Journal: PLoS ONE

Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants

doi: 10.1371/journal.pone.0073284

Figure Lengend Snippet: ( A ) DSB-triggered protein loadings at the proximal and distal regions. The regions, surrounding the site of I- Sce I, used for ChIP analysis were schematically illustrated. Numbers represent the distance from the site of I- Sce I in base pairs. The levels of I- Sce I expression, at different time points post-transfection, were analyzed by Western blotting with a α-HA antibody. Representative image of ChIP analysis of locus −303/−57 was shown, in which GAPDH was used as a positive control. PCR analysis (primer set: F13/IN2R1) of an unrelated region on 6p21.3 was included as an additional ChIP control. Arrows were used to mark the positions of the PCR products. ( B ) DSB-induced hMRE11, hRad51, hMSH5, hMSH4, and c-Abl loadings were analyzed at the proximal and distal loci. Error bars represent standard deviations from the means of triplicate measurements.

Article Snippet: Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .

Techniques: Expressing, Transfection, Western Blot, Positive Control, Control

Journal: PLoS ONE

Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants

doi: 10.1371/journal.pone.0073284

Figure Lengend Snippet: ( A ) ChIP analysis was performed in conjunction with RNAi-mediated gene silencing to determine the interdependency of DSB-triggered protein loadings at the proximal region. Controls without RNAi treatment were from Fig. 2B – the data is presented again on this graph for the purpose of comparison. ( B ) Knockdown efficiencies of shRNA encoding construct targeting hMRE11, hRad51, hMSH5, or hMSH4. Due to the difficulty in detecting endogenous hMSH4 in 293T cells by Western blotting, the hMSH4 knockdown efficiency was determined by the use of 293T/f45 cells. ( C ) ChIP analysis of the effects of RNAi on DSB-induced protein loadings at a distal region. Levels of protein loading in the absence of RNAi treatment were from Fig. 2B and included for the purpose of comparison. Error bars represent standard deviations from the means of triplicate measurements.

Article Snippet: Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .

Techniques: Comparison, Knockdown, shRNA, Construct, Western Blot

Journal: PLoS ONE

Article Title: MutS Homologue hMSH5: Recombinational DSB Repair and Non-Synonymous Polymorphic Variants

doi: 10.1371/journal.pone.0073284

Figure Lengend Snippet: ( A ) ChIP analysis of the effects of hMSH5 Y742F on DSB-triggered protein loading at both the proximal and distal regions was carried out with 293T reporter cells expressing hMSH5 or hMSH5 Y742F . Briefly, cells were transfected with pcDNA6/Flag-hMSH5 or Flag-hMSH5 Y742F and selected with 10 µg/ml blasticidin. ( B ) Expression of hMSH5 and hMSH5 Y742F in selected clones was validated by Western blot analysis of approximately equal numbers of hMSH5 and hMSH5 Y742F cells. ( C ) The effects of c-Abl kinase inhibition on DSB-induced protein loading at the proximal and distal regions. 293T reporter cells were pretreated with 4 µM imatinib for 48 hrs prior to the induction of DSB by I- Sce I transfection. ChIP analysis was performed to evaluate DSB-induced hRad51, hMSH5, and hMSH4 chromatin association. ( D ) ChIP analysis of GAPDH promoter performed with α-RNAPII or the mouse IgG in the presence or absence of imatinib treatment. Error bars represent standard deviations from the means of triplicate measurements. Asterisks indicate p<0.05 by Student’s t -test.

Article Snippet: Antibodies used in the experiments included α-hMRE11 (NB100–142, Novus Biologicals Inc., Littleton, CO), α-c-Abl, α-hRad51 (14B4) (NB100–148, Novus Biologicals Inc), α-hMSH5 , and α-hMSH4 .

