bt20  (ATCC)


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    Structured Review

    ATCC bt20
    RKIP regulates E-cadherin (E-cad) and subsequent cell invasion through ERK2. ( a ) Representative western blots with lysates prepared from control knockdown (shLUC) or RKIP knockdown (shRKIP) <t>BT20</t> as indicated. Numbers are shown for quantified phosphorylated ERK1 or ERK2 bands normalized with total ERK protein. ( b ) (Top left panel) Representative western blots of lysates prepared from control knockdown (shGFP), ERK1 (shERK1) or ERK2 (shERK2) BT20 cells. (Bottom left panel) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of BT20 cells shown in the left top panel. (Right panel) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. ( c ) (Left panel) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of BT20 cells with different combinations of control knockdown, RKIP knockdown (shRKIP) or ERK2 knockdown (shERK2). right) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. ( d ) (Left panel) Representative results of invasion assays with same set of cells used in ( c ) showing number of invaded cells through Matrigel in each indicated BT20 cell lines (mean ± SE). (Right panel) Representative images of the stained invaded cells in indicated cell lines shown at the left panel. Unpaired Student's t-test (two-tailed) was used for all analyses with p
    Bt20, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The RhoA dependent anti-metastatic function of RKIP in breast cancer"

    Article Title: The RhoA dependent anti-metastatic function of RKIP in breast cancer

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-96709-6

    RKIP regulates E-cadherin (E-cad) and subsequent cell invasion through ERK2. ( a ) Representative western blots with lysates prepared from control knockdown (shLUC) or RKIP knockdown (shRKIP) BT20 as indicated. Numbers are shown for quantified phosphorylated ERK1 or ERK2 bands normalized with total ERK protein. ( b ) (Top left panel) Representative western blots of lysates prepared from control knockdown (shGFP), ERK1 (shERK1) or ERK2 (shERK2) BT20 cells. (Bottom left panel) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of BT20 cells shown in the left top panel. (Right panel) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. ( c ) (Left panel) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of BT20 cells with different combinations of control knockdown, RKIP knockdown (shRKIP) or ERK2 knockdown (shERK2). right) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. ( d ) (Left panel) Representative results of invasion assays with same set of cells used in ( c ) showing number of invaded cells through Matrigel in each indicated BT20 cell lines (mean ± SE). (Right panel) Representative images of the stained invaded cells in indicated cell lines shown at the left panel. Unpaired Student's t-test (two-tailed) was used for all analyses with p
    Figure Legend Snippet: RKIP regulates E-cadherin (E-cad) and subsequent cell invasion through ERK2. ( a ) Representative western blots with lysates prepared from control knockdown (shLUC) or RKIP knockdown (shRKIP) BT20 as indicated. Numbers are shown for quantified phosphorylated ERK1 or ERK2 bands normalized with total ERK protein. ( b ) (Top left panel) Representative western blots of lysates prepared from control knockdown (shGFP), ERK1 (shERK1) or ERK2 (shERK2) BT20 cells. (Bottom left panel) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of BT20 cells shown in the left top panel. (Right panel) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. ( c ) (Left panel) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of BT20 cells with different combinations of control knockdown, RKIP knockdown (shRKIP) or ERK2 knockdown (shERK2). right) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. ( d ) (Left panel) Representative results of invasion assays with same set of cells used in ( c ) showing number of invaded cells through Matrigel in each indicated BT20 cell lines (mean ± SE). (Right panel) Representative images of the stained invaded cells in indicated cell lines shown at the left panel. Unpaired Student's t-test (two-tailed) was used for all analyses with p

