Journal: Cell Reports Methods

Article Title: Long-term imaging of individual mRNA molecules in living cells

doi: 10.1016/j.crmeth.2022.100226

Figure Lengend Snippet:

Article Snippet: 8×PP7 and 10×PP7 fragment were amplified from pLH-sgRNA-Sirius-8×PP7 (Addgene #121940 ( )) and CMV-10×PP7 oligo-library based (Addgene #158199 ( )) respectively and replacing 12×PP7 to create cmv-mCherry-8×PP7 and cmv-mCherry-10×PP7.

Techniques: Recombinant, Software

Journal: Nature cell biology

Article Title: LIMIT is an immunogenic lncRNA in cancer immunity and immunotherapy

doi: 10.1038/s41556-021-00672-3

Figure Lengend Snippet: a . A375 shFluc or shLIMIT cells were treated with IFNγ for 24 hours. RNA levels of LIMIT were determined by qRT-PCR. P value by 2-sided t-test. b . A375 shFluc or shLIMIT cells were treated with IFNγ for the indicated time. Protein levels of phospho-STAT1 (p-Y701), STAT1, and GAPDH were determined by Western blotting. 1 of 2 experiments is shown. c . A375 shFluc or shLIMIT cells were treated with IFNγ for 48 hours. Surface expression of HLA-ABC were determined by flow cytometry (FACS). P value by 2-sided t-test. d-g . YUMM1.7 ( d, e ) or CT26 ( f, g ) cells carrying shFluc or shLimit were treated with IFNγ. RNA levels of Limit were determined 24 hours after treatment ( d, f ). Surface staining of MHC-I (H2-D b ) was detected 48 hours after treatment ( e, g ). P value by 2-sided t-test. h-i . A375 WT or LIMIT promoter deletion cells were treated with IFNγ. RNA levels of LIMIT were determined 24 hours after treatment ( h ). Surface expression of HLA-ABC were determined 48 hours after treatment ( i ). P value by 2-sided t-test. j-k . B16 cells were transfected with dCas9-VPR, together with non-targeting sgRNA (sgNT) or sgRNA targeting the promoter of Limit (sgLimit). RNA levels of Limit were determined 24 hours after treatment ( j ). Surface expression of MHC-I (H2-D b ) or PD-L1 were determined 48 hours after treatment ( k ). P value by 2-sided t-test. l . B16-OVA cells carrying shFluc or shB2m were manipulated with Limit CRISPRa (sgLimit) for 48 hours. Surface expression of OVA-H2K b were determined by FACS. P value by 2-sided t-test. m-n . B16-OVA cells were manipulated with B2m knocking down (shB2m) or Limit CRISPRa (sgLimit), and co-cultured with OT-I cell at the ratio of 1:4. Cell death was measured by PI staining in CD45 − tumor cells. Dot plots ( m ) and statistical results ( n ) are shown. P value by 2-sided t-test. All data are mean ± SD. n = 3 biological independent samples in ( a, c, d, e, f, g, h, i, j, k, l, n ). Source data are provided in Soure_data_Fig2.xlsx and Unmodified_blots_Fig2.pdf.

Article Snippet: To apply CRISPR activation system to activate mouse Limit, phU6-sgRNAs were transfected together with SP-dCas9-VPR (Addgene #63798) into B16 cells.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Flow Cytometry, Staining, Transfection, Cell Culture

Journal: Nature cell biology

Article Title: LIMIT is an immunogenic lncRNA in cancer immunity and immunotherapy

doi: 10.1038/s41556-021-00672-3

Figure Lengend Snippet: a . Co-IP of GBP1-5 with HSP90 antibody in IFNγ-pretreated A375 cells. 1 of 3 experiments is shown. b . Co-IP of HSP90 with Flag antibody in Flag-GBP1-overexpressed A375 cells. * indicated the band shift of HSP90 upon GBP1 overexpression. 1 of 2 experiments is shown. c . Immunofluorescence staining of GBP1 and HSP90 in IFNγ-pretreated A375 cells. 1 of 4 images is shown. d . 293T cells were forced expression of Flag-HSF1 and increased doses of Flag-GBP1. Co-IP of HSF1 or GBP1 with HSP90 antibody were performed 24 hours afterwards. 1 of 2 experiments is shown. e . A375 cells were treated with HSP90 inhibitor, or forced expression of GBP1. Indicated proteins were detected 48 hours afterwards. 1 of 2 experiments is shown. f-g . A375 cells were treated with IFNγ and KRIBB11. RNA ( f ) or surface staining ( g ) levels of indicated genes were determined 48 hours afterwards. P value by 2-sided t-test. h . YUMM1.7 shFluc or shHsf1 cells were treated with IFNγ. Total protein ( h ) or surface expression ( i ) levels of indicated genes were determined 48 hours afterwards. 1 of 2 experiments is shown. P value by 2-sided t-test. j-k . Tumor growth curves of YUMM1.7 shFluc or shHsf1 cells in NSG mice ( j ) or wild type C57BL/6 mice ( k ). n = 5 ( j ) or 6 ( k ) animals, P value by 2-sided t-test for end point tumor volume. l . Percentages of CD3 + , Ki67 + , IFNγ + , and TNFα + T cells in YUMM1.7 shFluc or shHsf1 tumors. n = 5 biological independent samples, P value by 2-sided t-test. m . A375 shFluc or shLIMIT cells were transfected with HSE-luc and PRL-SV40 overnight, and then treated with IFNγ for additional 48 hours. HSF1 transcriptional activity is depicted as the relative luciferase activity. P value by 2-sided t-test. n . B16 cells were manipulated with Limit CRISPRa and treated with KRIBB11. Surface expression of MHC-I (H2-D b ) was determined 48 hours afterwards. P value by 2-sided t-test. All data are mean ± SD. n = 3 biological independent samples in ( f, g, i, m, n ). Source data are provided in Soure_data_Fig6.xlsx and Unmodified_blots_Fig6.pdf.

Article Snippet: To apply CRISPR activation system to activate mouse Limit, phU6-sgRNAs were transfected together with SP-dCas9-VPR (Addgene #63798) into B16 cells.

Techniques: Co-Immunoprecipitation Assay, Electrophoretic Mobility Shift Assay, Over Expression, Immunofluorescence, Staining, Expressing, Transfection, Activity Assay, Luciferase