Techniques: Expressing, Transfection, Clone Assay, Western Blot, Inhibition

Journal: Journal of Biological Chemistry

Article Title: Cellular Redistribution of Rad51 in Response to DNA Damage

doi: 10.1074/jbc.m109.024646

Figure Lengend Snippet: FIGURE 1. DNA damage induces an increase in nuclear levels of Rad51 in Brca2-proficient and Brca2-deficient cells. HeLa (A), HCT116 (B), and Capan-1 (C) cells grown at 37 °C were harvested at the indicated times following exposure to 2 Gy of IR and fractionated as described under “Experimental Procedures” to yield cytoplasmic (Cyto), nucleoplasmic (Nuc), and chromatin (Chrom) samples. D and E, HCT116 and Capan-1 cells, respectively, were treated with cyclo- heximide (CHX; 20 M) 1 h prior to exposure to 2 Gy of IR. A portion of each fraction (30 g of total protein) was loaded onto 4–12% SDS-polyacrylamide gels, andWesternblotsweredevelopedusingamouseanti-Rad51monoclonalantibody.Blotswerealsodevelopedusingthefollowingmarkersasloadingcontrols: glyceraldehyde-3-phosphate dehydrogenase (GAPDH; cytoplasmic), Sam68 (nucleoplasmic), and fibrillarin (chromatin). F, changes in levels of nuclear Rad51 as a function of time after IR treatment in A–E were quantified as described under “Experimental Procedures.” The data shown are representative of the results of at least three separate experiments, and the S.D. observed with quantification was 20%.

Article Snippet: Antibodies—The primary antibodies used were mouse monoclonal antibodies against Rad51 (clone 3C10, Upstate), Rad51B (ab3637, Abcam), Rad51C (NB 100-177, Novus), Rad51D (sc-53432, Santa Cruz Biotechnology), Xrcc2 (ab20253, Abcam), and Xrcc3 (NB 100-180D1, Novus).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Cellular Redistribution of Rad51 in Response to DNA Damage

doi: 10.1074/jbc.m109.024646

Figure Lengend Snippet: FIGURE 2. Levels of Capan-1 nuclear Rad51 increase in an IR dose-de- pendent manner. Capan-1 cells exposed to 5 or 8 Gy of IR were grown at 37 °C for 2 h. Cells were harvested and fractionated as described under “Experimental Procedures,” and portions of the cytoplasmic (Cyto) and nucle- oplasmic (Nuc) fractions (25 g of total protein) were loaded onto 4–12% SDS-polyacrylamide gels. Western blots were developed using a mouse anti- Rad51 monoclonal antibody, and levels of cytoplasmic and nuclear Rad51 were quantified as described under “Experimental Procedures” (supplemen- tal Table 1). The blot shown is representative of four separate experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Antibodies—The primary antibodies used were mouse monoclonal antibodies against Rad51 (clone 3C10, Upstate), Rad51B (ab3637, Abcam), Rad51C (NB 100-177, Novus), Rad51D (sc-53432, Santa Cruz Biotechnology), Xrcc2 (ab20253, Abcam), and Xrcc3 (NB 100-180D1, Novus).

Techniques: Western Blot

Journal: Journal of Biological Chemistry

Article Title: Cellular Redistribution of Rad51 in Response to DNA Damage

doi: 10.1074/jbc.m109.024646

Figure Lengend Snippet: FIGURE 4. Rad51C depletion decreases the steady-state level of nuclear Rad51 and diminishes its DNA damage-inducednucleartransportinBrca2-proficientandBrca2-deficientcells.HeLa(A)andCapan-1(B) cells were transfected with a nonspecific () or Rad51C-specific () siRNA pool (SMARTpool), grown for 42 h at 37 °C, exposed to 6 Gy of IR, and harvested 2 h later. Cytoplasmic (Cyto) and nuclear (Nuc) fractions were analyzed by Western blotting using an anti-Rad51 monoclonal antibody. Changes in levels of cytoplasmic and nuclear Rad51 were quantified as described under “Experimental Procedures.” GAPDH, glyceraldehyde-3- phosphate dehydrogenase.

Article Snippet: Antibodies—The primary antibodies used were mouse monoclonal antibodies against Rad51 (clone 3C10, Upstate), Rad51B (ab3637, Abcam), Rad51C (NB 100-177, Novus), Rad51D (sc-53432, Santa Cruz Biotechnology), Xrcc2 (ab20253, Abcam), and Xrcc3 (NB 100-180D1, Novus).

