Fig 2: Expression of trilineage differentiation markers. A) Matched iPSCs and differentiated endoderm, mesoderm, ectoderm cells were analyzed by immunostaining followed by flow cytometry for expression of markers specific for each germ layer (mean ± SEM, n = 7–10 cell lines). See also Fig. S1 for isotype control data, Fig. S3 for morphological changes after differentiation, Fig. S4 for paired data, and Fig. S5 for independent validation of iPSC NES staining. B) Representative flow cytometry plots showing co-immunostaining of pluripotency marker POU5F1 with each of the four candidate ectoderm markers in undifferentiated iPSCs. See also Fig. S6 for complete dataset. C, D) Expression of pluripotent marker POU5F1 (C) and four candidate ectoderm markers (D) quantified by qPCR in matched iPSCs and ectoderm differentiated cells (mean ± SEM, n = 8). Gene expression is shown as fold change of ectoderm differentiated cells compared to iPSCs, with dotted line indicating fold change of 1. * P < .05, *** P < .001, **** P < .0001, two-tailed paired t-tests.

Fig 3: Anti-tumor effect of XPC on A549 tumor-bearing nude mice. (A). Xenografts were generated by A549 cells (shCtrl), A549 cells with stable XPC knockdown (shXPC). Tumors were removed from mice after 32 days. (B). Tumor sizes were measured every 4 days. (C). The expression of CD133 and OCT4 were determined using Flow Cytometry. p < 0.0001(CD133), p < 0.0001(OCT4), A549-shXPC compared with A549-shCtrl. (D). The expression of XPC and CD133 were determined using Western blotting. The intensity of each band was quantified using Image J and normalized to. Protein extractions come from xenotransplanted tumor tissue of mice. ***p < 0.001 (XPC), *p < 0.05(CD133), A549-shXPC compared with A549-shCtrl. (n = 6).

Fig 4: The effect of XPC expression on stem cell markers. (A). The 3 cell lines A549, H1299, and H1650 were divided into two groups: siCtrl group and siXPC group. Western blot was used to detect the expression of stem cell markers in the siCtrl group and siXPC group. (B). In the 3 cell lines A549, H1299, and H1650, the expression of XPC in the siXPC group was lower than that in the siCtrl group (**p < 0.01, ***p < 0.001, ****p < 0.0001). (C). In the A549 and H1650 cell lines, the expression of OCT4 in the siXPC group was higher than that in the siCtrol group (*p < 0.05, ****p < 0.0001). (D). In the 3 cell lines A549, H1299, and H1650, the expression of CD133 in the siXPC group was higher than that in the siCtrl group (*p < 0.05, **p < 0.01). (E,F). The protein levels of CD133 and OCT4 were determined using Flow Cytometry. p < 0.0001(CD133), p < 0.0001(OCT4), A549-shXPC compared with A549-shCtrl. (G). Immunohistochemical detection of XPC and CD133 expression correlation in 140 cases of lung adenocarcinoma tissues (p < 0.0006, R = -0.2580).

Fig 5: Generation of Oct4-conditional knockout human iPS cells. (A) Wild-type cells (+/+) or cells bearing the artificial intron in exon 1 of Oct4 (Oct4-DECAI #1 and Oct4-DECAI #2, created with two independent gRNAs) were transfected with Cre-ERT2 recombinase and 4-OHT or left untreated. Cells were fixed in 4% PFA and stained for Oct4 and SSEA4 using specific antibodies. DAPI was used for nuclear staining. Images were acquired with an Olympus IX83 microscope (10X magnification, 100 µm scale bars). For bright field images see Supplementary Figure 5. (B) Flow cytometry analysis of the cells before (No Cre) and after Cre treatment (+Cre) stained with Oct4 and SSEA4 specific antibodies. (C) qRT-PCR analysis was performed before and after induction of Cre recombination. Expression levels of Oct4, two additional pluripotency markers (Nanog and Sox2), and two differentiation markers (Cdx2 and Eomes) are shown. qRT-PCR values are normalized to GAPDH and shown as relative to the highest value of that target across all samples.