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. 2024 May 31;25(11):6082. doi: 10.3390/ijms25116082

Figure 2.

Figure 2

Radiotherapy promotes ESCC-derived exosomal miR-143-3p secretion. (A–F) qRT-PCR analysis of the miR-143-3p, miR-181a-5p, and miR-223-3p levels in ESCC cells (KYSE150, KYSE510, and KYSE180) cell lines (A–C) or exosomes from culture medium (D–F) treated with or without 8 Gy of radiation and allowed to recover for 72 h; n = 3 samples per group. (G) Western blotting analysis to detect typical exosomal biomarkers (HSP70, TSG101, and CD9) in exosomes. KYSE150 and KYSE 510 cells were treated with 10 μM GW4869 (an inhibitor of exosome secretion) or dimethyl sulfoxide (DMSO) (as a control). After 72 h, the culture medium was collected. Exosomes were isolated through ultracentrifugation. (H) NanoSight particle tracking analysis of the exosome size distribution and numbers derived from the culture medium of KYSE150, KYSE510, and KYSE180 cells. (I) Phenotypic analysis of exosomes derived from the culture medium of KYSE150 cells using electron microscopy. (J) qRT-PCR analysis of miR-143-3p expression in the culture medium of KYSE150, KYSE510, and KYSE180 cells depleted of exosomes using GW4869 or DMSO (control). (K) qRT-PCR analysis of the miR-143-3p levels in the culture medium of KYSE150 and KYSE510 cells treated with RNaseA (2 mg/mL) alone or in combination with Triton X-100 (0.1%) for 30 min. Abbreviations: ESCC, esophageal squamous cell carcinoma; qRT-PCR, quantitative real-time reverse-transcription PCR; PBS, phosphate-buffered saline; ns, not significant. (***: p < 0.001, **: p < 0.01, *: p < 0.05, ns: not significant).