fig6

Figure 6. ST6GAL1 sustained CSLC-associated paclitaxel resistance through the EGFR-mTOR-SOX2/BMI1 axis. (A) RT-qPCR analysis of 7 N-glycosylation-related genes in A549-TR spheres treated with 0.1 or 0.5 μM TM, n = 3; (B) Representative western blots and quantification of ST6GAL1 in H1299 cells transduced with ST6GAL1. Statistical data were from three independent biological replicates (n = 3); (C and D) Representative western blots and quantification of p-mTOR (C), BMI1, and SOX2 (D) in H1299 cells transduced with ST6GAL1. Statistical data were from three independent biological replicates (n = 3); (E and F) Representative images (E) and quantification (F) of the SFA of H1299 cells transduced with ST6GAL1 and treated with 117 nM paclitaxel, n = 4; (G) Representative western blots and quantification of ST6GAL1 in A549-TR cells transduced with shST6GAL1. Statistical data were from three independent biological replicates (n = 3); (H and I) Representative western blots and quantification of p-mTOR (H), BMI1, and SOX2 (I) in A549-TR cells transduced with shST6GAL1. Statistical data were from three independent biological replicates (n = 3); (J and K) Representative images and quantification of the SFA of A549-TR cells transduced with shST6GAL1 and treated with 117 nM paclitaxel; n = 4; (L and M) Representative gating plots of EGFR (L) and MFI of EGFR expression (M) in A549-TR cells transduced with shST6GAL1, n =3; (N and O) Representative SNA lectin blots, corresponding EGFR immunoblots, and quantification of SNA signals normalized to immunoprecipitated EGFR in H1299 cells overexpressing ST6GAL1 (N) and A549-TR cells expressing shST6GAL1 (O). Normal IgG was used as a negative control for immunoprecipitation. Data were obtained from three independent biological replicates (n = 3); (P and Q) Representative gating plots (P) and quantification (Q) of CD104^-CD166⁺CD49f^hi CSLC subpopulations in A549-TR cells with ST6GAL1 knockdown, n = 3. n.s., not significant; ^*P < 0.05; ^**P < 0.01. CSLC: Cancer stem-like cell; EGFR: epidermal growth factor receptor; mTOR: mechanistic target of rapamycin; RT-qPCR: reverse transcription quantitative polymerase chain reaction; TM: tunicamycin; SFA: sphere-forming ability; MFI: mean fluorescence intensity; SNA: Sambucus nigra agglutinin; DMSO: dimethyl sulfoxide; sh1: shST6GAL1-1; sh2: shST6GAL1-2; IP: immunoprecipitation; CSC: cancer stem cell.