Journal: Cell reports
Article Title: RPL22 is a tumor suppressor in MSI-high cancers and a splicing regulator of MDM4
doi: 10.1016/j.celrep.2024.114622
Figure Lengend Snippet: (A) Immunoblot of MSS cell lines with CRISPR-Cas9 knockout of RPL22 . (B) RT-PCR analysis of MSS cell lines with CRISPR-Cas9 knockout of RPL22 . (C) Focus formation assay of MSS cell lines with CRISPR-Cas9 knockout of RPL22 . p values: ZR-75–1 RPL22 KO1 = 0.0377, ZR-75–1 RPL22 KO2 = 0.0464, MCF7 RPL22 KO1 < 0.0001, MCF7 RPL22 KO2 = 0.0089, C32 RPL22 KO1 = 0.0033, and C32 RPL22 KO2 = 0.0029. Error bars represent the mean ± SD of three replicates. (D) Immunoblot of RPL22 mutant MSI-H cell lines with overexpression of RPL22 . (E) Nutlin-3a treatment of MSS cell lines (ZR-75–1) with CRISPR-Cas9 knockout of RPL22 . Error bars represent the mean ± SD of six replicates. Half-maximal inhibitory concentrations (IC50) were as follows: GFP KO = 1.566 μM, RPL22 KO1 = 3.297 μM, and RPL22 KO2 = 2.574 μM. (F) Focus formation assay with Nutlin-3a treatment of MSS cell lines (ZR-75–1) with CRISPR-Cas9 knockout of RPL22 . p values: 0.1 μM Nutlin-3a: ZR-75–1 RPL22 KO1 = 0.0001 and ZR-75–1 RPL22 KO2 = 0.0126; 1.0 μM Nutlin-3a: ZR-75–1 RPL22 KO1 = 0.0008 and ZR-75–1 RPL22 KO2 = 0.0008; and 2.5 μM Nutlin-3a: ZR-75–1 RPL22 KO1 = 0.0010 and ZR-75–1 RPL22 KO2 = 0.0015. Error bars represent the mean ± SD of three replicates. (G) Immunoblot of MSS cell line (ZR-75–1) with CRISPR-Cas9 knockout of RPL22 with immunostaining for p21 after 1 week of 1 μM Nutlin-3a treatment. (H) RT-PCR analysis of RPL22 mutant MSI-H cell lines with overexpression of RPL22 . RPCR cells are TP53 wild type, while RICR cells are TP53 null. (I) Immunoblot of RPL22 mutant MSI-H cell lines with overexpression of RPL22 .
Article Snippet: Membranes were cut, blocked with Intercept blocking buffers (LI-COR) and probed for antibodies against RPL22 (1:100, Santa Cruz, sc-136413), RPL22L1 (1:100, 1:5000, MyBioSource, MBS7050452), HdmX/MDMX (1:500, 1:1000, Bethyl, A300–287A-M), MDM2 (1:200, Santa Cruz, sc-965), p53 (1:200, Santa Cruz, sc-126), p53 (1:1000, Cell Signaling, 2524S), p-p53 (1; 100, Santa Cruz, sc-sc-377567), p21 (1:2000, Cell Signaling, 2946S), UBAP2L/NICE-4 (1:1000, Thomas Scientific, A300–534A-T), B-actin (1:1000, Cell Signaling, 4970S), Vinculin (1:500, Sigma, V9131), or V5 (1:4000, Invitrogen, P/ N 46705).
Techniques: Western Blot, CRISPR, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Tube Formation Assay, Mutagenesis, Over Expression, Immunostaining
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