Fig 2: Hypoxia mediated regulation of key factors in the process of DNA dynamic methylation. (A) Proportion of 5-methylcytosine (5-mC) in total cytosine (Cyt) in 786-O and 769-P was detected after being treated with DAC for 72 h, in normoxia or hypoxia. The mRNA expression level of DNMT1, DNMT3, DNMT3b (B)and TETs (C)was significantly decreased during hypoxia in 786-O and 769-P compared with normoxia. Comparison of TET1 protein expression (D) and 5-hmC abundance (E) in 786-O, 769-P, Caki-1 and ACHN cell lines under hypoxia or normoxia by westernblot and dotblot separately. (F) The expression level of TET1 and OCT2 in 786-O and 769-P was detected by RT-PCR after TET1 knock-down and 72 h DAC treatment. (H) The expression level of OCT2 were assayed in 786-O and 769-P cell lines transfected with TET1 catalytic domain expression vector or empty vector. The mRNA expression level of TET1 (H) and the density of 5-hmC (G) were detected to ensure successful overexpression of TET1. TET1-CD, TET1 catalytic domain; N, normoxia; H, hypoxia.

Fig 3: Potential of miR-489-3p and miR-630 as candidate biomarkers. (A) Representative electron microscopy images of exosomes secreted by 786-O-OCT2 cells. Scale bar, 100 nm. (B) ZetaView particle tracking analysis of the size distributions and number of exosomes. (C) Western blot analysis of protein markers in cells and exosomes. GRP94 (ab3674, Abcam) was rarely expressed in exosomes but rich in cells. ALIX (ab117600, Abcam), TSG101 (sc-7694, Santa Cruz) and CD63 (ab59479, Abcam) were all often used as identification protein marker of exosomes. (D) MiRNAs expression in 786-O-OCT2-NC, 786-O-OCT2-489 and 786-O-OCT2-630 cell lines and respective exosomes.

Fig 4: Effects of re-oxygenation on hypoxia-induced DAC resistance. (A) Quantitative RT-PCR analysis of SLC22A2 mRNA in 786-O and 769-P, treated with 2.5µmol/L DAC for 72h in normoxia, after exposed under hypoxia for 24 or 48 h and The protein expression level of ENT1 in 786-O, 769-P and Caki-1, exposed under normoxia for indicated time, after exposed under hypoxia for 24 h. (B) Schematic of Hemoglobin-based nanocarrier preparation and structure. (C) Western blotting of ENT1 treated by H-NPs in 769-P under hypoxia. (D) MSRE-qPCR analysis of E-BOX methylation frequency in 769-P cell lines, treated with DAC, in the presence or absence of H-NPs, for 72 h in hypoxia separately. The mRNA (E) and protein (F) level expression of OCT2 and oxaliplatin uptake (G) in 769-P cell lines, treated with DAC, in the presence or absence of H-NPs, for 72 h in hypoxia separately. (H) Representative in-vivo and ex-vivo fluorescence images of nude mice treated with saline, Free ICG and ICG-H-NPs 5 h post injection. (I) The z-stacking images of the 769-P spheroid at different sections from 1 to 55 µm, sections from 20-40 µm had high fluorescence intensity and confocal microscope images of spheroid core and 3D graphics constructed by Imaris 9.3.1. The high fluorescence intensity at the core of 769-P spheroid which arrowed in 3D-plot indicated the H-NPs could alleviate hypoxic core of the solid tumor in vivo.

Fig 5: C-Myc upregulates miR-630 expression by binding to its promoter region. (A) and (B) Comparison of C-Myc mRNA and protein expression in human matched renal normal-tumour samples. (C) The expression of C-Myc, pri-miR-630, pre-miR-630 and OCT2 in c-Myc knockdown 786-O cells. (D) The uptake of MPP+ in c-Myc knockdown 786-O cells. (E) Chip-qPCR analysis of c-Myc at the promoter of miR-630 in c-Myc knockdown cell model and 786-O cells that transiently transfected with c-Myc expression plasmid or empty expression vector (pENTER). IgG, immunoglobulin G. (F) The expression of C-Myc, pri-miR-630 and abundance of c-Myc occupied around E2 in human matched renal normal-tumor samples. (G) Relative light unit (RLU) was detected in HEK293 cells transfected with wild pri-miR-630 promoter (gray) or E-Box element (CACGTG) mutated (white) reporter plasmid along with either c-Myc or empty expression vector (pENTER).