Figure S4.

MRCKβ is required for retinal integrity and function in vivo. (a) Subretinally injected lentiviral vectors specifically infect the RPE. Shown is GFP expression upon transduction with a control vector, combined with staining for the RPE marker RPE65 and DNA. (b) Frozen sections of mouse eyes 21 d after injection with either control or MRCKβ-targeting viruses were stained for RPE65 and DNA. Note, MRCKβ knockout leads to a pronounced retinal degeneration as determined by extensive thinning of the ONL. (c and d) MRCKβ expression upon transduction with control CRISPR or two distinct viruses targeting the MRCKβ gene. Note, apical membrane expression, highlighted by white arrowheads, of MRCKβ observed in control tissue samples is lost in both CRISPR MRCKβ 1 and 2. (e and f) Confocal immunofluorescence analysis of retinal sections from mice injected with either control or MRCKβ 2 knockout vector; as with MRCKβ 1 knockout vector, no significant difference in the overall retinal structure after 7 d but reduced apical F-actin staining, with reference to basal F-actin, in the MRCKβ-deficient RPE. INL, inner nuclear layer; IS, inner segments; OS, outer segments. White arrows highlight apical F-actin cortex. Protein staining was quantified as mean intensity (Int). Quantifications represented by bar graphs show means ± 1SD, n = 3, and P values derived from t tests. In d, a column scatter plot from three independent experiments shows the median and upper and lower quartiles. P values are derived from ANOVA tests.