JCS068924 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Knockdown of pRb inhibits calcium-mediated differentiation. (A) Western blot analysis shows knockdown of pRb levels in primary human foreskin keratinocytes (HFKs) using two different retrovirally expressed shRNA molecules; β-actin was used as a loading control. pRb knockdown using shRNA designed to the coding region (CR, upper blots) or the 3′ untranslated region (UTR, lower blots) resulted in inhibition of the differentiation process compared with control cells (scram), as ascertained by expression of the differentiation marker keratin 1 over a time-course of calcium treatment. (B) Control and knockdown keratinocytes were differentiated as in A for 72 hours. Knockdown of pRb reduced the expression of keratin 1 when assessed by immunofluorescence. (C) Staining of 72-hour differentiated cultures with transglutaminase 1 (TG1) and E-cadherin show that although Rb-depleted cells have a differentiated morphology, they do not express differentiation markers. Control cells prior to differentiation are also shown. Scale bar: 200 µm.

• Supplemental Figure 2 -

  Fig. S2. (A) Saos-2 cells infected with recombinant adenovirus encoding HA-tagged wild-type Rb (Ad-RbWT) or the acetylation mutant RbRR (Ad-RbRR) (MOI 200). Forty-eight hours after infection, cells were pulsed with BrdU and fixed. The number of cells in S-phase was determined by indirect immunofluorescence. (B) Saos-2 cells were infected with recombinant adenovirus encoding RbWT and RbRR with a C-terminal GFP tag (MOI200). The number of BrdU-positive cells was determined as in A.

• Supplemental Figure 3 -

  Fig. S3. Differentiation is restored by re-expression of RbWT but not the acetylation mutant RbRR. Five-day organotypic raft cultures of control and knockdown keratinocytes infected with control adenovirus (Ad-GFP) or Ad-Rb (WT or RR mutant) were sectioned and stained for the indicated differentiation markers. Knockdown of Rb did not dramatically alter the gross morphology of the rafts, although it was repeatedly observed that there was an expansion of spinous layer resulting in a thicker epithelium. Knockdown of Rb inhibited expression of the differentiation markers keratin 1 and keratin 10 and also the late marker loricrin. Rescue of Rb levels with AdRbWT restored expression of these differentiation markers but the acetylation mutant did not, although there was some intermittent loricrin staining. Staining for BrdU in the raft cultures identified cells just above the basal epithelium, which were positive (arrows), suggesting a delay in exit from the cell cycle. This was restored only by expression of wild-type Rb. Scale bar: 100 µm.

• Supplemental Figure 4 -

  Fig. S4. BrdU staining observed in morphologically differentiated si-Rb keratinocytes. Following 72 hours of differentiation, Rb levels were depleted using siRNA, and BrdU incorporation was measured 24 hours later. Cells were then stained for BrdU and E-cadherin. Confocal images were visualised in 3D using Volocity image analysis software (Perkin Elmer). Image A was cropped to focus on the BrdU-positive cell; the perpendicular image is shown in B. Arrows indicate E-cadherin at the edge of the cell. Images identify that BrdU-positive cells have differentiated morphology, suggesting that these cells have re-entered S-phase.