Fig. 3.

RB loss stimulates cell death in cerebral organoids. (A) Representative wild-type and KO organoids at 15 and 28 DIV sectioned and immunostained against AC3 and Sox2, and counterstained with DAPI. The graphs show the percentage of AC3⁺/DAPI cells and AC3⁺/Sox2⁺ cells at both time points. (B) Representative wild-type and KO organoids at 15 and 28 DIV sectioned and immunostained against AC3 and DCX, and counterstained with DAPI. The graphs show the percentage of AC3⁺/DCX⁺ cells at both time points. (C) Representative wild-type and KO organoids at 28 DIV sectioned and immunostained against AC3 and Tuj1, and counterstained with DAPI. Insets in wild-type and KO organoids show an enlarged image of AC3⁺ and Tuj1⁺ cells in the RB-KO organoid, which were reduced in the wild-type organoid. The graph shows the percentage of AC3⁺/Tuj1⁺ cells. (D) Bright-field and red fluorescence images showing representative PI-treated wild-type and KO organoids at 28 DIV. (E) Relative mRNA levels of genes of the RB family and genes involved in cell cycle regulation and apoptosis measured by RT-qPCR. The results are mean±s.e.m. of five or six organoids from three independent experiments (*P<0.05, Student's t-test; n.s., not significant). Scale bar: 50 µm in A-C; 200 µm in D.