Grhl1 is regulated upstream of Dsg1. (A and B) Retroviral transduction of shNT (non-targeting control) or shDsg1 knockdown vectors was performed in primary human keratinocytes and RNA or protein was collected for RT-PCR and Western blot validation of knockdown. No significant difference in Grhl1 mRNA expression or protein level was observed in response to Dsg1 knockdown. (C) Retroviral transduction of GFP or GRHL1 (Grhl1 OE) was performed in primary human keratinocytes and protein was collected for Western blot validation of expression (see Fig. 3 H). (D) 3D organotypic raft cultures comprises shNT or shDsg1 expressing keratinocytes were grown for 6 d and stained for Grhl1. Nuclear to cytoplasmic staining intensity of Grhl1 was not statistically different between the two cultures. Mean ± SEM depicted. n = 3, Student’s t test. (E) Similarly, 3D organotypic raft cultures composed of shNT or shDsg1 expressing keratinocytes were grown for 6 d and stained for Slug and the number of suprabasal Slug expressing cells was counted. The number of suprabasal Slug expressing cells was not different between the two cultures. n = 3. Student’s t test. Source data are available for this figure: SourceData FS2.