Techniques: Transfection, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Deficient Regulation of DNA Double-strand Break Repair in Fanconi Anemia Fibroblasts

doi: 10.1074/jbc.m213251200

Figure Lengend Snippet: FIG. 2. RAD51 protein levels are elevated in protein extracts from FA fibroblasts of complementation groups A, C, and G. A, RAD51 protein levels were examined by Western blot analysis of equal amounts of nuclear protein extracts prepared from normal diploid fi- broblasts (N) and patient-derived FA fibroblasts of complementation groups A, C, G, and D2. B, RAD51 Western blot analysis was performed on equal amounts of nuclear extracts prepared from normal diploid fibroblasts (PD.792.F (lane 1), PD.751.F (lane 2), PD.715.F (lane 3), PD.793.F (lane 4), CCD-1059Sk (lane 5), CCD-1056Sk (lane 6), and CCD-1108Sk (lane 7)) and HT1080 cells (H). C, the same amounts of the same nuclear protein extracts from A were examined by Western blot analysis for AP endonuclease-1 protein expression. D, RAD51 Western blot analysis was performed on equal amounts of whole cell protein extracts prepared from normal diploid fibroblasts; patient-derived FA fibroblasts of complementation groups A, C, and G and their retrovi- rally corrected counterparts (Acor, Ccor, and Gcor, respectively); and patient-derived FA fibroblasts of complementation group D2. E, the same amounts of the same whole cell protein extracts from D were examined by Western blot analysis for KU86 protein expression.

Article Snippet: Therefore, we examined the effect of cointroduced polyclonal anti-RAD51 primary antibody (Novus Biologicals Inc.) on intracellular HR activity in diploid FA fibroblasts.

Techniques: Western Blot, Derivative Assay, Expressing

Journal: Journal of Biological Chemistry

Article Title: Deficient Regulation of DNA Double-strand Break Repair in Fanconi Anemia Fibroblasts

doi: 10.1074/jbc.m213251200

Figure Lengend Snippet: FIG. 3. RAD51 and MRE11 protein levels are elevated in pro- tein extracts from FA fibroblasts of complementation groups A, C, and G. A, Western blot analysis of equal amounts of whole cell protein extracts from normal diploid fibroblasts (N); patient-derived FA fibroblasts of complementation groups A, C, and G and their retrovi- rally corrected counterparts (Acor, Ccor, and Gcor, respectively); and patient-derived FA fibroblasts of complementation group D2 was per- formed by probing the blot with both anti-RAD51 and anti-MRE11 antibodies. B, the same amounts of the same whole cell protein extracts from A were examined by Western blot analysis for AP endonuclease-1 expression.

Article Snippet: Therefore, we examined the effect of cointroduced polyclonal anti-RAD51 primary antibody (Novus Biologicals Inc.) on intracellular HR activity in diploid FA fibroblasts.

Techniques: Western Blot, Derivative Assay, Expressing

Journal: Journal of Biological Chemistry

Article Title: Deficient Regulation of DNA Double-strand Break Repair in Fanconi Anemia Fibroblasts

doi: 10.1074/jbc.m213251200

Figure Lengend Snippet: FIG. 4. Normal diploid fibroblasts expressing a dominant-negative FANCC gene have elevated RAD51 protein and high HR activity. A, RAD51 protein (RAD51p) levels were ex- amined by Western blot analysis of equal amounts of whole cell protein extracts prepared from normal diploid fibroblasts that were unmodified (N) or that overex- press the pL554P FANCC gene (NpL554P) and from FA fibroblasts from patients of complementation group C and their retrovirally corrected counter- parts (Ccor). Protein levels were quanti- tated in three independent experiments and are expressed as the -fold increase ver- sus unmodified normal diploid extracts. Er- ror bars represent S.E. *, p 0.05. B, HR frequency was examined in these same cells. Results are an average of at least three independent experiments. Error bars represent S.E. *, p 0.05.

Article Snippet: Therefore, we examined the effect of cointroduced polyclonal anti-RAD51 primary antibody (Novus Biologicals Inc.) on intracellular HR activity in diploid FA fibroblasts.

Techniques: Expressing, Dominant Negative Mutation, Activity Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Deficient Regulation of DNA Double-strand Break Repair in Fanconi Anemia Fibroblasts

doi: 10.1074/jbc.m213251200

Figure Lengend Snippet: FIG. 5. Cells from a mouse model of FA display elevated RAD51 protein and high levels of HR activity. A, left panel, Western blot analysis of RAD51 protein levels was performed on equal amounts of whole cell protein extracts prepared from embryonic fibroblasts derived from wild-type mice (N) and from mice homozygous for an inactivating deletion of the murine FancC gene (C); right panel, Coomassie Blue- stained gel of the same extracts. B, HR frequency was examined in these same cells. Results are an average of at least four independent experiments in each cell strain. Error bars represent S.E. *, p 0.05 compared with wild-type embryonic fibroblasts in the absence of antibody.

