Fig 2: Human telomerase reverse transcriptase (hTERT) increases NRF2 expression by recruiting YBX1 to bind to the NRF2 promoter. (A) Flow chart for screening of potential hTERT-recruited NRF2 transcription factors. (B) The YBX1 and NRF2 mRNA expression level was identified by qRT-PCR after downregulation of YBX1. (C) Luciferase activity of the NRF2 promoter was detected after downregulation of YBX1. (D) NRF2 promoter with a 5' biotin label was used to pull-down YBX1. Mock beads were used as a negative control. (E) Diagrammatic drawing of NRF2 P2 fragment. The P2 fragment was divided into 5 fragments. (F) YBX1 antibody was used to immunoprecipitate binding fragments of the NRF2 promoter under the condition of hTERT overexpression and fragments were identified by ChIP-qPCR with six primers (Left). Statistical analysis of ChIP-qPCR. (G) Luciferase activity of P2 fragment containing different mutant sites was detected after YBX1 overexpression. (H) HCT116 cell lysates were prepared for separate IP with hTERT and YBX1 antibody and then evaluated via western blotting. (I) The subcellular localization and the colocalization of hTERT and YBX1 were examined in SW620 cells via dual immunofluorescence using confocal microscopy. (J) YBX1 in cell nuclei and cytoplasm was identified by western blotting after overexpression of Flag-hTERT. (K) The NRF2 mRNA expression level was identified by qRT-PCR after overexpression of hTERT and simultaneous knockdown of YBX1. (L) Luciferase activity of the NRF2 promoter was detected after overexpression of hTERT and simultaneous knockdown of YBX1. (M) The NRF2 protein level was identified by western blotting after overexpression of hTERT and simultaneous knockdown of YBX1 (Left). Statistical analysis of western blotting (Right). *p < 0.05; **p < 0.01; ***p < 0.001; ns, no significance.

Fig 3: YBX1 is responsible for upregulation of NRF2 expression and CRC proliferation and migration. (A) The YBX1 and NRF2 mRNA expression level were identified by qRT-PCR after downregulation of YBX1 and simultaneous overexpression of NRF2. (B) The YBX1 and NRF2 protein expression level were identified via western blotting after downregulation of YBX1 and simultaneous overexpression of NRF2 (Left). Statistical analysis of western blotting (Right). (C) CCK8 assays were performed to detect cell proliferation after downregulation of YBX1 but an increase in NRF2 expression. (D) Colony formation assays were performed after downregulation of YBX1 but an increase in NRF2 expression (Left). Statistical analysis of the colony numbers (Right). (E) Migration and invasion assays were performed after downregulation of YBX1 but an increase in NRF2 expression. (F) Statistical analysis of the migration cell numbers. (G) Statistical analysis of the invasion cell numbers. (H) The YBX1 and NRF2 mRNA expression level were identified by qRT-PCR after overexpression of YBX1 and simultaneous downregulation of NRF2. (I) The YBX1 and NRF2 protein expression level were identified via western blotting after overexpression of YBX1 and simultaneous downregulation of NRF2 (Left). Statistical analysis of western blotting (Right). (J) CCK8 assays were performed to detect cell proliferation after overexpression of YBX1 and downregulation of NRF2. (K) Colony formation assays were performed after overexpression of YBX1 and downregulation of NRF2 (Left). Statistical analysis of the colony numbers (Right). (L) Migration and invasion assays were performed after overexpression of YBX1 and downregulation of NRF2. (M) Statistical analysis of the migration cell numbers. (N) Statistical analysis of the invasion cell numbers. *p < 0.05; **p < 0.01; ***p < 0.001; ns, no significance.

Fig 4: YBX1 is highly expressed in CRC and associated with poor prognosis. (A) YBX1 expression in CRC tissues and adjacent normal tissues was investigated in Oncomine database. (B) YBX1 mRNA expression in CRC tissues and paired adjacent normal tissues was identified by qRT-PCR. (C) Regulation analysis of the correlation between YBX1 and NRF2. Each point represents one cancer sample. (D) Representative immunohistochemical staining and expression level statistics of YBX1 in CRC tissues and paired adjacent normal tissues. (E) Regulation analysis of the correlation between YBX1 and NRF2. Each point represents one cancer sample. (F) Kaplan–Meier analysis of the overall survival of CRC patients with different YBX1 expression levels (p < 0.05, log-rank test). (G) Kaplan–Meier analysis of the overall survival of CRC patients with different YBX1 and NRF2 expression levels (p < 0.05, log-rank test). *p < 0.05;**p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no significance.

Fig 5: The effect of YB-1 on cell morphology, proliferation, and cell cycle in MCF-7 and MCF-7/ADR cells. (a) The expression of YB-1 in four group cells. (b) The expression of YB-1 in MCF-7 and MCF-7/ADR cells treated with YB-1 overexpression and knockdown. 1: MCF-7; 2: MCF-7/ADR; 3: MCF-7/ADR+YB-1 overexpression; 4: MCF-7/ADR+YB-1 knockdown; 5: MCF-7/ADR+YB-1 EV. (c) Cell morphology was observed by optical microscopy (magnification 100x). (d) Cell proliferation was detected by CCK-8. (e) Cell progression was examined by flow cytometry. Value was presented for three times to conduct statistical analysis. N = 3, * means P < 0.05, ** means P < 0.01, and *** means P < 0.001.