Fig 2: Generation and initial characterization of Tbc1d7 knockout mice(A and B) Scheme for CRISPR-Cas9 targeting of Tbc1d7, with the identified 35-bp out-of-frame deletion (A) denoted and (B) detected by PCR genotyping.(C and D) Immunoblots of tissue lysates from littermate Tbc1d7+/+ and Tbc1d7-/- mice at 6 weeks of age following a 6 h daytime fast. Phospho-S6 to total S6 ratios are quantified in (D) as mean ± SD. n = 3.(E) Genotypes of 368 offspring of crosses between Tbc1d7+/- mice.(F) Body weights from a cohort of littermate offspring from Tbc1d7+/- crosses over 13 months graphed as mean ± SD at each age. n = 13 Tbc1d7+/+, 19 Tbc1d7+/-, and 12 Tbc1d7-/-. Age 0 is the weight at weaning (21 days).(G) DEXA scan analysis of the mice in (F) at 6 months of age, with the lean-to-fat-mass ratio graphed as mean ± SD.Statistical analysis by one-way ANOVA, *p < 0.05.

Fig 3: Loss of TBC1D7 leads to brain overgrowth(A and B) Representative brains from each genotype at 13 months (A) with brain weights graphed as mean ± SD. (B). n = 12 Tbc1d7+/+, 8 Tbc1d7++/-, and 13 Tbc1d7-/-.(C) Brain weights at 6 weeks graphed as mean ± SD. n = 11 Tbc1d7+/+ and 13 Tbc1d7-/-.(D) Brain weights at E16.5 graphed as mean ± SD. n = 8 Tbc1d7+/+ and 7 Tbc1d7-/-.(E and F) Cortical thickness measured at the primary motor cortex in sagittal brain sections from 13-month-old mice is graphed as mean ± SD (E). n = 11 Tbc1d7+/+, 7 Tbc1d7+/- and 9 Tbc1d7-/-. Representative H&E-stained sections are shown, with the cortical region measured denoted with a dashed line (F, top). Scale bars, 500 µm.(G and H) Immunofluorescence imaging of brain sections from 8-month-old mice stained as indicated (G and H). Scale bars, 100 (G) and 50 µm (H).(I–O) Measurement of thickness for layer IV/V (I), layer VI (J), and total cortex (K) and ratio of layer IV/V (L) or layer VI (M) to total cortex, and quantification of neuronal density in these layers (N and O) were graphed as mean ± SEM. n = 7 Tbc1d7+/+ and 5 Tbc1d7-/-.(P) Immunofluorescence imaging of brain sections from 8-month-old mice stained as indicated. Inset shows a 5× magnification. Scale bar, 100 µm.Statistical analysis by one-way ANOVA (B and E) and Student’s t test (C, D, and I–K), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: Muscle and gait analysis of Tbc1d7 knockout mice(A–C) Histological cross-sections of muscle from 6-week-old mice stained as indicated. Scale bars, 100 µm. (B) Cross-sectional muscle-fiber area graphed as mean ± SD.(C) Quantification of muscle-fiber types graphed as mean percentage of total ± SD. n = 4.(D and E) Front-limb (D) and hind-limb (E) grip strength measured at 6 weeks and 6 months of age graphed as mean ± SD. 6 week: n = 9 Tbc1d7+/+ and 13 Tbc1d7-/- 6 month: n = 13 Tbc1d7+/+ and 12 Tbc1d7-/-.(F and G) Open-field test of 6-month-old mice, with (F) distance traveled and (G) speed graphed as mean ± SD. n = 12.(H–K) Video-recorded gait analysis of 6-month-old mice with (I) tail angle, (J) stride length, and (K) stride offset graphed as mean ± SD. n = 5.Statistical analysis (B, E–G, and I–K) by Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: KIF2C expression is upregulated by Wnt/β-catenin signaling in HCC. (A) Western blot analyses showing that activation of Wnt/β-catenin by Wnt3a increases the expression level of KIF2C protein in HepG2 and SNU387 cells. (B) The effect of LiCl treatment on the expression of KIF2C in HepG2 and SNU387 cells as examined by Western blot. (C) Western blot analysis of the expression level of KIF2C protein in SK-Hep1 cells treated with Wnt antagonists (including Dvl-PDZ domain inhibitor II, XAV939 and iCRT3). (D) Schematic illustration of the luciferase reporters driven by the KIF2C promoter and its mutants. (E) Dual-luciferase assay in 293T cells suggests that the putative TCF4 binding site is essential for the responsiveness of KIF2C promoter to Wnt3a treatment. Data are presented as mean ± SD of three independent experiments, ns, non-significant, **P < 0.01, Student’s t-test. (F) ChIP assay performed in SK-Hep1 cells with anti-TCF4 antibody or control IgG. c-Myc acts as a positive control. Data are presented as mean ± SD of three independent experiments, ***P < 0.001, Student’s t-test. (G) Western blot assay revealing that overexpression of KIF2C restores iCRT3-inhibited phosphorylation of mTOR1, p70 S6K and 4EBP1in HepG2 cells. (H) Wnt3a-enhanced mTORC1 signaling is dismissed by knockdown of KIF2C in SK-Hep1 cells. (I) The proposed model of the function of KIF2C in the activation of mTORC1 signaling during HCC pathogenesis. KIF2C is transcriptionally activated by Wnt/β-catenin signaling, then interacts with TBC1D7 to disrupt the stability of the TSC complex, thereby enhancing mTORC1 signaling and HCC progression. (J and K) Cell viability assay (CCK-8 assay) shows the effect of INK128 on KIF2C-depleted SK-Hep1 (J) and KIF2C-overexpressing HepG2 (K) cells. Cells were treated with INK128 for 72 h and measured by CCK-8. (L and M) The effect of INK128 on KIF2C-depleted SK-Hep1 (L) and KIF2C-overexpressing HepG2 (M) cells was detected by colony formation assay