Article Snippet: Therefore, we examined the effect of cointroduced polyclonal anti-RAD51 primary antibody (Novus Biologicals Inc.) on intracellular HR activity in diploid FA fibroblasts.

Techniques: Activity Assay, Western Blot, Derivative Assay, Staining

Journal: Journal of Biological Chemistry

Article Title: Deficient Regulation of DNA Double-strand Break Repair in Fanconi Anemia Fibroblasts

doi: 10.1074/jbc.m213251200

Figure Lengend Snippet: FIG. 6. Protein levels of the RAD51 paralogs are not altered in FA fibroblast extracts. Equal amounts of whole cell protein extracts prepared from FANCC fibroblasts (C) and retrovirally corrected FANCC fibroblasts (Ccor) were examined by Western blot analysis using polyclonal anti-RAD51B, anti-RAD51C, anti-RAD51D, anti-XRCC2, and anti-XRCC3 antibodies.

Article Snippet: Therefore, we examined the effect of cointroduced polyclonal anti-RAD51 primary antibody (Novus Biologicals Inc.) on intracellular HR activity in diploid FA fibroblasts.

Techniques: Western Blot

Journal: Nature Communications

Article Title: Breast cancer cell-derived extracellular vesicles promote CD8 + T cell exhaustion via TGF-β type II receptor signaling

doi: 10.1038/s41467-022-31250-2

Figure Lengend Snippet: a A representative TEM image of purified EVs from MDA-MB-231 cells (left), and nanoparticle tracking of purified EVs (right). Scale bar (left panel), 100 nm. b Mass-spectrometry analysis of purified EVs secreted by MDA-MB-231 cells, showing results for Annexin A2, Integrin α6, HSP90, TβRII (TGFBR2, red), TSG101, CD9, CD63 and CD81 (left). TβRII peptide identified by mass-spectrometry analysis of purified EVs from MDA-MB-231 (right panel). c Immunoblot detection of TβRII in purified EVs (E) and whole lysates (W) from different breast cancer cell lines. d TEM images of different breast cancer cell lines-derived EVs immunogold-labeled with anti-TβRII antibodies (left panel), and quantification of number of gold particles by TEM (right panel). Gold particles are depicted as black dots. Scale bar, 50 nm. e Density gradient centrifugation confirming that TβRII secreted by MDA-MB-231 cells co-fractionated with exosome markers Alix and TSG101. f FACS analysis and quantification of the percentage of TβRII positive (TβRII + ) EVs from different breast cancer cell lines (left panel). n = 3 biological replicates per group (right panel). The percentage was referred to as the percentage of beads with TβRII + EVs. g Co-localization of endogenous TβRII and Alix in MDA-MB-231 cells. Scale bars, 10 μm. h Immunoblot analysis (left) and quantification (right) of TβRII in whole cells lysate and EVs derived from control cells and TGF-β-treated cells. i Schematic diagram of biotin-labeling assay (left) and immunoblot analysis (right) measuring TβRII levels on the cell membrane and secreted EVs. j Schematic diagram (left) of ELISA to measure TβRII concentration (right) on the surface of EVs derived from 4T1 and MDA-MB-231 cells, with or without TGF-β treatment. TMB, 3,3’,5,5’-tetramethylbenzidine; SA-HRP, streptavidin-horseradish peroxidase. k ELISA of TβRII on the surface of EVs from indicated cell types. l , ELISA of TβRII on EVs isolated from MDA-MB-231 cells pre-treated with or without TGF-β (2.5 ng/ml), TβRII neutralizing antibody (10 µg/ml) or TGF-β neutralizing antibody (10 µg/ml) for 24 h as indicated. * p < 0.05 (two-tailed Student’s t test d , f , h , k , l or two-way ANOVA j ). Data are analyzed of three independent experiments and shown as mean ± SD ( d , f , h , j , k and l ). Source data are provided as a Source Data file.

