Figure S5.

Additional data to Fig. 9. (A and B) Overexpression of PKC isoforms in human breast cancers. (A) The overexpression of PKC isoforms was evaluated in the METABRIC and TGCA (Pan Cancer Atlas) BC datasets, as described in Materials and methods. Parameters used were: mRNA expression z-scores relative to all samples (log microarray); z-score threshold + 2.0. The percentage of alterations in the two datasets (METABRIC, N = 1904; TGCA, N = 994) is reported for the three classes of PKCs and further subdivided for each gene (A, PRKCA or α; B, PRKCB or β; G, PRKCG or γ; D, PRKCD or δ; E, PRKCE or ε; H, PRKCH or η; Q, PRKCQ or θ; Z, PRKCZ or ζ; I, PRKCI or ι). (B) Matrix showing the co-occurrence of overexpression of PKC isoforms. The matrix shows the co-occurrence (P < 0.05 by one-tailed Fisher’s exact test). Color code: black, co-occurrence in both datasets (METABRIC and TGCA); gray, co-occurrence in only one dataset. (C–H) Various controls for the specificity of the effects of TPA and of PKC inhibitors. (C) The indicated MEC transfectants were stimulated with TPA (1 μM for 12′) followed by IB with the indicated Ab. GAPDH is a loading control. S.e, short exposure; l.e, long exposure. The blot shows that also in the absence of PKCζ, TPA can efficiently phosphorylate Numb. (D) Numb confocal immunofluorescence in mouse MECs treated with TPA (1 μM TPA, 12 min), BIS (3 μM, 6 h) or a combination of TPA + BIS. In the combination treatment, cells were pretreated with BIS for 6 hr and then induced with TPA for 1 μM TPA for 12 min. Numb (green), DAPI (blue). Bar, 100 µm. (E) Numb confocal immunofluorescence in mouse MECs stably overexpressing Venus (Venus) or PKCζ-Venus (PKCζ -OE) treated with BIS (3 μM, 6 h). Numb (red), epifluorescence (Venus), DAPI (blue). Bars, 100 µm. (F) The indicated BC cultures were treated or not with BIS (3 μM o/n) and immunoblotted as shown on the right. Vinculin, loading control; l.e., long exposure. The figure shows that in BC displaying high pNumb, BIS significantly reduces the level of pNumb. (G) MS from WT mice were dispersed and infected with Venus (EV) or PKCζ-Venus (PKCζ -OE), and treated with BIS (3 μM o/n) or Sotrastaurin (0.5 μM o/n), another PKC inhibitor (Albert et al., 2022) and subjected to IB as indicated. (H) Cells as in G were treated with BIS (3 μM for the duration of the experiment) or Sotrastaurin (0.5 μM for the duration of the experiment), and subjected to a MS assay, as indicated. Data are expressed relative to control (EV untreated) cells ± SD (see also Table S2). Statistical analysis was with the Student’s t test two-tailed. The combined results of G and H show that treatment with two different PKC inhibitors cannot revert the biochemical and biological effects of PKCζ -OE. Source data are available for this figure: SourceData FS5.