Supplementary Figure 3 : Characterization of shRNA knockdown efficiency in vitro and in vivo.

From: Par3–mInsc and Gαi3 cooperate to promote oriented epidermal cell divisions through LGN

Supplementary Figure 3

(a) Quantification of mInsc knockdown efficiency in keratinocytes stably-transduced with retroviral mInsc, necessary due to low endogenous levels of mInsc in low calcium conditions. (b) Quantification of Pard3 knockdown in keratinocytes. Red letters indicate those with the highest levels of knockdown (94–97%), subsequently used for in vivo studies. (c) Quantification of mRNA knockdown efficiency in keratinocytes for Gnai2 (left) and Gnai3 (right) shRNA clones. Those with the strongest knockdown (>97%) are shown in red letters, and were subsequently used in vivo. Bars are the mean ± s.d. (d) Back skin sections from E17.5 epidermis showing specific loss of Par3 expression in mosaic Pard3 knockout (top left) or knockdown tissue. (e) Confirmation of knockdown efficiency by immunofluorescence in E17.5 back skin. In addition to its polarized localization in mitotic basal cells, Gαi3 is also localized to cell membranes suprabasally, like Par3 (top panel). This localization is specifically lost in RFP+ regions of mosaic knockdown tissue (bottom panel). (f) Gαii3 is normally enriched apically in mitotic basal cells (left, see also Fig. 5b), but following Gnai3 knockdown, LGN cortical expression is frequently reduced (middle row, weaker hairpin) or delocalized (bottom row, stronger hairpin).