Article Snippet: The antibodies used for immunoprecipitation (IP), immunoblotting (IB), immunofluorescence (IF), and immunohistochemistry (IHC) were as follows: TβRII (sc-400, Santa Cruz Biotechnology, 1:1000 for IB, 1:50 for IP, 1:100 for IHC, 1:50 for immunogold label, 1:100 for IF), human TβRII (ab184948, Abcam, 1:1000 for IB), CD63 (ab216130, Abcam, 1:2000 for IB), TSG101 (sc-7964, Santa Cruz Biotechnology, 1:1000 for IB), Alix (sc-53540, Santa Cruz Biotechnology, 1:1000 for IB, 1:100 for IF), CD9 (A19027, ABclonal, 1:2000 for IB), CD81(sc-166029, Santa Cruz Biotechnology, 1:1000 for IB), Calnexin (A4846, ABclonal, 1:2000 for IB), Hrs (A1790, ABclonal, 1:2000 for IB), Rab27a (sc-74586, Santa Cruz Biotechnology, 1:1000 for IB), N-cadherin (610920, BD Bioscience, 1:50000 for IB), E-cadherin (610181, BD Bioscience, 1:10000 for IB, 1:100 for IF), SMAD4 (sc-7966, Santa Cruz Biotechnology, 1:1000 for IB), SMAD2-3 (610842, BD Bioscience, 1:2500 for IB, 1:500 for IP, 1:100 for IF), phospho-SMAD2 (#3101, Cell Signaling, 1:5000 for IB, 1:50 for IHC), SMAD3 (A19115, ABclonal, 1:100 for IP, 1:2000 for IB), phospho-SMAD3 (AP0727, ABclonal, 1:2000 for IB), SMAD4 (A19116, ABclonal, 1:2000 for IB), Ub (sc-8017, Santa Cruz Biotechnology, 1:1000 for IB), fibronectin (SAB4500974, Sigma, 1:1000 for IB), SMA (SAB5500002, Sigma, 1:1000 for IB), vimentin (#5741, Cell Signaling, 1:1000 for IB), TCF1/7 (#2203, Cell Signaling, 1:1000 for IB), EOMES (#4540, Cell Signaling, 1:1000 for IB), GZMB (sc-8022, Santa Cruz Biotechnology, 1:1000 for IB), CD8a (ab22378, Abcam, 1:400 for IHC), K48-linkage specific polyubiquitin (#8081 S, Cell Signaling, 1:1000 for IB), K63-linkage specific polyubiquitin (#A18164, ABclonal, 1:1000 for IB), β-actin (#A5441, Sigma, 1:10000 for IB), Phalloidin (#93042, Sigma, 1:1000 for IF), polyclonal HA(Y-11) (sc-805, Santa Cruz Biotechnology, 1:1000 for IB), monoclonal HA(12CAS5, home-made, 1:5000 for IB), polyclonal Myc (A-14) (sc-789, Santa Cruz Biotechnology, 1:1000 for IB), monoclonal Myc (9E10) (sc-40, Santa Cruz Biotechnology, 1:1000 for IB), Flag (M2, Sigma, 1:10000 for IB, 1:200 for IF), Protein A–HRP (Sigma-Aldrich GENA9120, 1:10000 for IB), HRP-conjugated secondary antibodies to mouse (NA931) or rabbit (NA934) (both from Amersham Biosciences, 1:10000 for IB), AlexaFluor488-labeled secondary antibody to rabbit (Molecular Probes R37116, 1:300 for IF) or AlexaFluor593-labeled antibody to mouse (Molecular Probes R3712, 1:300 for IF).

Techniques: Purification, Mass Spectrometry, Western Blot, Derivative Assay, Labeling, Gradient Centrifugation, Control, Membrane, Enzyme-linked Immunosorbent Assay, Concentration Assay, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: Breast cancer cell-derived extracellular vesicles promote CD8 + T cell exhaustion via TGF-β type II receptor signaling

