Fig 2: Lzts1 overexpression positions cells outside the VZ. a Experimental design. b Lzts1 overexpression (1.0 µg µl-1) promotes the detachment of cells from the VZ surface. Cell morphology was visualized by Cre-mediated Lyn-EGFP (membrane-targeted form of EGFP), which was co-electroporated (2 days after E13 electroporation). c An example of GFP+ Sox2+ cells in the SVZ that have long basal processes, which is the typical morphology of oRGs. d–m The Lzts1 expression vector (1.0 µg µl-1, indicated as “Lzts1 1.0” or 0.2 µg µl-1, indicated as “Lzts1 0.2”) was in vivo electroporated at E13, and E15 brain sections were examined by IHC with anti-Ki67 e, anti-Sox2 f, anti-Tbr2 g and l, anti-PH3 h and i, anti-BrdU (30 min of BrdU pulse labeling) j, and anti-Pax6 k antibodies. d Distribution of Lzts1-overexpressing cells in 10 bins throughout the cerebral wall. f Forced expression of Lzts1 at both high (1.0 µg µl-1) and moderate (0.2 µg µl-1) levels increased the number of Sox2+ GFP+ cells in the SVZ. h and i Lzts1 significantly increases the percentage of non-apical PH3+ mitotic cells in a dose-dependent manner (Fisher’s exact test, two-sided, with Bonferroni-adjusted P value p < 0.05/6 = 0.0083). The asterisk in h indicates a PH3+ GFP+ cell. m The mitotic index (MI) indicates the percentage of Ki67+ cells among BrdU+ GFP+ cells that received BrdU 20 h before fixation. d N = 8 sections from 4 embryos per experiment; iN = 343, 56, 46, and 91 cells from 11, 9, 6, and 12 brain sections from 7, 4, 3, 4 embryos, respectively; j N = 8, 6; k N = 8, 6; l N = 6, 6; and m N = 4, 6 sections from three embryos per experiment, d *p < 0.05 and **p < 0.01, d and j–m Wilcoxon rank sum test. Bars, 50 µm in b, and f–h, 30 µm in c, 100 µm in e. Means ± s.d. Source data are provided as a Source Data file

Fig 3: Variable expression of Lzts1 is responsible for generation of oRG-like cells. a lzts1 is expressed in a subset of aRGs within the developmental time window. Single-cell transcriptome profiles24, 25 revealed that 7/33 aRGs are lzts1+ (Log value > 6.0) at E14. Variances of the values are significantly different between E11 and E14, as well as between E14 and E16 aRGs (F-test, p = 4.1 × 10-8, p = 0.0038, N = 24, 33 and 17, respectively, bars indicate means). b–d Variations in gene expression in lzts1+ aRGs (N = 7) and lzts1- aRGs (N = 26). Significantly lower hes1 expression was observed in lzts1+ aRGs than in lzts1- aRGs (p = 0.045), but pax6 (p = 0.36) (c) and neurog2 (p = 0.68) d expression were not significantly altered (permuted Brunner–Munzel test). As the references, data from IPs are also shown. Data from single-cell transcriptome profiles24. See also Supplementary Figure 8. e Lzts1 KO decreases the division angles (?) of aRGs. N = 193 and 193 cells from 10 and 9 embryos, respectively, Brunner–Munzel test, medians with Q1 and Q3 values. f Lzts1 KO reduces the oblique division of aRGs. hCas9, the guide RNA for Lzts1, ZO1-EGFP, and PACT1-mCyRFP were expressed by electroporation at E13, and after 2 days, en face imaging of the apical surface was performed to observe the aRG division pattern (images were obtained from four and three embryos, two-sided Fisher’s exact test). g–i Lzts1 KD impairs the generation of Sox2+ GFP+ cells outside the VZ. The percentage of Sox2+ cells among GFP+ cells present in the SVZ (and IZ) was examined 2 days after electroporation. Bar, 30 µm. Arrowheads in the magnified views (g, h; Bar, 10 µm) indicate Sox2+ GFP+ cells. i The percentage of Sox2+ GFP+ cells in the SVZ (and IZ) was significantly reduced by Lzts1 KD following electroporation at E14, and this reduction was rescued by the introduction of the siRNA-resistant Lzts1, but not wild-type Lzts1 (N = 12, 9, 12, 12, 14, and 12 sections from 4, 3, 4, 4, 5, and 4 embryos, respectively; medians with Q1 and Q3 values are shown; Wilcoxon exact test at E13 and Steel–Dwass test at E14). Source data are provided as a Source Data file

Fig 4: The phosphorylation of PTK6 promotes CRC cell stemness. A Western blot analyses were performed to verify the successful construction of FLAG-tagged empty vector, wild-type (WT), kinase-dead (KM) and constitutively active (YF) PTK6 recombinant mutants overexpression CRC cells. B In vitro limiting dilution assays show the effects of phosphorylated PTK6 on the formation of CSC spheres (mean ± SD, n = 12), likelihood ratio test. C Tumor sphere formation assays were performed to detect the sphere-forming ability of vector, PTK6WT, PTK6KM and PTK6YF overexpression CRC cells (mean ± SD, n = 3). D Real-time q-PCR analysis shows the effects of phosphorylated PTK6 on the expression of stem cell markers in CRC cells (mean ± SD, n = 3). E Western blot assays were performed to detect the effects of phosphorylated PTK6 on the expression of stem cell markers in CRC cells. F The number of CD133 + or SOX2 + cells was evaluated in the vector, PTK6WT, PTK6KM and PTK6YF overexpression CRC cells by flow cytometry (mean ± SD, n = 3). G Co-immunofluorescent staining of PTK6 (red), CD133 (green) and EPACAM (green) in CRC cells. Scale bar represents 5 µm. H Co-immunofluorescent staining of PTK6 (red), CD133 (green) and EPACAM (green) in CRC tumor spheres. Scale bar = 5 µm. I In vivo limiting dilution analysis was performed to detect the tumorigenicity of vector, PTK6WT, PTK6KM and PTK6YF overexpression CRC cells in nude mice (mean ± SD, n = 7). J IHC staining was used to detect the expression of PY342, SOX2 and Ki67 in indicated subcutaneous tumors of nude mice. Scale bar represents 50 µm. *P < 0.05, **P < 0.01, ***P < 0.001, # indicates P > 0.05

Fig 5: Infant gastric digestion kills all cell types equally, except for immune cells, which are less resilient to digestion. A) Cell populations were identified using antibody staining and subsequent flow cytometry analysis. The relative percentages of defined viable cell populations in milk before and after digestion did not change, with the exception of immune cells, which did not survive digestion. (n = 6 replicates from milk expressed on different days). B) The ALDEFLUOR assay measured stem cell populations before and after simulated digestion. High ALDH signal indicates stem-like phenotypes. ALDEFLUOR staining found no change in stemness following digestion. DEAB is an inhibitor of ALDH and was used to control for background fluorescence (n = 5). C) Digested and undigested milk cells were analyzed by flow cytometry for the presence of stem and progenitor cell markers NANOG, SOX2, CD49f, and TRA-1-60. No changes in stemness were detected following digestion (n = 5). D) Digested and undigested milk cells were analyzed by qPCR for the presence of stem cell markers. No significant changes were detected (n = 5). In all panels, significance was determined by Student's t test compared to undigested control cells, with *p < 0.05. Error bars represent standard deviation.