Fig 1: 2'-O-methylation on Pxdn mRNA requires fibrillarin. a 2'-O-methylation (Nm) can occur in combination with any base. Nucleoside Am is shown, compared to base-modified m6A. b UPLC-MS/MS quantification of modified nucleosides on total RNA, mRNA after two rounds of oligo-dT selection, and mRNA further purified by rRNA depletion. c Predicted interaction between human snoRNA U32A (U32A) and peroxidasin (Pxdn). A3150 is the predicted Nm target (red), which is the first position of a lysine codon (K). d Illustration of RTL-P method. The amount of qPCR product from the low dNTP reactions of two different samples can be compared to establish the relative “RTL-P efficiency (RQ)”. RQ = relative quantity. See Methods for detail. e–f Fbl knockdown (KD) in HeLa cells, vs. negative control (ctrl) using siRNA for 48 h. n = 3 independent experiments, SEM error bars, *p < 0.05 by unpaired t-test. e Fbl KD leads to loss of Nm on Pxdn mRNA (p = 0.0005). f Fbl KD reduces Pxdn mRNA levels, normalized to Rplp0 control transcript (p = 0.0002)
Fig 2: Knockdown of FBL suppresses hepatocellular carcinoma cell growth in vivo. (A) The knockdown efficiency of FBL knockdown lentivirus (shFBL) on FBL in Huh7 cells was detected by Western blotting analysis. shNC is the control group. (B) Gross tumors excised from the nude mice subcutaneously injected with Huh7 cells stably transfected with shNC or shFBL.. The smallest scale division of the ruler is 0.1 mm. (C) Tumor growth curve chart, tumor sizes in nude mice subcutaneously injected with Huh7 cells stably transfected with shNC or shFBL were measured at days 14, 21, 24, 27, and 30. (D) Weights of gross tumors excised from the nude mice subcutaneously injected with Huh7 cells stably transfected with shNC or shFBL. * p < 0.05, ** p < 0.01, *** p < 0.001. (A,C,D) Student’s t test.
Fig 3: FBL is highly expressed in hepatocellular carcinoma, and high expression of FBL is positively correlated with the occurrence of lung metastasis, indicating a poor prognosis in our cohort. (A) Western blotting analysis of FBL protein expression in hepatocellular carcinoma and adjacent liver tissues. GAPDH was used as an internal reference. In each band, normal tissue is shown on the left, and tumor tissue is shown on the right. (B) RT-qPCR analysis of FBL mRNA expression in hepatocellular carcinoma tissue and adjacent liver tissue. The ordinate is -?t (the number of PCR cycles of FBL minus the number of PCR cycles of the internal control GAPDH). (C) Representative images of hepatocellular carcinoma paraffin specimens stained by the immunohistochemical method that display the different expression levels of FBL. (D) Comparison of the incidence of lung metastasis in hepatocellular carcinoma patients with high or low expression of FBL. (E, F) Overall survival (OS) and disease-free survival (DFS) between FBL high expression and low expression groups of hepatocellular carcinoma patients in our cohort. * p < 0.05, ** p < 0.01, *** p < 0.001. (A) Student’s t test. (B) paired t-test. (D) ?2 test. (E,F) Log-rank test.
Fig 4: Knockdown of FBL suppresses migration and invasion in HCC cell lines. (A, B) The effect of FBL knockdown on the migration ability of Huh7 and PLC/PRF/5 cells was verified by a scratch wound healing assay. Forty-eight hours after scratching, the differences in the degree of wound healing between the two knockdown FBL experimental groups (siFBL#1 and siFBL#2) and the control group (siNC) transfected with control siRNA were detected. (C) The effect of FBL knockdown on the migration ability of Huh7 and PLC/PRF/5 cells was verified by the Transwell migration assay. Scale bar: 100 μm. (D) The effect of FBL knockdown on the invasion ability of Huh7 and PLC/PRF/5 cells was verified by the Transwell invasion assay. Scale bar: 100 μm. These experiments were performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001. (A–D) random block design analysis.
Fig 5: Schematic summarizing key observations of the current study.NIC4 mobility in the nucleus and nucleolus is dynamic with NIC4 localization to the nucleolus, guided by its NoLS. NIC4 associates with NCL and NPM, and protection from genomic stressors is dependent on nucleolar proteins NCL and FBL. Nucleolar functions can be uncoupled from nuclear activities of NIC4. Not to scale.
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