doi: 10.1038/s41467-022-31250-2

Figure Lengend Snippet: a Schematic diagram (left panel), confocal microscopy (middle panel) and quantification (right panel) of MCF7 cells incubated with EVs derived from MDA-MB-231 cells stably expressing TβRII-GFP for indicated times. Scale bar, 20 μm. b Schematic diagram (left panel), FACS analysis (middle panel) and quantification (right panel) of MCF7 cells incubated with MDA-MB-231 cells stably expressing TβRII-GFP for indicated times. c Transcriptional analysis of TGF-β-induced (2.5 ng/ml for 16 hrs) CAGA 12 -Luc response in TβRII-deficient mink lung epithelial DR26 cells pre-treated for 24 h with empty liposome (as a control; Co.EVs), TβRII + (RII+) EVs (40 μg) derived from untransfected MDA-MB-231 cells or TβRII − (RII−) EVs (40 μg) derived from TβRII knockout MDA-MB-231 cells. d Immunoblot analysis of total lysates of DR26 cells pre-incubated with empty liposome (as a control; Co.EVs) or EVs (40 μg) derived from TβRII + (RII+) or TβRII − (RII−) MDA-MB-231 cells, and stimulated with TGF-β (2.5 ng/ml) for 1 h as indicated. e Transcriptional analysis of CAGA 12 -Luc response in TβRII-deficient DR26 cells incubated with empty liposome (as a control; Co.EVs) or EVs (40 μg) derived from TβRII + (RII+) MDA-MB-231 cells (MDA EVs) and treated with control DMSO, SB431542 (10 μM) or LY-364947 (10 μM) as indicated. f Immunofluorescence and DAPI staining of CAF cells pre-incubated with Co.EVs, TβRII + or TβRII − EVs (40 μg) and treated with control DMSO, SB431542 (10 μM) or LY-364947 (10 μM) as indicated. Scale bar, 20 μm. g Transcriptional analysis of CAGA 12 -Luc response in HEK293T cells as indicated pre-treated for 24 h with EVs (40 μg) derived from TβRII + (RII+) or TβRII − (RII−) MDA-MB-231 cells. h qPCR analysis in MCF7 cells pre-incubated for 48 h with Co.EVs, TβRII + or TβRII − EVs (40 μg), followed by no stimulation (−) or stimulation (+) for 16 h with TGF-β (2.5 ng/ml). Relative mRNA levels are shown as a heatmap. * p < 0.05 (two-tailed Student’s t test ( a , b , c , e , g )). Data are analyzed of three independent experiments and shown as mean + SD ( a , b , c , e , g ). Source data are provided as a Source Data file.

Article Snippet: The antibodies used for immunoprecipitation (IP), immunoblotting (IB), immunofluorescence (IF), and immunohistochemistry (IHC) were as follows: TβRII (sc-400, Santa Cruz Biotechnology, 1:1000 for IB, 1:50 for IP, 1:100 for IHC, 1:50 for immunogold label, 1:100 for IF), human TβRII (ab184948, Abcam, 1:1000 for IB), CD63 (ab216130, Abcam, 1:2000 for IB), TSG101 (sc-7964, Santa Cruz Biotechnology, 1:1000 for IB), Alix (sc-53540, Santa Cruz Biotechnology, 1:1000 for IB, 1:100 for IF), CD9 (A19027, ABclonal, 1:2000 for IB), CD81(sc-166029, Santa Cruz Biotechnology, 1:1000 for IB), Calnexin (A4846, ABclonal, 1:2000 for IB), Hrs (A1790, ABclonal, 1:2000 for IB), Rab27a (sc-74586, Santa Cruz Biotechnology, 1:1000 for IB), N-cadherin (610920, BD Bioscience, 1:50000 for IB), E-cadherin (610181, BD Bioscience, 1:10000 for IB, 1:100 for IF), SMAD4 (sc-7966, Santa Cruz Biotechnology, 1:1000 for IB), SMAD2-3 (610842, BD Bioscience, 1:2500 for IB, 1:500 for IP, 1:100 for IF), phospho-SMAD2 (#3101, Cell Signaling, 1:5000 for IB, 1:50 for IHC), SMAD3 (A19115, ABclonal, 1:100 for IP, 1:2000 for IB), phospho-SMAD3 (AP0727, ABclonal, 1:2000 for IB), SMAD4 (A19116, ABclonal, 1:2000 for IB), Ub (sc-8017, Santa Cruz Biotechnology, 1:1000 for IB), fibronectin (SAB4500974, Sigma, 1:1000 for IB), SMA (SAB5500002, Sigma, 1:1000 for IB), vimentin (#5741, Cell Signaling, 1:1000 for IB), TCF1/7 (#2203, Cell Signaling, 1:1000 for IB), EOMES (#4540, Cell Signaling, 1:1000 for IB), GZMB (sc-8022, Santa Cruz Biotechnology, 1:1000 for IB), CD8a (ab22378, Abcam, 1:400 for IHC), K48-linkage specific polyubiquitin (#8081 S, Cell Signaling, 1:1000 for IB), K63-linkage specific polyubiquitin (#A18164, ABclonal, 1:1000 for IB), β-actin (#A5441, Sigma, 1:10000 for IB), Phalloidin (#93042, Sigma, 1:1000 for IF), polyclonal HA(Y-11) (sc-805, Santa Cruz Biotechnology, 1:1000 for IB), monoclonal HA(12CAS5, home-made, 1:5000 for IB), polyclonal Myc (A-14) (sc-789, Santa Cruz Biotechnology, 1:1000 for IB), monoclonal Myc (9E10) (sc-40, Santa Cruz Biotechnology, 1:1000 for IB), Flag (M2, Sigma, 1:10000 for IB, 1:200 for IF), Protein A–HRP (Sigma-Aldrich GENA9120, 1:10000 for IB), HRP-conjugated secondary antibodies to mouse (NA931) or rabbit (NA934) (both from Amersham Biosciences, 1:10000 for IB), AlexaFluor488-labeled secondary antibody to rabbit (Molecular Probes R37116, 1:300 for IF) or AlexaFluor593-labeled antibody to mouse (Molecular Probes R3712, 1:300 for IF).