    Techniques Used: Western Blot, Staining, Software, Two Tailed Test

    RKIP functions through downstream RhoA to promote E-cadherin (E-cad) localization to cell-to-cell junctions. ( a ) (Left) Representative western blots of lysates prepared from control knockdown (shLUC), two different RKIP knockdowns (shRKIP 175/186 ), empty vector control, or RKIP expressing BT20 cells. (Middle) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of BT20 cells shown in the left panel. (Right) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the middle panel using MetaMorph analysis software. ( b ) (Left) Representative western blots of lysates prepared from control knockdown (shLUC), RKIP knockdowns (shRKIP 175 ), empty vector control (EVC), or RKIP expressing SUM149 cells. (Middle) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of SUM149 cells shown in the left panel. (Right) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the middle panel using MetaMorph analysis software. ( c ) (Left) Representative western blots of lysates prepared from control knockdown (shLUC), RKIP knockdowns (shRKIP 175 ), empty vector control (EVC), or RKIP expressing MDA-MB468 cells. (Middle) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 (Nucleus) of the same set of MDA-MB468 cells shown in the left panel. (Right) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the middle panel using MetaMorph analysis software. ( d ) (Left) Representative western blots of lysates prepared from control knockdown (shLUC), or RKIP knockdowns (shRKIP 175 ) MCF7 cells. (Middle) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of MCF7cells shown in the left panel. (Right) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the middle panel using MetaMorph analysis software. ( e ) (Left) Representative western blots of lysates prepared from control knockdown (shLUC), or RKIP knockdowns (shRKIP 175 ) T47D cells. (Middle) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of T47D cells shown in the left panel. (Right) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the middle panel using MetaMorph analysis software. ( f ) (Left, top) Representative western blots of lysates prepared from control knockdown (shCTR), two different RhoA knockdowns (shRhoA#4/#5), empty vector control, or RhoAQL expressing BT20 cells. (Left, bottom) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of BT20 cells shown in the left top panel. (Right) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. ( g ) (Left, top) Representative western blots of lysates prepared from BT20 cells with different combinations of controls, RKIP knockdown (shRKIP 175 ) and RhoAQL expression as indicated. (Left, bottom) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of BT20 cells shown in the left top panel. (Right) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. Unpaired Student's t-test (two-tailed) was used for all analyses with p
    Figure Legend Snippet: RKIP functions through downstream RhoA to promote E-cadherin (E-cad) localization to cell-to-cell junctions. ( a ) (Left) Representative western blots of lysates prepared from control knockdown (shLUC), two different RKIP knockdowns (shRKIP 175/186 ), empty vector control, or RKIP expressing BT20 cells. (Middle) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of BT20 cells shown in the left panel. (Right) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the middle panel using MetaMorph analysis software. ( b ) (Left) Representative western blots of lysates prepared from control knockdown (shLUC), RKIP knockdowns (shRKIP 175 ), empty vector control (EVC), or RKIP expressing SUM149 cells. (Middle) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of SUM149 cells shown in the left panel. (Right) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the middle panel using MetaMorph analysis software. ( c ) (Left) Representative western blots of lysates prepared from control knockdown (shLUC), RKIP knockdowns (shRKIP 175 ), empty vector control (EVC), or RKIP expressing MDA-MB468 cells. (Middle) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 (Nucleus) of the same set of MDA-MB468 cells shown in the left panel. (Right) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the middle panel using MetaMorph analysis software. ( d ) (Left) Representative western blots of lysates prepared from control knockdown (shLUC), or RKIP knockdowns (shRKIP 175 ) MCF7 cells. (Middle) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of MCF7cells shown in the left panel. (Right) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the middle panel using MetaMorph analysis software. ( e ) (Left) Representative western blots of lysates prepared from control knockdown (shLUC), or RKIP knockdowns (shRKIP 175 ) T47D cells. (Middle) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of T47D cells shown in the left panel. (Right) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the middle panel using MetaMorph analysis software. ( f ) (Left, top) Representative western blots of lysates prepared from control knockdown (shCTR), two different RhoA knockdowns (shRhoA#4/#5), empty vector control, or RhoAQL expressing BT20 cells. (Left, bottom) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of BT20 cells shown in the left top panel. (Right) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. ( g ) (Left, top) Representative western blots of lysates prepared from BT20 cells with different combinations of controls, RKIP knockdown (shRKIP 175 ) and RhoAQL expression as indicated. (Left, bottom) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of BT20 cells shown in the left top panel. (Right) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. Unpaired Student's t-test (two-tailed) was used for all analyses with p

    Techniques Used: Western Blot, Plasmid Preparation, Expressing, Staining, Software, Multiple Displacement Amplification, Two Tailed Test

    RKIP-mediated inhibition of cell invasion depends on the E-cadherin (E-cad) cell–cell junction localization. ( a ) (Top left panel) Representative western blots of lysates prepared from control knockdown (shGFP), or four different E-cad knockdowns (shEcad#2–5) 4T1 cells. (Bottom left panel) Representative results of invasion assays with same set of cells showing number of invaded cells through Matrigel in each indicated 4T1 cell lines (mean ± SE). (Right panel) Representative images of the stained invaded cells in indicated cell lines shown in the bottom left panel. ( b ) (Top left panel) Representative western blots of lysates prepared from control knockdown (shGFP), or E-cad knockdowns BT20 cells. (Bottom left panel) Representative results of invasion assays with shGFP control knockdown or titrated E-cad knockdown showing number of invaded cells through Matrigel in each indicated BT20 cell lines (mean ± SE). (Right panel) Representative images of the stained invaded cells in indicated cell lines shown in the bottom left panel. ( c ) (Top left panel) Representative western blots of lysates prepared from BT20 cells with different combinations of controls, E-cad knockdown (shEcad) and RKIP expression as indicated. (Bottom left panel) Representative results of invasion assays with same set of cells showing number of invaded cells through Matrigel in each indicated BT20 cell lines (mean ± SE). (Right panel) Representative images of the stained invaded cells in indicated cell lines shown at the bottom left panel. ( d ) (Left panel) Representative western blots of lysates prepared from BT20 cells with different combinations of controls, RKIP knockdown (shRKIP 175 ) and RhoAQL expression as indicated. (Left, bottom) Representative images of immunofluorescent staining with β-Catenin Ab and DRAQ5 (Nucleus) of BT20 cells with control knockdown (shLUC), RKIP knockdown (shRKIP), empty vector control (EVC) or RKIP expression. (right panel) β-Catenin intensity quantification on cell–cell junctions of immunofluorescent images shown in the left panel using MetaMorph analysis software. Representative images of immunofluorescent staining with p120 Ab and DRAQ5 (Nucleus) of BT20 cells with control knockdown (shLUC), RKIP knockdown (shRKIP), empty vector control (EVC) or RKIP expression. (Right panel) p120 intensity quantification on cell–cell junctions of immunofluorescent images shown in the left panel using MetaMorph analysis software. Unpaired Student's t-test (two-tailed) was used for all analyses with p
    Figure Legend Snippet: RKIP-mediated inhibition of cell invasion depends on the E-cadherin (E-cad) cell–cell junction localization. ( a ) (Top left panel) Representative western blots of lysates prepared from control knockdown (shGFP), or four different E-cad knockdowns (shEcad#2–5) 4T1 cells. (Bottom left panel) Representative results of invasion assays with same set of cells showing number of invaded cells through Matrigel in each indicated 4T1 cell lines (mean ± SE). (Right panel) Representative images of the stained invaded cells in indicated cell lines shown in the bottom left panel. ( b ) (Top left panel) Representative western blots of lysates prepared from control knockdown (shGFP), or E-cad knockdowns BT20 cells. (Bottom left panel) Representative results of invasion assays with shGFP control knockdown or titrated E-cad knockdown showing number of invaded cells through Matrigel in each indicated BT20 cell lines (mean ± SE). (Right panel) Representative images of the stained invaded cells in indicated cell lines shown in the bottom left panel. ( c ) (Top left panel) Representative western blots of lysates prepared from BT20 cells with different combinations of controls, E-cad knockdown (shEcad) and RKIP expression as indicated. (Bottom left panel) Representative results of invasion assays with same set of cells showing number of invaded cells through Matrigel in each indicated BT20 cell lines (mean ± SE). (Right panel) Representative images of the stained invaded cells in indicated cell lines shown at the bottom left panel. ( d ) (Left panel) Representative western blots of lysates prepared from BT20 cells with different combinations of controls, RKIP knockdown (shRKIP 175 ) and RhoAQL expression as indicated. (Left, bottom) Representative images of immunofluorescent staining with β-Catenin Ab and DRAQ5 (Nucleus) of BT20 cells with control knockdown (shLUC), RKIP knockdown (shRKIP), empty vector control (EVC) or RKIP expression. (right panel) β-Catenin intensity quantification on cell–cell junctions of immunofluorescent images shown in the left panel using MetaMorph analysis software. Representative images of immunofluorescent staining with p120 Ab and DRAQ5 (Nucleus) of BT20 cells with control knockdown (shLUC), RKIP knockdown (shRKIP), empty vector control (EVC) or RKIP expression. (Right panel) p120 intensity quantification on cell–cell junctions of immunofluorescent images shown in the left panel using MetaMorph analysis software. Unpaired Student's t-test (two-tailed) was used for all analyses with p

