Fig 2: Loss of BIG1 does not change the proportion of NPCs.(A-C), Comparison of the numbers of Pax6+ and DAPI+ cells between Arfgef1geo/geo and control embryos at E13.5 (A), E15.5 (B) and E17.5 (C). The numbers of cells from the edge of the lateral ventricle within 200 µm cortical width were counted. Scale bars, 200 µm. n = 3 per group. (D) The ratio of Pax6+ NPCs and DAPI+ cells did not have significant difference at E13.5 (p = 0.09), E15.5 (p = 0.10), or E17.5 (p = 0.41). n = 3 per group. (E) The total number of cells in Arfgef1geo/geo neocortex is reduced at E15.5 (*p = 0.03) and E17.5 (*p = 0.01) but not E13.5 (p = 0.09). n = 3 per group. (F) The thickness of Arfgef1geo/geo neocortex is reduced at E17.5 (***p = 6.10E-06) but not at E13.5 (p = 0.09) and E15.5 (p = 0.41). n = 3 per group. Student’s T-test was used for comparisons between groups. Data are shown as the mean ± SD. n = 3 per group for each time point.

Fig 3: Loss of BIG1 delays early axonal differentiation in neurons.(A-B) In Arfgef1geo/geo primary hippocampal neuron cultures at DIV 2, the number of stage I (*p = 0.02) and stage II (*p = 0.02) neurons were significantly higher at the expense of stage III neurons (*p = 0.01). Scale bars, 200 µm. n = 50 to 100 neurons per group, from 3 embryos. (C-D), At DIV 2, in Arfgef1geo/geo neuron cultures, significantly fewer Golgi complexes were localized adjacent to the primary axons (***p = 0.0002) compared to those localized lateral to the primary axons (**p = 0.003). The Golgi complexes localized at basal quadrant were not significantly different between groups (p = 0.31). Scale bars, 20 µm. n = 50 neurons per group from 3 embryos. (E) At DIV 10, the Arfgef1geo/geo neurons established dendrites and axons as seen in control neurons. Scale bars, 50 µm. n = 50 neurons per group from 3 embryos. For all bar graphs, Student’s T-test was used for comparisons between groups. Data are shown as the mean ± SD.

Fig 4: Loss of BIG1 causes cell death of excitatory neurons.(A-C), Activated Caspase-3 colocalized with Tbr1 (white arrowheads) at (A) E13.5, (B) E15.5 and (C) E17.5 in Arfgef1geo/geo brains. Scale bars, 200 µm; *insets, 50 µm. n = 3 per group. Fro, apoptosis front; Max, maximum apoptosis area.

Fig 5: Myeloid cell-specific BIG1 KO protected mice from LPS-induced endotoxemic shock.WT and myeloid cell-specific BIG1 KO (BIG1 cKO) mice were intra-peritoneally administered with either LPS (50 mg/kg) or isometric saline. a Survival curves of LPS-treated WT and BIG1 cKO mice were calculated according to the Kaplan–Meier method. Survival analysis was performed using log-rank test (**P < 0.01; n = 10). b Body temperature of WT and BIG1 cKO mice were measured at the indicated time period. Mean ± SEM is presented and analyzed by Student’s t-test (***P < 0.001; n = 6). c Body weight loss of WT and BIG1 cKO mice were measured at the indicated time period. Mean ± SEM is presented and analyzed by Student’s t-test (***P < 0.001; n = 6). d The mRNA levels of the indicated cytokines in peritoneal macrophages from WT and BIG1 cKO mice injected intra-peritoneally with LPS (50 mg/kg) were measured by RT-qPCR at the indicated time period. Mean ± SEM is presented and analyzed by Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001; n = 6). e Blood was collected from WT and BIG1-cKO mice injected intra-peritoneally with LPS (50 mg/kg) at the indicated time points. Serum levels of the indicated cytokines from WT and BIG1 cKO mice were measured by ELISA at the indicated time point. Mean ± SEM is presented and analyzed by Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001; n = 6). f, g Liver and lung sections from WT and BIG1 cKO mice were subjected to H&E staining at the indicated time point. The representative images were shown (original magnification, ×400;Scale bars, 50 µm). The inflammatory infiltration score was quantified. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; n = 6; Student’s t-test.