Journal: International journal of cancer

Article Title: The impact of SAMHD1 expression and mutation status in mantle cell lymphoma: An analysis of the MCL Younger and Elderly trial.

doi: 10.1002/ijc.33202

Figure Lengend Snippet: Figure 2. The impact of SAMHD1 expression on the failure-free survival of

Article Snippet: After heat-induced epitope retrieval at pH 9.0 the rabbit polyclonal SAMHD1 antibody (1:200; Proteintech) was incubated for 30 min, followed by washing and detection according to the manufacturer’s protocol.

Techniques: Expressing

Journal: Nature Communications

Article Title: CRISPR activation screen identifies BCL-2 proteins and B3GNT2 as drivers of cancer resistance to T cell-mediated cytotoxicity

doi: 10.1038/s41467-022-29205-8

Figure Lengend Snippet: a Schematic of the CRISPRa screen. NY-ESO-1 + and HLA-A2 + A375 melanoma cells were transduced with the pooled sgRNA library targeting more than 23,000 coding isoforms. A375 cells were exposed to primary CD4 + and CD8 + T cells expressing the T cell receptor (TCR) specific for the NY-ESO-1 antigen. Deep sequencing identified candidate genes. b Average MAGeCK analysis P -values for the acute and chronic exposure screens. Top candidate genes are annotated and the two most enriched genes from each screening strategy are highlighted in red. c Most significant pathways enriched among the 576 candidate genes. d Heatmap showing Pearson’s correlation between expression of the top four candidate genes and cytolytic activity across patient tumors from TCGA. Only significant (FDR < 0.05) correlations are shown. e Box plots showing single-sample Gene Set Enrichment Analysis (ssGSEA) of 576 candidate genes in 308 patient tumor samples – . Patient samples were collected prior to treatment with checkpoint inhibitors and classified as responders ( n = 83) or nonresponders ( n = 225) to immunotherapy. Box plots indicate median (middle line), 25th, 75th percentile (box), and 5th and 95th percentile (whiskers). Two-tailed t tests were performed. f Cell survival of A375 cells transduced with ORFs encoding candidate genes against ESO T cell cytotoxicity at different effector to target (E:T) ratios. Cell survival was measured using a luminescent cell viability assay and normalized to paired control cells that were not cultured with T cells. T cells were derived from three donors used in the CRISPRa screen, with n = 4 replicates per donor for n = 12 total. All values are mean ± s.e.m. Two-tailed t tests with adjustments for multiple comparisons were performed. Source data are provided in Source Data 1.

Article Snippet: The human SAM CRISPR activation library (Addgene 1000000057) was used for CRISPR-Cas9 activation screening.

Techniques: Transduction, Expressing, Sequencing, Activity Assay, Two Tailed Test, Cell Viability Assay, Control, Cell Culture, Derivative Assay

Journal: Theranostics

Article Title: Blockade of phosphotyrosine pathways suggesting SH2 superbinder as a novel therapy for pulmonary fibrosis

doi: 10.7150/thno.72269

Figure Lengend Snippet: High pY levels might affect the proliferation, migration and differentiation of lung fibroblasts. A , Changes in cell proliferation between hnLFs and hfLFs determined by CCK-8 kit (n = 7). *** P < 0.001. B-C , Assessing cell proliferation by EdU assay between hnLFs and hfLFs (n = 6). *** P < 0.001. D-G , Comparison of migration between hnLFs and hfLFs (n = 6). *** P < 0.001. H , Representative images of hnLFs and hfLFs under TEM. Arrows pointed to the lipid droplets in cytoplasm. I , Representative fluorescence images of Nile Red in hnLFs and hfLFs. J-K , The contractility of hnLFs and hfLFs in 3D collagen matrices (n = 6). *** P < 0.001. L , Phospho-RTK array analysis with 200 μg of lysates from hnLFs and hfLFs, which were serum-starved for 6 h. M , Quantification of phospho-RTK levels (average of two drops fold average of positive control). N-O , Western blot and quantification of p-VEGR2, p-PDGFRβ, p-EGFR and p-FGFR1 in hnLFs and hfLFs (n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001. P-Q , Western blot and quantification of p-PLCγ1, p-GAB1, p-SHC and p-SRC in hnLFs and hfLFs (n = 6). ** P < 0.01, *** P < 0.001. R-S , Western blot and quantification of p-AKT, p-ERK1/2 and p-STAT3 in hnLFs and hfLFs (n = 6). ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against FN (Cat#15613-1-AP), COL1A1 (Cat#67288-1-lg), α-SMA (Cat#55135-1-AP, Cat#67735-1-AP), FGFR1 (Cat#60325-1-lg), CD31 (Cat#60287-1-lg), CD45 (Cat#11265-1-lg), EpCAM (Cat#60287-1-lg), Vimentin (Cat#10366-1-AP) and GAPDH (Cat#60004-1-lg) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Migration, CCK-8 Assay, EdU Assay, Comparison, Fluorescence, Positive Control, Western Blot

Journal: Theranostics

Article Title: Blockade of phosphotyrosine pathways suggesting SH2 superbinder as a novel therapy for pulmonary fibrosis

doi: 10.7150/thno.72269

Figure Lengend Snippet: SH2 superbinder blocked multiple fibrosis associated pathways through interrupting pY-SH2 combination in pY mediated signal transmission. A-B , Phospho-RTK array analysis with 200 μg of lysates from hfLFs treated with 1 μM GST-SH2 WT or GST-SH2 TrM for 24 h. C , Western blot of p-VEGR2, p-PDGFRβ, p-EGFR and p-FGFR1 in hnLFs and hfLFs after GST, GST-SH2 WT and GST-SH2 TrM incubation. D , Western blot of p-PLCγ1, p-GAB1, p-SHC and p-SRC in hnLFs and hfLFs after GST, GST-SH2 WT or GST-SH2 TrM incubation. E , Western blot of p-AKT, p-ERK1/2 and p-STAT3 in hnLFs and hfLFs after GST, GST-SH2 WT or GST-SH2 TrM incubation. F , hnLFs and hfLFs were incubated with 1 μM GST, GST-SH2 WT or GST-SH2 TrM for 24 h. The immunoprecipitation of EGFR and SHC was detected. G , Representative fluorescence images of EGFR and SHC colocalization in hnLFs and hfLFs after GST, GST-SH2 WT or GST-SH2 TrM treatment. H , Immunoprecipitations of GRB2 with GAB1, SHC, EGFR and PDGFRβ. I , GST pull down assay showed the binding capacity of SH2 superbinder with pY in hnLFs and hfLFs. J , Representative fluorescence images of GST tag and pY colocalization in hnLFs and hfLFs after incubation with GST, GST-SH2 WT or GST-SH2 TrM. K-L , GST pull down assay showed the binding capacity of SH2 superbinder with RTKs (such as VEGFR2, PDGFRβ, EGFR and FGFR1) and adaptor proteins (such as GAB1, SHC and SRC) in hfLFs and hfLFs.

Article Snippet: Antibodies against FN (Cat#15613-1-AP), COL1A1 (Cat#67288-1-lg), α-SMA (Cat#55135-1-AP, Cat#67735-1-AP), FGFR1 (Cat#60325-1-lg), CD31 (Cat#60287-1-lg), CD45 (Cat#11265-1-lg), EpCAM (Cat#60287-1-lg), Vimentin (Cat#10366-1-AP) and GAPDH (Cat#60004-1-lg) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Transmission Assay, Western Blot, Incubation, Immunoprecipitation, Fluorescence, Pull Down Assay, Binding Assay