    Techniques Used: Inhibition, Western Blot, Staining, Expressing, Plasmid Preparation, Software, Two Tailed Test

    RKIP activates RhoA through GEF-H1. ( a ) Representative western blots of VAV2 GEF activity pull-down assays with lysates prepared from BT20 cells. Numbers are shown for quantified active VAV2 bands normalized with total VAV2 protein. ( b ) (Left panel) Representative western blots of lysates prepared from control knockdown (shCTR), or five different VAV2 knockdowns (shVAV2 2.1–2.5 ) BT20 cells. (Middle panel) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of BT20 cells shown in the left top panel. (Right panel) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. ( c ) (Left panel) Representative western blots of lysates prepared from control knockdown (shCTR), or two different GEF-H1 knockdowns (shGEF-H1 2.2–2.3 ) BT20 cells. (Middle panel) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of BT20 cells shown in the left top panel. (Right panel) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. ( d ) (Top left panel) Representative images of immunofluorescent staining with E-Cad Ab and DRAQ5 of BT20 cells with different combinations of control knockdown, GEF-H1 knockdown (shGEF-H1), empty vector control (EVC) or RKIP expression. (Bottom left panel) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. (Right panel) Representative western blots of lysates prepared from the same set of cells shown in the left panel. ( e ) (Left panel) Representative results of invasion assays with same set of cells used in ( d ) showing number of invaded cells through Matrigel in each indicated BT20 cell lines (mean ± SE). (Right panel) Representative images of the stained invaded cells in indicated cell lines shown at the left panel. Unpaired Student's t-test (two-tailed) was used for all analyses with p
    Figure Legend Snippet: RKIP activates RhoA through GEF-H1. ( a ) Representative western blots of VAV2 GEF activity pull-down assays with lysates prepared from BT20 cells. Numbers are shown for quantified active VAV2 bands normalized with total VAV2 protein. ( b ) (Left panel) Representative western blots of lysates prepared from control knockdown (shCTR), or five different VAV2 knockdowns (shVAV2 2.1–2.5 ) BT20 cells. (Middle panel) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of BT20 cells shown in the left top panel. (Right panel) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. ( c ) (Left panel) Representative western blots of lysates prepared from control knockdown (shCTR), or two different GEF-H1 knockdowns (shGEF-H1 2.2–2.3 ) BT20 cells. (Middle panel) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of BT20 cells shown in the left top panel. (Right panel) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. ( d ) (Top left panel) Representative images of immunofluorescent staining with E-Cad Ab and DRAQ5 of BT20 cells with different combinations of control knockdown, GEF-H1 knockdown (shGEF-H1), empty vector control (EVC) or RKIP expression. (Bottom left panel) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. (Right panel) Representative western blots of lysates prepared from the same set of cells shown in the left panel. ( e ) (Left panel) Representative results of invasion assays with same set of cells used in ( d ) showing number of invaded cells through Matrigel in each indicated BT20 cell lines (mean ± SE). (Right panel) Representative images of the stained invaded cells in indicated cell lines shown at the left panel. Unpaired Student's t-test (two-tailed) was used for all analyses with p

    Techniques Used: Western Blot, Activity Assay, Staining, Software, Plasmid Preparation, Expressing, Two Tailed Test

