Journal: Cell Biology International
Article Title: FLOT1 Is a Novel Serum Biomarker of Ovarian Cancer Targeted by N6‐methyladenosine Modification Inhibition
doi: 10.1002/cbin.70015
Figure Lengend Snippet: Effect of m 6 A modification on FLOT1 expression and tumor formation. (A) Heatmap showing unsupervised clustering for 26 genes of m 6 A RNA modification in OC patients ( n = 376). Each column represents patients, and each row represents an m 6 A RNA modification regulator. (B) Heatmap of the correlation coefficient for the interaction between FLOT1 and the m 6 A RNA modification regulator. Red indicates a positive correlation, and green indicates a negative correlation. (C) Detection of FLOT1 mRNA expression in two human ovarian cancer cell lines (OVCAR‐3 and A2780) and a normal human ovarian surface epithelial cell line (IOSE‐80) by RT‐qPCR. Two‑way ANOVA followed by the Sidak test was used. The level of FLOT1 mRNA expression was higher in OVCAR‐3 and A2780 cells than in IOSE‐80 cells ( n = 3). (D) The levels of total m 6 A methylated RNAs in OC cells were verified by colorimetrically quantified ( n = 3). (E) Detection of the m 6 A modification level of FLOT1 mRNA in IOSE‐80, OVCAR‐3, and A2780. (F) Prediction of FLOT1 m 6 A modification sites by SRAMP. (G) Measurement of FLOT1 mRNA expression in OVCAR‐3 and A2780 cells detected by RT‐qPCR after 3‐deazaadenosine (3‐DAA) treatment. A Student t ‐test was used ( n = 3). (H) Detection of the m 6 A modification level of FLOT1 mRNA by MeRIP‑qPCR after 3‐DAA treatment. A Student t ‐test was used ( n = 3). (I) The workflow of ovarian cancer xenograft model construction and 3‐DAA treatment. (J) Xenograft tumor formation in nude mice. OVCAR‐3 cells were subcutaneously implanted in nude mice ( n = 5/group). Animals were photographed on day 30. Measurement of mouse weight. (K) Photo of executed tumors. Live measurement of tumor volume. (L) Images of the H&E and immunohistochemistry staining of FLOT1 and Ki67. Original magnification x200; Scale bar, 200 µm. Partial magnification x600; Scale bar, 50 µm. (M) IHC staining score (ISS) of FLOT1 and Ki67 protein expression. (N) Evaluation of FLOT1 protein expression by Western blot and semi‐quantification of the blot in the 3‐DAA treated (+) and untreated (−) groups. The data are shown in the form of the mean ± SD. A Student t ‐test was used. ns, not sigificance; * p < 0.05; ** p < 0.01; **** p < 0.0001.
Article Snippet: Co. Ltd., Fuzhou, Fujian, China) for 40 min at room temperature, the sections were incubated with a primary rabbit monoclonal anti‐human FLOT1 antibody (1:100 dilution; RRID: AB_11156367, Cat# ab133497, Abcam, Melbourne, Australia) at 4°C overnight, followed by a HRP‐conjugated anti‐rabbit secondary antibody (1:500 dilution; RRID: AB_2811189; Cat# GB23303, Servicebio, Wuhan, China) for 1 h at room temperature or a rabbit monoclonal anti‐human Ki67 antibody (1:100 dilution; RRID: AB_2834152; Cat# AF0198, Affinity Biosciences, OH, United States).
Techniques: Modification, Expressing, RNA modification, Quantitative RT-PCR, Methylation, Immunohistochemistry, Staining, Western Blot
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