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. 2024 Sep 19;25:246. doi: 10.1186/s13059-024-03392-7

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — ZBTB48 inhibits cellular proliferation. A Effects of ZBTB48 KD (left) or overexpression (right) on cell proliferation in HEK293 cells (error bars denote SEM, n = 4, ∗∗p ≤ 0.01, ∗p ≤ 0.05, two-way ANOVA). Cell counts are presented as log2. B Equal numbers of HCT-116 cells were transfected with the indicated siRNAs in biological triplicates and seeded at the same time in 6-well plates. Cells were separately transfected and seeded for each time point in biological triplicates. Error bars denote SEM. ∗∗∗p ≤ 0.001, ∗∗p ≤ 0.01, ∗p ≤ 0.05 (two-way ANOVA). C Representative genome browser snapshot for MTA1 locus showing the read coverage for ZBTB48 iCLIP-seq, miCLIP-seq (GFP samples), and previously published miCLIP-seq data in HEK293 cells. D Left: CLIP-qRT-PCR to examine binding of ZBTB48 to MTA1 transcripts in HCT-116 cells. Control IPs were performed using IgG. HPRT1 was used as a negative control. Right: CLIP-qPCR to examine binding of FTO to MTA1 transcripts in siNT or siZBTB48 HCT-116 cells. Data are represented as % input. E Bar plot showing the relative m6A levels for MTA1 transcripts estimated using m6A-RIP-qPCR in ZBTB48 knockdown (left), siFTO (right), or siNT-treated HCT-116 cells. Data are represented as % input. F qRT-PCR analysis of MTA1 mRNA in siZBTB48 or siNT-treated HCT-116 cells after treating cells with Actinomycin D for the indicated times. G Western blotting analysis in whole cell lysates prepared from either ZBTB48 KD or control cells. ZBTB48 KD was carried out using two different siRNAs, i.e., siZBTB48 #1 and #2 (third and fourth lanes, respectively). Blots were probed with the indicated antibodies. H qRT-PCR analysis of MTA1 mRNA in cells treated with siNT, siZBTB48 (siRNA#2), siIGF2BP2 (siRNA#2), or a combination of siZBTB48 and siIGF2BP2. For each time point, relative transcript levels were normalized to 18S rRNA and time point “0”. I Wound healing assay using ZBTB48-knockdown or control cells was recorded and quantitatively analyzed (right) (∗p ≤ 0.05, two-way ANOVA). Note: For all qPCR experiments: at least 3 biological replicates, student’s t-test, ∗∗∗p ≤ 0.001, ∗∗p ≤ 0.01, ∗p ≤ 0.05, n.s.: non-significant, error bars denote SEM. See also Additional file 1: Fig. S11 and S12.