    RKIP activates RhoA to suppress breast cancer cell invasion. ( a ) Representative western blots with the indicated Abs of GTPase pull-down assays with lysates prepared from control knockdown (shLUC) or RKIP knockdown (shRKIP 369 ) 168 FARN cells as indicated. Numbers are shown for quantified active GTPase bands normalized with total protein. ( b ) Representative western blots of GTPase pull-down assays with lysates prepared from control knockdown (shLUC) or RKIP knockdown (shRKIP 175 ) BT20 (left) and MDA-MB231 (right) cells as indicated. Numbers are shown for quantified active RhoA bands normalized with total protein. ( c ) Representative western blots of GTPase pull-down assays with lysates prepared from control (EVC) or RKIP expressing (RKIP) 4T1(left), BT20 (middle) and MDA-MB231 (right) as indicated. Numbers are shown for quantified active RhoA bands normalized with total protein. ( d ) (Top) Representative western blots of lysates prepared from BT20 cells with different combinations of controls, RKIP knockdown (shRKIP 175 ) and RhoAQL expression as indicated. (Left, bottom) Representative results of invasion assays with same set of cells showing number of invaded cells through Matrigel in each indicated BT20 cell lines (mean ± SE). (Right, bottom) Representative images of the stained invaded cells in indicated cell lines shown at the bottom left panel. ( e ) (Left) Number of invaded 4T1 cells with different combinations of controls, RhoA knockdown (shRhoA 34 ) and RKIP expression as indicated through Matrigel (mean ± SE). (Right) Representative images of the stained invaded cells in indicated cell lines shown in the left panel. Unpaired Student's t-test (two-tailed) was used for all analyses with p
    Figure Legend Snippet: RKIP activates RhoA to suppress breast cancer cell invasion. ( a ) Representative western blots with the indicated Abs of GTPase pull-down assays with lysates prepared from control knockdown (shLUC) or RKIP knockdown (shRKIP 369 ) 168 FARN cells as indicated. Numbers are shown for quantified active GTPase bands normalized with total protein. ( b ) Representative western blots of GTPase pull-down assays with lysates prepared from control knockdown (shLUC) or RKIP knockdown (shRKIP 175 ) BT20 (left) and MDA-MB231 (right) cells as indicated. Numbers are shown for quantified active RhoA bands normalized with total protein. ( c ) Representative western blots of GTPase pull-down assays with lysates prepared from control (EVC) or RKIP expressing (RKIP) 4T1(left), BT20 (middle) and MDA-MB231 (right) as indicated. Numbers are shown for quantified active RhoA bands normalized with total protein. ( d ) (Top) Representative western blots of lysates prepared from BT20 cells with different combinations of controls, RKIP knockdown (shRKIP 175 ) and RhoAQL expression as indicated. (Left, bottom) Representative results of invasion assays with same set of cells showing number of invaded cells through Matrigel in each indicated BT20 cell lines (mean ± SE). (Right, bottom) Representative images of the stained invaded cells in indicated cell lines shown at the bottom left panel. ( e ) (Left) Number of invaded 4T1 cells with different combinations of controls, RhoA knockdown (shRhoA 34 ) and RKIP expression as indicated through Matrigel (mean ± SE). (Right) Representative images of the stained invaded cells in indicated cell lines shown in the left panel. Unpaired Student's t-test (two-tailed) was used for all analyses with p

    Techniques Used: Western Blot, Multiple Displacement Amplification, Expressing, Staining, Two Tailed Test

    2) Product Images from "Vitamin C sensitizes triple negative breast cancer to PI3K inhibition therapy"

    Article Title: Vitamin C sensitizes triple negative breast cancer to PI3K inhibition therapy

    Journal: Theranostics

    doi: 10.7150/thno.53225

    The impact of vitamin C on the transcriptome of BT20 cells. (A) Vitamin C (100 μM) changes the transcriptome of BT20 cells as shown by heatmap. (B) A total of 4,862 transcripts is changed by vitamin C treatment, which are called by both DESeq2 and edgeR. (C) Two of the top 5 downregulated pathways can be merged as one PI3K-mTOR pathway. (D) Vitamin C changes the expression of genes in the PI3K pathway, which predicts a downregulation of PI3K-mTOR signaling as shown by pathway enrichment analysis.
    Figure Legend Snippet: The impact of vitamin C on the transcriptome of BT20 cells. (A) Vitamin C (100 μM) changes the transcriptome of BT20 cells as shown by heatmap. (B) A total of 4,862 transcripts is changed by vitamin C treatment, which are called by both DESeq2 and edgeR. (C) Two of the top 5 downregulated pathways can be merged as one PI3K-mTOR pathway. (D) Vitamin C changes the expression of genes in the PI3K pathway, which predicts a downregulation of PI3K-mTOR signaling as shown by pathway enrichment analysis.

