JCS065243 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Table. S1. Summary of effects of JNK inhibition on mammary acini. Inhibition of JNK with SP600125 blocks acinus formation by primary and MCF10A mammary epithelial cells.

• Supplemental Figure S1 -

  Fig. S1. PKCζ kinase inhibition using the specific pseudosubstrate inhibitor does not impact PKCζ phosphorylation at T410. Confocal microscopy analysis of phospho-PKCζ (red) localising at apical puncta in developing primary mammary acini (24 hours), where it colocalises with ZO-1 (green) (a, b, c). After PKCζ kinase activity inhibition, phospho-T410-PKCζ is still found in dysmorphic primary acini, however it no longer colocalises with ZO-1 (d, e, f). Scale bar: 20 uM.

• Supplemental Figure S2 -

  Fig. S2. Anti-Par3 antibodies detect two bands by western analysis of primary mammary acinus extracts. Par 3 antibodies pull down PKCζ as early as 4 hours into primary mouse mammary acinus development by immunoprecipitations. Sometimes two forms of the Par 3 protein are observed by western blot analysis (detected at 100 and ∼130 kDa); the band at 100 kDa being the most prominent.

• Supplemental Figure S3 -

  Fig. S3. Total PKCζ and phospho-PKCζ have distinct cellular localisation patterns in primary mammary acini. (A) Unlike apically localised phospho-T410 PKCζ (Fig. 2Ae, Fig. S1), total PKCζ (red) distributes to the peri-nuclear region of polarised primary mammary epithelial cells during acinus formation, showing no colocalisation with ZO-1 (green). Merged confocal images taken at 4, 17, 24 and 48 hours after plating is shown. (B) The effect JNK inhibition using SP600125 (SP), is shown on localization of total PKCζ (red). DAPI, blue. Scale bar: 10 µM.

• Supplemental Figure S4 -

  Fig. S4. Complete blot of cropped Fig. 7e also displaying extracts of WT PKCζ overexpressing MCF10A acini.

• Supplemental Figure S5 -

  Fig. S5. Total PKCζ and ZO-1 distribution in mature virgin, pregnant, lactating and involuting mammary gland. Confocal microscopy analysis of in vivo mammary glands from virgin, (V 12 week), early pregnancy (day 8-p), pregnant (D14-p, D17-p), lactating (Lac D3) and 2 days post-involution (Inv 2), showing total-PKCζ staining in epithelial cells, somewhat apical but more diffuse in the cytoplasm than phosho-PKCζ (green, a−f). DAPI labels nuclei blue. ZO-1 does not colocalise (red). Scale bar: 20 uM.