Techniques: Confocal Microscopy, Incubation, Derivative Assay, Stable Transfection, Expressing, Control, Knock-Out, Western Blot, Immunofluorescence, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Breast cancer cell-derived extracellular vesicles promote CD8 + T cell exhaustion via TGF-β type II receptor signaling

doi: 10.1038/s41467-022-31250-2

Figure Lengend Snippet: a Immunoblot analysis of total cells lysates from MCF7 cells (upper panel) or 4T07 cells (lower panel) pre-treated for 24 h with TβRII + EVs or TβRII − EVs (40 μg) derived from MDA-MB-231 cells, and stimulated with or without TGF-β (5 ng/ml) for 1 h as indicated. b Immunoblot analysis of total cell lysate of MCF10A-RAS cells pre-treated for 48 h with TβRII + (RII+) EVs or TβRII − (RII−) EVs (40 μg) derived from MDA-MB-231 cells, and treated with or without SB431542 (10 μM) for 48 h as indicated. c Immunofluorescence and DAPI staining of HaCaT cells pre-treated for 48 h with TβRII + (RII+) EVs or TβRII − (RII−) EVs (40 μg) derived from MDA-MB-231 cells, and treated with or without TGF-β (5 ng/ml) and SB431542 (10 μM) for 48 h as indicated. Scale bars, 20 μm. d MCF10A-RAS cells pre-treated with control EVs, TβRII + (RII+) EVs, or TβRII − (RII−) EVs (40 μg) derived from MDA-MB-231 cells were analyzed in a tumor sphere assay. Quantification of the number of tumor spheres per 5 × 10 3 seeded cells (left panel) and pictures show representative images of tumor spheres (right panel). Scale bar, 2 mm. e FACS analysis (left panel) and Quantification (right panel) of the CD44 high /CD24 low population in MCF10A-RAS cells treated with Co.EVs, TβRII + or TβRII − EVs (40 μg) for 48 h. f Cytotoxic crystal violet staining (left) and dose-response curves (right) to paclitaxel (PTX) of MDA-MB-231 cells pre-incubated with Co.EVs, TβRII + or TβRII − EVs (40 μg) for 48 h. The dose is presented on the x axis, while cell viability (% vs control) is presented on the y axis. g Cytotoxic crystal violet staining (left) and dose-response curves (right) to Doxorubicin (Doxo) of MDA-MB-231 cells pre-incubated with Co.EVs, TβRII + or TβRII − EVs (40 μg) for 48 h. The dose is presented on the x axis, while cell viability (% vs control) is presented on the y axis. h – k EVs administration and experimental analysis in vivo: 4T07 cells pre-treated with Co.EVs, TβRII + or TβRII − EVs (40 μg) were tail vein-injected into nude mice ( n = 8 mice per group). Mice were also given injection (into the tail vein) of corresponding EVs (50 μg per mouse every other day) before the tumor injection h ; the percentage of metastasis-free mice in each experimental group followed in time i ; Lung metastasis was measured by BLI. Normalized photon flux (left panel), representative images (right panel) in each experimental group followed in time j ; Representative images of bright view (upper), BLI signal (scale bars, 2 mm, middle upper), HE (scale bars, 1 mm, middle lower) and IHC staining (scale bars, 2 μm, lower) in each group k . ns not significant ( p > 0.05) and * p < 0.05 (unpaired two-tailed Student’s t test d , e or two-way ANOVA f , g , j ). Data are analyzed from three independent experiments and shown as mean ± SD d , e , f , g , j . Source data are provided as a Source Data file.