    Techniques Used: Expressing

    Vitamin C and buparlisib cooperatively decrease the expression of genes critical to cancer metastasis. (A) Western blot of fascin and FILIP1L in BT20 cells treated with vitamin C and buparlisib. (B) Semi-quantification of band density shows that vitamin C (100 μM) alone reduces fascin while buparlisib (0.5 μM) alone has no effect on fascin. (C) Semi-quantification of band density shows that vitamin C (100 μM) alone, or buparlisib (0.5 μM) alone, increases FILIP1L in BT20 cells. (D) Western blot of fascin and FILIP1L in BT20 cells treated with TETs siRNA followed by vitamin C. (E) Semi-quantification of band density shows that TETs siRNA abolishes the downregulation of fascin by vitamin C. (F) Semi-quantification of band density shows that TETs siRNA does not affect the upregulation of FILIP1L by vitamin C. (G) Western blot of fascin and FILIP1L in BT20 cells treated with vitamin C and CPI-455. (H) Semi-quantification of band density shows that CPI-455 does not affect the downregulation of fascin by vitamin C. (I) Semi-quantification of band density shows that CPI-455 abolishes the upregulation of FILIP1L by vitamin C.
    Figure Legend Snippet: Vitamin C and buparlisib cooperatively decrease the expression of genes critical to cancer metastasis. (A) Western blot of fascin and FILIP1L in BT20 cells treated with vitamin C and buparlisib. (B) Semi-quantification of band density shows that vitamin C (100 μM) alone reduces fascin while buparlisib (0.5 μM) alone has no effect on fascin. (C) Semi-quantification of band density shows that vitamin C (100 μM) alone, or buparlisib (0.5 μM) alone, increases FILIP1L in BT20 cells. (D) Western blot of fascin and FILIP1L in BT20 cells treated with TETs siRNA followed by vitamin C. (E) Semi-quantification of band density shows that TETs siRNA abolishes the downregulation of fascin by vitamin C. (F) Semi-quantification of band density shows that TETs siRNA does not affect the upregulation of FILIP1L by vitamin C. (G) Western blot of fascin and FILIP1L in BT20 cells treated with vitamin C and CPI-455. (H) Semi-quantification of band density shows that CPI-455 does not affect the downregulation of fascin by vitamin C. (I) Semi-quantification of band density shows that CPI-455 abolishes the upregulation of FILIP1L by vitamin C.

    Techniques Used: Expressing, Western Blot

    Vitamin C improves the response of TNBC xenografts to buparlisib treatment. (A) Photograph of human BT20 TNBC xenografts from female NSG mice treated with or without buparlisib (25 mg/kg body weight) and supplemented with or without vitamin C (3.3 g/L) in the drinking water. (B) Quantification of xenograft weights shows that xenografts in buparlisib alone group are similar to non-treated group ( P > 0.05). Xenografts in buparlisib and vitamin C co-treatment group are smaller compared to non-treated group ( P
    Figure Legend Snippet: Vitamin C improves the response of TNBC xenografts to buparlisib treatment. (A) Photograph of human BT20 TNBC xenografts from female NSG mice treated with or without buparlisib (25 mg/kg body weight) and supplemented with or without vitamin C (3.3 g/L) in the drinking water. (B) Quantification of xenograft weights shows that xenografts in buparlisib alone group are similar to non-treated group ( P > 0.05). Xenografts in buparlisib and vitamin C co-treatment group are smaller compared to non-treated group ( P

    Techniques Used: Mouse Assay

    Vitamin C and buparlisib cooperatively reduce H3K4 methylation. (A) Western blot of H3K4 methylation marks in BT20 cells treated with vitamin C. (B) Semi-quantification of band density shows that vitamin C (100 μM) reduces H3K4me3 and H3K4me2, but not H3K4me1, in BT20 cells. (C) Western blot of H3K4me3 in BT20 and MDA-MB-453 cells treated with different concentrations of vitamin C. (D) Semi-quantification of band density shows that compared to 50 μM, vitamin C at 100 μM further decreases H3K4me3 in BT20 and MDA-MB-453 cells. (E) Western blot of H3K4me3 in BT20 and MDA-MB-453 cells treated with vitamin C and buparlisib. (F) Semi-quantification of band density shows that vitamin C (100 μM) alone or buparlisib (0.5 μM) alone reduces H3K4me3 in BT20 and MDA-MB-453 cells. Co-treatment with vitamin C and buparlisib further reduces H3K4me3 in these TNBC cells.
    Figure Legend Snippet: Vitamin C and buparlisib cooperatively reduce H3K4 methylation. (A) Western blot of H3K4 methylation marks in BT20 cells treated with vitamin C. (B) Semi-quantification of band density shows that vitamin C (100 μM) reduces H3K4me3 and H3K4me2, but not H3K4me1, in BT20 cells. (C) Western blot of H3K4me3 in BT20 and MDA-MB-453 cells treated with different concentrations of vitamin C. (D) Semi-quantification of band density shows that compared to 50 μM, vitamin C at 100 μM further decreases H3K4me3 in BT20 and MDA-MB-453 cells. (E) Western blot of H3K4me3 in BT20 and MDA-MB-453 cells treated with vitamin C and buparlisib. (F) Semi-quantification of band density shows that vitamin C (100 μM) alone or buparlisib (0.5 μM) alone reduces H3K4me3 in BT20 and MDA-MB-453 cells. Co-treatment with vitamin C and buparlisib further reduces H3K4me3 in these TNBC cells.

    Techniques Used: Methylation, Western Blot, Multiple Displacement Amplification

    Vitamin C improves the response of TNBC cells to buparlisib in a KDM5 dependent manner. (A) Western blot of H3K4me3 and PI3K pathway genes in BT20 cells treated with vitamin C and KDM5 inhibitor CPI-455. (B) Semi-quantification of band density shows that vitamin C (100 μM) decreases the protein level of AKT2, mTOR, GSK3α, mLST8 as well as H3K4me3 in BT20 cells. This effect of vitamin C on H3K4me3 and the PI3K pathway proteins is largely abolished by CPI-455 (25 μM). (C) Western blot of phosphorylated AKT (Ser473, pAKT) in BT20 cells treated with vitamin C and buparlisib. (D) Semi-quantification of band density shows that buparlisib indeed inhibits pAKT, while vitamin C alone only marginally reduced pAKT ( P = 0.056), when normalized with total AKT. (E) When normalized with histone H3 since vitamin C decreases AKT2 expression, vitamin C alone also reduces pAKT ( P
    Figure Legend Snippet: Vitamin C improves the response of TNBC cells to buparlisib in a KDM5 dependent manner. (A) Western blot of H3K4me3 and PI3K pathway genes in BT20 cells treated with vitamin C and KDM5 inhibitor CPI-455. (B) Semi-quantification of band density shows that vitamin C (100 μM) decreases the protein level of AKT2, mTOR, GSK3α, mLST8 as well as H3K4me3 in BT20 cells. This effect of vitamin C on H3K4me3 and the PI3K pathway proteins is largely abolished by CPI-455 (25 μM). (C) Western blot of phosphorylated AKT (Ser473, pAKT) in BT20 cells treated with vitamin C and buparlisib. (D) Semi-quantification of band density shows that buparlisib indeed inhibits pAKT, while vitamin C alone only marginally reduced pAKT ( P = 0.056), when normalized with total AKT. (E) When normalized with histone H3 since vitamin C decreases AKT2 expression, vitamin C alone also reduces pAKT ( P

