Figures & data
Figure 1. Co-culture with M2-polarized macrophages enhanced the resistance of gastric carcinoma cells to OXA. Human monomyelocytic leukemia (THP-1) cells were stimulated to obtain M2 polarized macrophages. (A) the differentiation of macrophages was detected by flow cytometry. (B) the mRNA expression levels of CD206, Arg1, IL-10, and TGF-β in macrophages were detected by qRT-PCR. *P <0.05 vs. Un-Mac. (C) the cell viability was determined by CCK8. *P <0.05 vs. 24 h or 48 h. (D) the cell apoptosis was detected by flow cytometry. *P <0.05 vs. Un-Mac. B:Unpaired t test; C: Two-way ANOVA followed by Tukey's multiple comparisons test; D: Brown-Forsythe and Welch's ANOVA test followed by Unpaired t with Welch's correction.
Figure 2. Exosomes derived from M2-polarized macrophages can be absorbed by gastric carcinoma cells. Exosomes were isolated from M2 macrophages. (A) the morphology of exosomes was observed in transmission electron microscopy. (B) the size of exosomes was assessed by nanoparticle tracking analysis. (C) the markers of exosomes were detected using Western blot. (D) Immunofluorescence staining was performed.
Figure 3. Exosomes derived from M2-polarized macrophages induced OXA resistance in gastric carcinoma cells. Gastric carcinoma cells (KATO III and MKN45) were incubated with exosomes (50 μg/ml) from M2 macrophages for 48 h, followed by treatment with OXA (15 μM) for 48 h. (A) the cell viability was determined by CCK8. (B) the cell apoptosis was detected by flow cytometry. Gastric carcinoma cells were co-cultured with exosomes from M2 macrophages, followed by injected subcutaneously to male nude mice as well as OXA treatment. mice were grouped: Con, M2-Exo, OXA, and OXA + M2-Exo. N = 5. (C) the tumor growth was analyzed. *P <0.05. A/C: Brown-Forsythe and Welch's ANOVA test followed by Unpaired t with Welch's correction; B: Unpaired t test.
Figure 4. Circ 0008253 was transferred from exosomes from M2 macrophages to gastric carcinoma cells. (A) the candidate circRNA hsa_circ_0008253 was screened by bioinformatics analysis. (B) Levels of circ 0008253 in cells were detected using qRT-PCR. *P <0.05 vs. KATO III, or MKN45, or Un-Mac. (C) Levels of circ 0008253 in clinical samples (n = 10) of gastric cancer patients were detected using qRT-PCR. *P <0.05 vs. adjacent. (D) Levels of circ 0008253 in macrophages were detected using qRT-PCR. *P <0.05 vs. Un-Mac Exo. Gastric carcinoma cells were co-cultured with M2-polarized macrophages or M2-Exos, followed by treatment with or without ActD (an inhibitor of endogenous RNA production). (E) Levels of circ 0008253 in cells were detected using qRT-PCR. *P <0.05 vs. KATO III or MKN45. B/E: Brown-Forsythe and Welch's ANOVA test followed by Unpaired t with Welch's correction; C: Paired t test; D: Unpaired t test.
Figure 5. Circ 0008253 induced OXA resistance in gastric carcinoma cells. Gastric carcinoma cells (KATO III and MKN45) were transfected with circ 0008253 overexpression vector or its negative control. (A) Levels of circ 0008253 in gastric carcinoma cells were detected using qRT-PCR. (B) CCK-8 assay was performed. (C)the colony formation assay was performed in gastric carcinoma cells KATO III and MKN45. Gastric carcinoma cells MKN45 were transfected with circ 0008253 overexpression vector or its negative control, followed by subcutaneously injected into nude mice. One week later, mice were injected with 5 mg/kg OXA. Mice were grouped: NC, NC + OXA, circ, circ + OXA. N = 5. (D) the tumor growth was analyzed. (E) Levels of ABCB1 and ABCG2 in gastric carcinoma cells KATO III and MKN45 were determined. ns P >0.05; *P <0.05. A/B: Brown-Forsythe and Welch's ANOVA test followed by Unpaired t with Welch's correction; C/D: Two-way ANOVA followed by Tukey's multiple comparisons test; E: Unpaired t test or Unpaired Welch's t test.