Article Snippet: The antibodies used for immunoprecipitation (IP), immunoblotting (IB), immunofluorescence (IF), and immunohistochemistry (IHC) were as follows: TβRII (sc-400, Santa Cruz Biotechnology, 1:1000 for IB, 1:50 for IP, 1:100 for IHC, 1:50 for immunogold label, 1:100 for IF), human TβRII (ab184948, Abcam, 1:1000 for IB), CD63 (ab216130, Abcam, 1:2000 for IB), TSG101 (sc-7964, Santa Cruz Biotechnology, 1:1000 for IB), Alix (sc-53540, Santa Cruz Biotechnology, 1:1000 for IB, 1:100 for IF), CD9 (A19027, ABclonal, 1:2000 for IB), CD81(sc-166029, Santa Cruz Biotechnology, 1:1000 for IB), Calnexin (A4846, ABclonal, 1:2000 for IB), Hrs (A1790, ABclonal, 1:2000 for IB), Rab27a (sc-74586, Santa Cruz Biotechnology, 1:1000 for IB), N-cadherin (610920, BD Bioscience, 1:50000 for IB), E-cadherin (610181, BD Bioscience, 1:10000 for IB, 1:100 for IF), SMAD4 (sc-7966, Santa Cruz Biotechnology, 1:1000 for IB), SMAD2-3 (610842, BD Bioscience, 1:2500 for IB, 1:500 for IP, 1:100 for IF), phospho-SMAD2 (#3101, Cell Signaling, 1:5000 for IB, 1:50 for IHC), SMAD3 (A19115, ABclonal, 1:100 for IP, 1:2000 for IB), phospho-SMAD3 (AP0727, ABclonal, 1:2000 for IB), SMAD4 (A19116, ABclonal, 1:2000 for IB), Ub (sc-8017, Santa Cruz Biotechnology, 1:1000 for IB), fibronectin (SAB4500974, Sigma, 1:1000 for IB), SMA (SAB5500002, Sigma, 1:1000 for IB), vimentin (#5741, Cell Signaling, 1:1000 for IB), TCF1/7 (#2203, Cell Signaling, 1:1000 for IB), EOMES (#4540, Cell Signaling, 1:1000 for IB), GZMB (sc-8022, Santa Cruz Biotechnology, 1:1000 for IB), CD8a (ab22378, Abcam, 1:400 for IHC), K48-linkage specific polyubiquitin (#8081 S, Cell Signaling, 1:1000 for IB), K63-linkage specific polyubiquitin (#A18164, ABclonal, 1:1000 for IB), β-actin (#A5441, Sigma, 1:10000 for IB), Phalloidin (#93042, Sigma, 1:1000 for IF), polyclonal HA(Y-11) (sc-805, Santa Cruz Biotechnology, 1:1000 for IB), monoclonal HA(12CAS5, home-made, 1:5000 for IB), polyclonal Myc (A-14) (sc-789, Santa Cruz Biotechnology, 1:1000 for IB), monoclonal Myc (9E10) (sc-40, Santa Cruz Biotechnology, 1:1000 for IB), Flag (M2, Sigma, 1:10000 for IB, 1:200 for IF), Protein A–HRP (Sigma-Aldrich GENA9120, 1:10000 for IB), HRP-conjugated secondary antibodies to mouse (NA931) or rabbit (NA934) (both from Amersham Biosciences, 1:10000 for IB), AlexaFluor488-labeled secondary antibody to rabbit (Molecular Probes R37116, 1:300 for IF) or AlexaFluor593-labeled antibody to mouse (Molecular Probes R3712, 1:300 for IF).

Techniques: Western Blot, Derivative Assay, Immunofluorescence, Staining, Control, Incubation, In Vivo, Injection, Immunohistochemistry, Two Tailed Test