    Techniques Used: Western Blot, Expressing

    Vitamin C increases the efficacy of buparlisib. (A) Vitamin C (50 μM) decreases the EC 50 of buparlisib in BT20 cells. (B) Vitamin C (50 μM) decreases the EC 50 of buparlisib in MDA-MB-453 cells. GSH has no obvious effect on the EC 50 of buparlisib. (C) Both 100 and 300 μM vitamin C decrease the EC 50 of buparlisib further by ~3.5-fold compared to 50 μM vitamin C in BT20 cells. (D) Both 100 and 300 μM vitamin C decrease the EC 50 of buparlisib further by ~3.5-fold compared to 50 μM vitamin C in MDA-MB-453 cells.
    Figure Legend Snippet: Vitamin C increases the efficacy of buparlisib. (A) Vitamin C (50 μM) decreases the EC 50 of buparlisib in BT20 cells. (B) Vitamin C (50 μM) decreases the EC 50 of buparlisib in MDA-MB-453 cells. GSH has no obvious effect on the EC 50 of buparlisib. (C) Both 100 and 300 μM vitamin C decrease the EC 50 of buparlisib further by ~3.5-fold compared to 50 μM vitamin C in BT20 cells. (D) Both 100 and 300 μM vitamin C decrease the EC 50 of buparlisib further by ~3.5-fold compared to 50 μM vitamin C in MDA-MB-453 cells.

    Techniques Used: Multiple Displacement Amplification

    3) Product Images from "Astaxanthin Reduces Stemness Markers in BT20 and T47D Breast Cancer Stem Cells by Inhibiting Expression of Pontin and Mutant p53"

    Article Title: Astaxanthin Reduces Stemness Markers in BT20 and T47D Breast Cancer Stem Cells by Inhibiting Expression of Pontin and Mutant p53

    Journal: Marine Drugs

    doi: 10.3390/md18110577

    Effects of pontin-targeting siRNAs on expression levels of Oct4, Nanog, and mutp53 in T47D and BT20 breast cancer cell lines. Cells were transfected with pontin-targeting or control siRNA. ( A ) Cell lysates were prepared from T47D and BT20 cells, and the expression levels of Oct4, Nanog, and mutp53 were detected by Western blotting. ( B ) Total RNA was extracted, and the expression levels of Oct4, Nanog, and mutp53 were evaluated via qRT-PCR. All experiments were normalized by comparison with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data represent the mean ± SD of three independent experiments (* p
    Figure Legend Snippet: Effects of pontin-targeting siRNAs on expression levels of Oct4, Nanog, and mutp53 in T47D and BT20 breast cancer cell lines. Cells were transfected with pontin-targeting or control siRNA. ( A ) Cell lysates were prepared from T47D and BT20 cells, and the expression levels of Oct4, Nanog, and mutp53 were detected by Western blotting. ( B ) Total RNA was extracted, and the expression levels of Oct4, Nanog, and mutp53 were evaluated via qRT-PCR. All experiments were normalized by comparison with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data represent the mean ± SD of three independent experiments (* p

    Techniques Used: Expressing, Transfection, Western Blot, Quantitative RT-PCR

    Pontin knockdown attenuated the proliferation of T47D and BT20 cells. ( A ) Cell cycle analyses of T47D and BT20 cells after targeted pontin knockdown. Cells were harvested 3 days after transfection of pontin siRNAs or control siRNA. Similar results were obtained from three independent experiments. ( B ) Ki67 incorporation was used to determine the proportions of cells in each cell cycle phase. Cells were harvested 3 days after transfection of control siRNA or pontin siRNAs. Proportions of Ki67-positive cells are shown. Results are expressed as the mean ± SD of three independent experiments. * p
    Figure Legend Snippet: Pontin knockdown attenuated the proliferation of T47D and BT20 cells. ( A ) Cell cycle analyses of T47D and BT20 cells after targeted pontin knockdown. Cells were harvested 3 days after transfection of pontin siRNAs or control siRNA. Similar results were obtained from three independent experiments. ( B ) Ki67 incorporation was used to determine the proportions of cells in each cell cycle phase. Cells were harvested 3 days after transfection of control siRNA or pontin siRNAs. Proportions of Ki67-positive cells are shown. Results are expressed as the mean ± SD of three independent experiments. * p

