Mitotic WNT signalling orchestrates neurogenesis in the developing neocortex (2021)
Figure 5

Figure 5. Sox4/11 protein levels are decreased in DKO NPCs, predominantly during mitosis.

(A) Immunoblot analysis with Sox4 antibody of protein lysates extracted from control (Co) (Ccny+/-; Ccnyl1-/-; shRNA Co) and mutant (Ccny+/-; Ccnyl1-/-; shRNA Ccny) NPCs. Numbers are fold change vs. controls of Sox4 protein levels normalized to GAPDH (n=3). Immunoblot image cropped from Figure EV5H. Quantification performed on immunoblot shown in Figure EV5H and two additional immunoblots (not shown).

(B) IF for Sox4 and pHH3 on neocortex sections of E13.5 control and DKO embryos. The level of Sox4 immunoreactivity is reduced in pHH3+ cells (white arrowheads).

(C) Quantification of Sox4 staining in SVZ mitotic cells from E13.5 control and DKO embryos. Mitotic Sox4 staining was defined as high if the pixel intensity was similar to, or higher than, non-mitotic cells, or low if the levels were below those observed in non-mitotic cells. Data are means ± SEM for ratios of cells with high vs. low Sox4, and are displayed as fold change vs. controls (n=4 embryos, 3 litters).

(D) Strategy to generate Sox4 S316 phospho-antibody (pSox4) by phosphopeptide injection into rabbits.

(E) Flag-Sox4 transfection in 293T cells followed by 24-hour nocodazole (100 ng/ml) and BIO (1 µM) treatments. Samples analyzed by immunoblot using pSox4 antibody; numbers above blot are quantification of Sox4 phospho-band normalized to total Sox4 (FLAG) for nocodazole-treated and non-treated cells and are expressed as fold change vs. controls (n=6, experiment performed 2x in triplicate).

(F) Immunoblot analysis with pSox4 antibody of cell lysates extracted from E13.5 control and DKO forebrains. Red arrowheads indicate Sox4 phosphorylation band; red asterisks are non-specific bands. Numbers above blot are quantification of Sox4 phosphorylation levels normalized to total Sox4 and are expressed as fold change vs. controls (n=4 embryos, 3 litters).

(G) Immunoblot analysis with a Sox11 antibody of protein lysates extracted from control and mutant E13.5 NPCs. Numbers are fold change vs. controls of Sox11 protein levels normalized to GAPDH (n=3 biological replicates).

(H) IF for Sox11 and pHH3 in E13.5 control and DKO neocortices. Sox11 staining intensity is decreased in mitotic cells (white arrowheads, insets).

(I) Quantification of Sox11 protein levels in SVZ mitotic cells from E13.5 control and DKO embryos. Quantification performed as in (C). Data are means ± SEM for ratios of high vs. low Sox11 and are displayed as fold change vs. controls (n=5 embryos, 3 litters).

(J) Immunoblot analysis with a Sox11 antibody of protein lysates extracted from E13.5 control and DKO forebrains. Numbers are fold change vs. controls of Sox11 protein levels normalized to Tuj1 (n=5 embryos, 4 litters). ERK1/2 immunoblot shown to document equal loading.

(K) Co-IF for Sox11 and Tbr2 in sections of neocortex of E13.5 control and DKO embryos. Sox11 immunoreactivity is reduced in some Tbr2+ cells (asterisks, insets).

(L) Quantification of percentage of Tbr2+ cells that show with high Sox11 staining intensity, in control and DKO E13.5 neocortices. High or low staining intensity was judged based on visual inspection, taking background staining levels into account. Data are means ± SEM and are shown as fold change vs. controls (n=5 embryos, 3 litters).

Data information: All scale bars 20 µm. Protein ladder numbers in italics indicate estimated size. Unpaired two-tailed t-test for all statistical analyses: ns, not significant; * p < 0.05, ** p < 0.01 *** p < 0.001. p = 0.0012 (A); p = 0.00020 (C); p = 0.0031 (E, 2nd lane); p = 0.0010 (E, 4th lane); p = 0.047 (F, 2nd lane); p = 0.047 (G); p = 0.045 (I); p = 0.031 (J); p = 0.0010 (L).