fig4

Figure 4. mTOR and AMPK signaling were modulated by N-glycosylation inhibition in paclitaxel-resistant lung cancer cells. (A and B) Scatter plots showing the correlation between the N-glycosylation index and selected oncogenic signaling pathways, including the mTOR (A) and AMPK (B) pathways; (C and D) Representative western blots (C) and quantification (D) of p-mTOR and p-AMPK levels in A549 and A549-TR cells, n = 3; (E and F) Representative western blots (E) and quantification (F) of p-mTOR and p-AMPK in A549-TR cells treated with TM (0.1 or 0.5 μM) for 48 h, n = 3; (G and H) Relative cell growth of A549-TR (G) and H1299-TR (H) cells following treatment with RA or DO alone or in combination with 117 nM paclitaxel for 48 h, n = 3; (I and J) SFAs of A549-TR (I) and H1299-TR (J) cells after treatment with RA or DO alone or in combination with paclitaxel at the indicated concentrations, n = 4; (K and L) RT-qPCR analysis (K) and forest plot of gene-specific SMDs (L) of the expression of stemness-associated genes in A549-TR spheres treated with 1 µM RA or DO for 48 h; n = 3; (M and N) Representative western blots (M) and quantification (N) of stemness-associated proteins in A549-TR cells treated with 1 or 10 µM DO for 48 h. Statistical data were from three independent biological replicates (n = 3); (O and P) Representative western blots (O) and quantification (P) of stemness-associated proteins in A549-TR cells treated with 1 or 10 µM RA for 48 h. Statistical data were from three independent biological replicates (n = 3). n.s., not significant; ^*P < 0.05; ^**P < 0.01. mTOR: Mechanistic target of rapamycin; AMPK: AMP-activated protein kinase; TM: tunicamycin; RA: rapamycin; DO: dorsomorphin; SFAs: sphere-forming abilities; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SMDs: standardized mean differences; DMSO: dimethyl sulfoxide; n.d.: not detected; CIs: confidence intervals.