Fig 1: Circ_0013958 silencing hampered OC tumor growth in vivo. SKOV3 cells stably expressing sh-NC or sh-circ_0013958 were subcutaneously injected into BALB/c nude mice (n = 6). (A) Representative images of xenograft tumor tissues. (B) Tumor volume was measured every 7 days for 28 days. (C) Tumor weight was measured at 28-day post injection. (D) QRT-PCR analysis for the expression of circ_0013958 and miR-637 in xenograft tumor tissues. (E) Western blot assay for the protein level of PLXNB2 in xenograft tumor tissues. *P < 0.05; **P < 0.01.
Fig 2: Effect of circPLXNB2 expression on the levels of PLXNB2 and cell cycle-related proteins. a CircPLXNB2 OE significantly decreased the proportion of HL-60 cells in G0/G1 phase and increased the proportion of HL-60 cells in S phase. b The circPLXNB2 shRNA significantly increased the proportion of OCI-AML3 cells in G0/G1 phase and decreased the proportion of OCI-AML3 cells in S phase. c Representative FCM plots of the effects of circPLXNB2 expression on cell cycle progression. The expression of circPLXNB2 (d) and the PLXNB2 mRNA (e) was detected using qRT-PCR. f Representative Western blots showing the expression of BCL2, BAX, cyclin D1 and PLXNB2 in AML cells; ß-actin was used as a loading control. Nontransfected cells were used as a control, and the vector or shRNA NC was used as a negative control. Each experiment was repeated three times. ***P < 0.001 compared with the control. CircPLXNB2 OE circPLXNB2 overexpression, circPLXNB2 shRNA circPLXNB2 short hairpin RNA, NC negative control
Fig 3: Expression of circPLXNB2 and PLXNB2 mRNA, and characterization and distribution of circPLXNB2 in AML cells. The expression of circPLXNB2 (a) and the PLXNB2 mRNA (b) in different AML cell lines was detected using qRT-PCR; BMMCs from healthy volunteers served as controls. qRT-PCR analysis was used to verify that transfection of OCI-AML3 cells (c) and HL-60 cells (d) with circPLXNB2 shRNA or shRNA NC or vector or circPLXNB2 OE plasmid was successful. e AML cells were treated with or without RNase R, and qRT-PCR was used to assess circPLXNB2 amplification with divergent primers and convergent primers using the template cDNA and gDNA derived from AML cells. f qRT-PCR assay of the expression of circPLXNB2 and the PLXNB2 mRNA in AML cells treated with the transcriptional inhibitor actinomycin D (2 µg/mL) at the indicated time points. The PLXNB2 mRNA was used as a control. g Identification of the circPLXNB2 distribution in OCI-AML3 cells and HL-60 circPLXNB2 OE cells using RNA-FISH. h Quantification of nuclear and cytoplasmic levels of circPLXNB2 staining in AML cells. Nuclei were stained with DAPI. The original magnification was 400×. Each experiment was repeated three times. *P < 0.05, **P < 0.01, and ***P < 0.001. CircPLXNB2 OE circPLXNB2 overexpression, circPLXNB2 shRNA circPLXNB2 short hairpin RNA, NC negative control, DAPI 4,6-diamidino-2-phenylindole, cDNA complementary DNA, gDNA genomic DNA, ns no significance
Fig 4: Effect of circPLXNB2 expression on PLXNB2, apoptosis and proteins associated with cell cycle in vivo. a TUNEL-positive cells were measured in 3 randomly chosen fields from each biopsy (3 × 104 µm/field). qRT-PCR was used to detect the expression of circPLXNB2 (b) and the PLXNB2 mRNA (c) in vivo. d IHC staining of xenograft tumour tissues obtained from three mice randomly selected from each group (n = 6). Representative IHC images with an original magnification of 400× show the expression of PLXNB2 in different murine AML xenograft models. e Image software measured the average optical density of the selected area in sections subjected to IHC staining for PLXNB2. f Representative Western blots showing the expression of BCL2, BAX, cyclin D1 and PLXNB2 in different AML xenograft models. Western blotting was performed on the xenograft tumour tissues obtained from three mice randomly selected from each group (n = 6) and ß-actin was used as a loading control. The vector or shRNA NC was used as a control. Each experiment was repeated three times. **P < 0.01 and ***P < 0.001. CircPLXNB2 OE circPLXNB2 overexpression, circPLXNB2 shRNA circPLXNB2 short hairpin RNA
Fig 5: Circ_0013958 positively regulated PLXNB2 expression by absorbing miR-637. (A,B) Western blot assay for the protein level of PLXNB2 in SKOV3 and CAOV3 cells transfected with si-NC, si-circ_0013958, si-circ_0013958 + in-miR-con, or si-circ_0013958 + in-miR-637. *P < 0.05; **P < 0.01.
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