    Techniques Used: Transfection

    Rottlerin reduced expression levels of Oct4, Nanog, and mutp53 in T47D and BT20 breast cancer cell lines. Cell lysates were prepared from T47D and BT20 cells that had been subjected to rottlerin treatment. Expression levels of Oct4, Nanog, and mutp53 were detected via Western blotting analyses and qRT-PCR. ( A ) Cell lysates were prepared from T47D and BT20 cells, and the expression levels of Oct4, Nanog, and mutp53 were detected by Western blotting. ( B ) Total RNA was extracted, and the expression levels of Oct4, Nanog, and mutp53 were evaluated by qRT-PCR. All expression levels were normalized to GAPDH. Data represent the mean ± SD of three independent experiments (* p
    Figure Legend Snippet: Rottlerin reduced expression levels of Oct4, Nanog, and mutp53 in T47D and BT20 breast cancer cell lines. Cell lysates were prepared from T47D and BT20 cells that had been subjected to rottlerin treatment. Expression levels of Oct4, Nanog, and mutp53 were detected via Western blotting analyses and qRT-PCR. ( A ) Cell lysates were prepared from T47D and BT20 cells, and the expression levels of Oct4, Nanog, and mutp53 were detected by Western blotting. ( B ) Total RNA was extracted, and the expression levels of Oct4, Nanog, and mutp53 were evaluated by qRT-PCR. All expression levels were normalized to GAPDH. Data represent the mean ± SD of three independent experiments (* p

    Techniques Used: Expressing, Western Blot, Quantitative RT-PCR

    Pontin knockdown reduced migration and invasion in breast cancer cells. Pontin knockdown by siRNA attenuated migration of ( A ) T47D and ( B ) BT20 cells, as determined by transwell migration assays. Left panel, representative images from a portion of field; right panel, quantification of average number of migrated cells/field (×200 magnification). Pontin knockdown by siRNA attenuated invasion of ( C ) T47D and ( D ) BT20 cells, determined using Matrigel-coated transwell invasion assays. Left panel, representative images from a portion of field; right panel, quantification of average number of migrated cells/field (×200 magnification). Data represent the mean ± SD of three independent experiments (* p
    Figure Legend Snippet: Pontin knockdown reduced migration and invasion in breast cancer cells. Pontin knockdown by siRNA attenuated migration of ( A ) T47D and ( B ) BT20 cells, as determined by transwell migration assays. Left panel, representative images from a portion of field; right panel, quantification of average number of migrated cells/field (×200 magnification). Pontin knockdown by siRNA attenuated invasion of ( C ) T47D and ( D ) BT20 cells, determined using Matrigel-coated transwell invasion assays. Left panel, representative images from a portion of field; right panel, quantification of average number of migrated cells/field (×200 magnification). Data represent the mean ± SD of three independent experiments (* p

    Techniques Used: Migration

    Pontin knockdown reduced colony and spheroid formation abilities in T47D and BT20 cells. Pontin knockdown with siRNA attenuated colony formation by ( A ) T47D and ( B ) BT20 cells. Left panel, representative images from a portion of field; right panel, quantification of average number of migrated cells/field. Pontin knockdown with siRNA attenuated spheroid formation by ( C ) T47D and ( D ) BT20 cells. Left panel, representative images from a portion of field; right panel, quantification of average number of migrated cells/field. Data represent the mean ± SD of three independent experiments (* p
    Figure Legend Snippet: Pontin knockdown reduced colony and spheroid formation abilities in T47D and BT20 cells. Pontin knockdown with siRNA attenuated colony formation by ( A ) T47D and ( B ) BT20 cells. Left panel, representative images from a portion of field; right panel, quantification of average number of migrated cells/field. Pontin knockdown with siRNA attenuated spheroid formation by ( C ) T47D and ( D ) BT20 cells. Left panel, representative images from a portion of field; right panel, quantification of average number of migrated cells/field. Data represent the mean ± SD of three independent experiments (* p

    Techniques Used:

    AST reduced the expression levels of pontin, mutp53, Oct4, and Nanog in T47D and BT20 breast cancer cell lines, thereby inhibiting cell proliferation. ( A ) Spheroid formation abilities of SKBR3, T47D, and BT20 breast cancer cell lines. ( B ) Expression levels of mutp53, pontin, Oct4, and Nanog were determined via Western blotting of T47D and BT20 cells after AST treatment. Actin was used as a loading control. ( C ) Cells were incubated for 48 h at 37 °C at the indicated concentrations of AST in a 96-well plate. Then, CCK-8 was added and the cells were incubated for 3 h at 37 °C. Absorbance was measured with a spectrophotometer at 450 nm. Data are expressed as the mean ± standard deviation (SD) of three independent experiments. * p
    Figure Legend Snippet: AST reduced the expression levels of pontin, mutp53, Oct4, and Nanog in T47D and BT20 breast cancer cell lines, thereby inhibiting cell proliferation. ( A ) Spheroid formation abilities of SKBR3, T47D, and BT20 breast cancer cell lines. ( B ) Expression levels of mutp53, pontin, Oct4, and Nanog were determined via Western blotting of T47D and BT20 cells after AST treatment. Actin was used as a loading control. ( C ) Cells were incubated for 48 h at 37 °C at the indicated concentrations of AST in a 96-well plate. Then, CCK-8 was added and the cells were incubated for 3 h at 37 °C. Absorbance was measured with a spectrophotometer at 450 nm. Data are expressed as the mean ± standard deviation (SD) of three independent experiments. * p

    Techniques Used: AST Assay, Expressing, Western Blot, Incubation, CCK-8 Assay, Spectrophotometry, Standard Deviation

    4) Product Images from "PGRMC1 Inhibits Progesterone-Evoked Proliferation and Ca2+ Entry Via STIM2 in MDA-MB-231 Cells"

    Article Title: PGRMC1 Inhibits Progesterone-Evoked Proliferation and Ca2+ Entry Via STIM2 in MDA-MB-231 Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21207641

