Fig 2: Cav-1 was negatively regulated by vimentin filaments. (A) Representative immunofluorescence images showed the subcellular distribution of endogenous vimentin and Cav-1. The white dotted outline showed the perinuclear vimentin-rich region. Bars, 10 µm. (B) The correlation analysis indicated by Pearson’s coefficient of the fluorescence signals of endogenous vimentin and Cav-1. The correlation between exogenous expressed Cav-1-mCherry and endogenous antibody stained Cav-1 was used as a positive control. (C) Representative images from time-lapse videos (Supplementary Materials) of U2OS cells expressing vimentin-GFP and Cav-1-mCherry. Magnified panels showed representative images at different time points in the yellow box of the left panel, and white arrows indicated the positions of individual Cav-1 vesicle observed in the video (Supplementary Materials). Bars, 10 µm in the left panels and 2 µm in the right magnified panels, respectively. (D) The extracts of U2OS cells with wild-type and vimentin-knockout (VIM KO) were subjected to Western blot analysis. (E) The quantified relative levels of phosphorylated Cav-1 (P-Cav-1) protein normalized to the internal control GAPDH in wild-type and vimentin-knockout cells from Western blots. The data was from five independent experiments (n = 5). (F,G) The intensity of P-Cav-1 was increased in vimentin-knockout U2OS cells and was able to recover in vimentin full-length (FL) reintroducing cells. In (F), it was showed the representative images of wild-type (arrowheads in WT and VIM KO Mix), vimentin-knockout and vimentin-knockout/full-length (arrowheads in VIM KO and FL Mix) cells. In (G), it was showed the quantification of normalized relative fluorescence intensities in wild-type (n = 21), vimentin-knockout cells (n = 13) and full-length rescue cells (n = 22). Bars, 10 µm. All the quantified data was presented as mean ± s.e.m. ** p = 0.01, *** p = 0.001 (t-test).

Fig 3: Impaired cerebral organoid morphology, ventricular zone, and laminar organization in PCDH12-KO cerebral organoids(A) Timeline of cerebral organoid formation.(B) Representative bright-field images of WT (left) and PCDH12-KO (right) embryoid bodies (EBs) at day 7 showing brightening edges, indicative of ectodermal differentiation.(C) Quantification of EB coefficient of circularity, defined as 4p(area/perimeter2) at day 7 (WT n = 38; PCDH12-KO n = 39; mean ± SD) and day 10 (WT n = 32; PCDH12-KO n = 34; mean ± SD) p = 0.0042; comparison of fits.(D) Representative bright-field images of WT (left) and PCDH12-KO (right) EBs at day 10 showing surface budding, indicative of neuroepithelial expansion.(E) Percentages of EBs in developmental categories 1 to 4 (right). Category 1: uniform and pronounced surface budding, clear edges; category 2: smoother surface budding, with occasional non-neuroepithelial outgrowths; category 3: few surface buddings with recurrent non-neuroepithelial outgrowths; category 4: no surface budding, lack of clear edges.(F) Representative category 1 WT (left) and category 3 PCDH12-KO (right) cerebral organoids at day 21.(G) Percentages of organoids in developmental categories 1 to 4. Category 1: expanded cerebral tissue with ventricle-like structures; category 2: expanded cerebral tissue with ventricle-like structures, alongside non-neuronal outgrowths; category 3: minimal cerebral expansion with fewer and smaller ventricle-like structures; category 4: no cerebral expansion, with cell processes extending into the Matrigel.(H) Immunofluorescence images of day 21 WT (left) and category 3 (middle) and category 4 (right) PCDH12-KO organoids stained with N-cadherin (red).(I) Quantification of N-cadherin-defined cortical rosettes in day 21 organoids. Each point represents the number of rosettes per organoid (WT n = 7; PCDH12-KO n = 12; mean + SD; unpaired t test with Welch’s correction).(J) Immunofluorescence images of day 21 WT (left) and category 3 (middle) and category 4 (right) PCDH12-KO organoids stained with phospho-vimentin (p-Vim; red), and phospho-histone H3 (pH-H3; green).(K) Average number of anchored mitotic cells per cortical rosette in day 21 organoids. Each point represents the average number per organoid (WT n = 6; PCDH12-KO n = 11; mean + SD; Mann-Whitney test).(L) Total number of mitotic cells per rosette in day 21 organoids. Each point represents the average number per organoid (WT n = 7; PCDH12-KO n = 12; mean + SD; Mann-Whitney test).(M) Immunofluorescence images of day 21 WT (left) and category 3 (middle) and category 4 (right) PCDH12-KO organoids stained with Sox2 (red).(N) Quantification of ventricular zone thickness in day 21 organoids, defined by the thickness of the SOX2+ layer. Each point represents the average ventricular zone thickness per cortical rosette (n = 27 for WT and PCDH12-KO; mean + SD; unpaired t test with Welch’s correction).(O) Immunofluorescence images of day 40 WT (left) and PCDH12-KO (right) organoids stained with PAX6 (red) and TBR2 (green). Examples of neural rosettes are highlighted by dashed lines.(P) Average ratio of TBR2+ relative to PAX6+ cells per rosette in day 40 organoids (WT n = 12; PCDH12-KO n = 6; mean + SD; unpaired t test).(Q) Immunofluorescence images of day 40 WT (left) and PCDH12-KO (right) organoids stained with CTIP2 (green) and TBR1 (red). Examples of rosettes are highlighted by dashed lines.(R) Average CTIP2/TBR1 Pearson’s correlation coefficients showing degree of colocalization in day 40 organoids (WT n = 6; PCDH12-KO n = 4; mean + SD; Mann-Whitney test).Data were obtained from 2 different cell lines for each genotype and from 3 independent differentiations.Scale bars represent 100 µm (B, D, H, O, and Q), 50 µm (J and M), and 1 mm (F).