac1 overexpressing tsA201 cells (means ± SEM), n = 6; t test; *, P < 0.05). (B) Mass spectrometry analysis for PIP₂. Same experiment as (A) but summarizing PIP₂ species. (means ± SEM; n = 6; t test; *, P < 0.05). (C, left) Normalized change in RFP-PH_PLCδ1 intensity from the TIRF footprint of a tsA201 cell after activation of the M₁R. (right) Summary (means ± SEM, n = 7; t test; *, P < 0.05). (D) Same experiment as C, but with GFP-Sac1 overexpressed. (E and F) Same experiment as A and B, except with siRNA against endogenous Sac1 (means ± SEM, n = 6; *, P < 0.05). (G) Normalized, averaged time course of RFP-PH_PLCδ1 within a TIRF footprint, after the activation of the M₁R, in control (black line) or Sac1 siRNA-treated (red line) cells. (right) Summary histograms (n = 9; *, P < 0.05.). (H, left) Normalized, mean time courses of PH_PLCδ1 FRET photometry signals in control, Sac1-overexpressing, and catalytically inactive (C389S) Sac1-expressing tsA201 cells. (right) Summary (means ± SEM, n = 6; t test; *, P < 0.05.). (I, left) Normalized, mean time courses of LibravIII FRET photometry signals in control or Sac1-overexpressing tsA201 cells. (right) Summary (means ± SEM, n = 6; t test; *, P < 0.05.). (J, left) two tsA201 cells expressing mCherry-P4M. (middle) Same experiment as left panel, except GFP-Sac1 is overexpressed. (right) Same experiments as left panel except cells have had hSac1tr recruited to their trans-Golgi network to deplete PI(4)P. (K) Ratio of the Golgi/cellular intensity in each experimental condition (scatter plot, means ± SEM n = 8–11; *, P < 0.05). Bars, 5 µm.