Supplemental Material to: Xiaomu Qiao, Isabelle Roth, Eric Féraille and Udo Hasler Different effects of ZO-1, ZO-2 and ZO-3 silencing on kidney collecting duct principal cell proliferation and adhesion

Supplemental Figure 1 ZO protein intracellular distribution in the kidney cortex. Confocal z-stacks of ZO-1, ZO-2 and ZO-3 immunostaining of kidney cortex slices depicted in Figure 1. Shown below are overexposed images of regions outlined by a white rectangle that illustrate intracellular distribution of each ZO protein. Bar, 20 µm. Supplemental Figure 2 ZONAB immunofluorescence signal control. Confocal z-stacks of ZONAB (grey) and Hoechst (blue) in mCCD cl1 cells transfected with scrambled siRNA (sc) or siRNA against ZONAB (siZONAB), performed as described in the legend of Figure 4. A signal corresponding to cytosolic ZONAB and nuclear ZONAB aggregates was significantly reduced by siZONAB. On the other hand, a signal that defined the boundaries of cells was unaffected by siZONAB and may be non-specific or may reflect stable ZONAB protein at the cell boundary. One of three similar experiments is shown. Bar, 10 µm. ZONAB sc siZONAB

Supplemental Figure 3 ZO-3 nuclear localization is dramatically increased in confluent mCCD cl1 cells. Cytosolic and nuclear extracts were prepared from cells of varying cell density (low to high) and ZO-1, ZO-2, ZO-3, ZONAB (both long and short isoforms), CycD1, p21 and PCNA expression in both cell fractions was analyzed by Western blot. a-tubulin was used as a loading control for cytosolic extracts and histone deacetylase 3 (HDAC3) was used as a loading control for nuclear extracts. ER-resident calreticulin was used as an additional control for nuclear extract purity. Quantification of data, shown at right, is represented as fold difference of cytosolic and nuclear protein expression over values obtained in low-density cells and is expressed as the mean ±SEM of three independent experiments.

Supplemental Figure 4 Decreased ZO-3 protein expression in the absence of ZO-1 does not result from increased protein degradation. Cells were transfected with scrambled siRNA (sc) or siRNA against ZO-1 (siZO-1) or ZO-3 (siZO-3). 2 days post transfection, cells were challenged or not (Ctl) with either the lysosomal inhibitor cholorquine (100 µM) or the proteasomal inhibitor epoxomicine (100 nM) for 1 h, 6 h or 24 h. Protein was isolated and Western blot was performed against ZO-1, ZO-3 and claudin-4, used as a positive control for decreased protein degradation by either chemical. b-actin was used as a loading control. Supplemental Figure 5 Effects of ZO silencing on cell cycle progression. Cells were transfected 1 day after seeding with scrambled siRNA (sc) or siRNA against ZO-1 (siZO-1), ZO-2 (siZO-2) or ZO-3 (siZO-3) and cell cycle analysis was performed by flow cytometry 1 – 6 days post transfection (T1 – T6). Data is expressed as the mean percentage of G 0 /G 1, S and G 2 /M phase cells ±SEM of four independent experiments. Significant change of cell cycle phase (p ≤ 0.05) is depicted by an asterisk. Supplemental Figure 6 Effects of ZO silencing on CycD1 and ZONAB mRNA. Cells were transfected with scrambled siRNA (sc) or siRNA against ZO-1 (siZO-1), ZO-2 (siZO-2), ZO-3 (siZO-3) or ZONAB (siZONAB) and mRNA was isolated 1-3 days post- transfection. Data is represented as fold difference of mRNA expression over values obtained in cells transfected with scrambled siRNA 1 day post transfection and is expressed as the mean ±SEM of three independent experiments. Significant change of expression (p ≤ 0.05) within a same experimental group is depicted by an asterisk.

Supplemental Figure 7 Effects of p21 silencing on mCCD cl1 detachment and analysis of claudin-4 and claudin-8 expression in proliferating cells. (A) Cells were transfected 1 day after seeding with scrambled siRNA (sc), siRNA against either ZO-3 (siZO-3), p21 (sip21) or both siZO-3 and sip21 and cell detachment was analyzed by flow cytometry two days later. (B) Immunofluorescence of low- and high-density cells. Shown are confocal z-stacks of claudin-4 (green) and claudin-8 (green) depicting their expression at intercellular junctions in low (day 3) and high (day 7) density cells. Enlarged single-plane images of Hoechst (blue) or immunofluorescence staining of cells are also shown below. Bar, 10 µm. (C) Double-stained confocal z-stacks of high-density cells 6 h after scratch. Shown are claudin-4 (green) / ZO-3 (red) and claudin-8 (green) / ZO-1 (red). Contrary to occludin (Figure 7G), a signal corresponding to claudin-4 and claudin-8 was apparent in cells bordering scratch wounds. Bar, 30 µm. (D) Western blot of whole-cell occludin, claudin-4 and claudin-8. b-actin was used as a loading control. Quantification of data is shown below and is represented as fold difference of protein expression over values obtained 3 days after seeding and is expressed as the mean ±SEM of 4 independent experiments. (E) Double-stained confocal z- stacks of occludin (green) and ZO-3 (red) of cells transfected with scrambled siRNA (sc) or siRNA against ZO-3 (siZO-3) depicting decreased expression of occludin by ZO-3 silencing. Bar, 10 µm. For all experiments, one of three similar experiments is shown.