Fig. 3

AHCYL1 expression in 3D-culture in A549 cell line and cell differentiation states of stably silenced AHCYL1 cells. A Representative phase-contrast microscopy images of human A549 LC cells grown as monolayers (2D) and spheroids (3D) culture enriched in LCSCs at 3 and 7 days. Ref: 1 mm. B Western Blot analysis of AHCYL1 (60 kDa), POU5F1 (48 kDa), and CD44 (75 kDa) protein levels of 2D and 3D culture of A549 lung adenocarcinoma cell line. GAPDH (37 kDa) was used as a loading control. The samples correspond to spheroids of 7 days. The blot corresponds to a representative experiment of three. C FACS analysis of CD133 expression in A549 monolayer vs spheres cultures. A549 spheres are enriched in CD133 expression. D qRT-PCR analysis of AHCYL1, stem cell markers POU5F1 and CD44 and lung marker MUC5B. Gene expression levels in sphere were normalized to their expression in monolayer cultures. RPL19 was used as a normalization control. T-test with Welch's correction (n = 3–5). E qRT-PCR analysis of KD-AL1-1, AL1-2, AL-1-3, and AL-1-4 A549 cells lines showing significantly decreased expression of AHCYL1 mRNA levels compared to non-targeting (NT) control cells. RPL19 was used as a normalization control. ANOVA followed by Dunnet's test (n = 3). Western blot analysis showing AHCYL1 protein level decreased for each line and increased POU5F1 (48 kDa) protein level. GAPDH (37 kDa) was used as a loading control. The blot corresponds to a representative experiment of three. F RT-qPCR analyzing the expression of the pluripotency markers POU5F1, AHCY, CD133, and MUC5B as differentiation marker in the lung in KD-AL1-2 and KD-AL1-4 cells compared to NT control cells. RPL19 was used as a normalization control. ANOVA followed by Dunnet’s test (n = 3). G Stem cell frequency was calculated using online Extreme Limiting Dilutions Assay (ELDA) analysis program. Significant differences in stem cell frequencies was determined between NT (1/49.83) and KD-AL1-2 (1/13.10) or KD-AL-1–4 (1/7.46) cells. The graph corresponds to a representative test (n = 3, p ≤ 0.001, in six replicates). The solid line shows the mean and the dotted lines show the confidence interval. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001