Journal: Nature Communications

Article Title: SOD1 regulates ribosome biogenesis in KRAS mutant non-small cell lung cancer

doi: 10.1038/s41467-021-22480-x

Figure Lengend Snippet: a Dox-induced expression of NoLS-Sod1-Flag (NoLS-Sod1) in mouse Sod1 knockout KP NSCLC cells. Mouse KP Sod1 F/F NSCLC cells were treated with 4OHT to knock out endogenous SOD1 in the absence or presence of Dox-induced NoLS-Sod1 expression. NoLS-Sod1-Flag was stained by IF with anti-Flag antibody. The nucleolus was stained by IF with an anti-Npm1 antibody, and the nucleus was stained with Hoescht 33342. A representative field from the analysis performed on 50–60 cells is shown. Scale bars, 10 μm. b NoLS-SOD1 is sufficient to suppress growth defects due to the knockout of endogenous Sod1 in mouse KP NSCLC cells. Same as ( a ) except plate growth assay was performed. The number of cells plated per well is indicated on the left. c NoLS-SOD1 is sufficient to suppress pre-rRNA processing defects due to the knockout of endogenous Sod1 in mouse KP NSCLC cells. Same as ( a ) except Northern blot analysis of pre-rRNAs was performed using a mouse ITS2 probe (right panel). Total 18S and 28S rRNAs were stained with methylene blue. d Quantification of results from ( c ). The ratios of 12S/32S and 12S/[47-43S] pre-rRNAs decreased by knockout of endogenous Sod1 are restored by expression of NoLS-SOD1. Bars show mean values ± SEM in biological replicates ( n = 3 for Sod1 +/+ , n = 4 for Sod1 F/F ), significant differences are indicated ( p < 0.01, unpaired two-tailed multiple t tests with Holm–Sidak correction).

Article Snippet: For immunohistochemistry and IF : SOD1 (Abcam, ab16831), 1:500, validated for immunohistochemical (IHC) staining by the manufacturer on human placenta tissue; Ki-67 (Thermo Scientific, MA5-14520), 1:200, validated for IHC staining by the manufacturer on high-grade invasive breast cancer tissue; cleaved caspase-3 (Cell Signaling, 9664), 1:100, validated for IHC staining by the manufacturer on mouse embryo; phospho-histone H2A.X (Cell Signaling, 9718), 1:100, validated for IHC staining by the manufacturer on paraffin-embedded ultraviolet (UV)-treated HT-29 cells; WDR12 antibody, A302-651A (Abcam, ab111955), 1:100, used for IF, validated for IF staining by the manufacturer on A431 cells; BOP1 antibody (Santa Cruz Biotechnology, sc-390672), 1:100, used for IF, validated for IF staining by the manufacturer on HeLa cells; Flag rabbit monoclonal antibody (mAb) (Cell Signaling, 14793), 1:800, used for IF, validated for IF staining by the manufacturer on 293T cells transfected with a GFP-DYKDDDDK-Tag; NPM1 monoclonal antibody (FC-61991) (Thermo Fisher, 32-5200), 1:200, used for IF, validated for IF staining by the manufacturer on A549 and HeLa cells; ApopTag® Plus Peroxidase In Situ Apoptosis Kit (EMD Millipore, S7101) (as per the user guide provided by the manufacturer), validated for IHC staining by the manufacturer on paraffin-embedded human lymph node; biotinylated goat anti-rat IgG antibody, mouse adso–rbed (Vector Labs, BA-9401), validated by the manufacturer against rat heavy- and light-chain IgG.

Techniques: Expressing, Knock-Out, Staining, Growth Assay, Northern Blot, Two Tailed Test