    Protein kinase A (PKA) activation by progesterone (P 4 ) impaired calcium (Ca 2+ ) entry in triple-negative breast cancer cells. MDA-MB-231 cells ( A.1 ), MDA-MB-468 ( A.2 ), and BT20 cells ( A.3 ) were loaded with fura-2/AM. Single-cell imaging experiments were done using fura-2 loaded triple-negative breast cancer cells that were previously incubated for 30 min with the vehicle (black solid traces) or with 3 μM of the PKA antagonist, KT5720 (PKAi, black dotted traces). Following this, the triple-negative breast cancer cells were stimulated for 4 min with P 4 (1 μM) in a Ca 2+ -free HBS medium (75 μM of ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) was added, as indicated by arrowheads), and subsequently, P 4 -evoked Ca 2+ entry was visualized by adding 1 mM of CaCl 2 to the extracellular medium. Areas under the curves evoked by P 4 and CaCl 2 administration to the cells were determined, and they were used for comparison between both experimental conditions. The histogram represents the average of the percentage ± Standard error of the Mean (S.E.M.), resulting in the analysis of the areas under the curves corresponding to the P 4 -evoked Ca 2+ release and Ca 2+ entry evoked in the absence or presence of the PKA antagonist. The 20-30 cells from 6 independent experiments were considered during the analysis. *, **, ***: represent p
    Figure Legend Snippet: Protein kinase A (PKA) activation by progesterone (P 4 ) impaired calcium (Ca 2+ ) entry in triple-negative breast cancer cells. MDA-MB-231 cells ( A.1 ), MDA-MB-468 ( A.2 ), and BT20 cells ( A.3 ) were loaded with fura-2/AM. Single-cell imaging experiments were done using fura-2 loaded triple-negative breast cancer cells that were previously incubated for 30 min with the vehicle (black solid traces) or with 3 μM of the PKA antagonist, KT5720 (PKAi, black dotted traces). Following this, the triple-negative breast cancer cells were stimulated for 4 min with P 4 (1 μM) in a Ca 2+ -free HBS medium (75 μM of ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) was added, as indicated by arrowheads), and subsequently, P 4 -evoked Ca 2+ entry was visualized by adding 1 mM of CaCl 2 to the extracellular medium. Areas under the curves evoked by P 4 and CaCl 2 administration to the cells were determined, and they were used for comparison between both experimental conditions. The histogram represents the average of the percentage ± Standard error of the Mean (S.E.M.), resulting in the analysis of the areas under the curves corresponding to the P 4 -evoked Ca 2+ release and Ca 2+ entry evoked in the absence or presence of the PKA antagonist. The 20-30 cells from 6 independent experiments were considered during the analysis. *, **, ***: represent p

    Techniques Used: Activation Assay, Multiple Displacement Amplification, Imaging, Incubation

    Progesterone receptor membrane component 1 (PGRMC1) downregulated the progesterone (P 4 )-evoked Ca 2+ entry. ( A ) Resting MCF10A, MCF7, MDA-MB-231, and BT20 cells were suspended at the same concentration, and they were subsequently lysed with NP40 buffer. The resulting protein samples were denaturalized by mixing with an equal volume of Laemmli’s buffer (5% sodium dodecyl sulphate, SDS). Subsequent Western blotting was performed by using the anti-PGRMC1 antibody, as described in Materials and Methods. The histogram represents the average of the fold increase ± S.E.M. of the amount of PGRMC1 found in MCF10A after they were previously normalized by considering the actin content in each gel lane. **, ***: represent p
    Figure Legend Snippet: Progesterone receptor membrane component 1 (PGRMC1) downregulated the progesterone (P 4 )-evoked Ca 2+ entry. ( A ) Resting MCF10A, MCF7, MDA-MB-231, and BT20 cells were suspended at the same concentration, and they were subsequently lysed with NP40 buffer. The resulting protein samples were denaturalized by mixing with an equal volume of Laemmli’s buffer (5% sodium dodecyl sulphate, SDS). Subsequent Western blotting was performed by using the anti-PGRMC1 antibody, as described in Materials and Methods. The histogram represents the average of the fold increase ± S.E.M. of the amount of PGRMC1 found in MCF10A after they were previously normalized by considering the actin content in each gel lane. **, ***: represent p

    Techniques Used: Multiple Displacement Amplification, Concentration Assay, Western Blot

    5) Product Images from "Blockade of CCL2 expression overcomes intrinsic PD-1/PD-L1 inhibitor-resistance in transglutaminase 2-induced PD-L1 positive triple negative breast cancer"

    Article Title: Blockade of CCL2 expression overcomes intrinsic PD-1/PD-L1 inhibitor-resistance in transglutaminase 2-induced PD-L1 positive triple negative breast cancer

    Journal: American Journal of Cancer Research

    doi:

    PD-L1 is an unpredictable biomarker of response to PD-1/PD-L1 Inhibition. (A) PD-L1 mRNA and protein expression levels and (B and C) surface PD-L1 expression in MCF7, BT20, and MDA-MB-231 cells; (D) western blot analysis for PD-1 expression and ELISA assay for IL-2 and IFNγ levels in activated Jurkat T cells after PMA (50 ng/mL) and Ionomycin (1 µg/mL) treatment; (E) Caspase 3/7 activity of PD-L1(-) MCF7 cells, co-cultured with activated Jurkat T cells; Caspase 3/7 activity of PD-L1(+) (F) BT20 and (G) MDA-MB-231 cells, co-cultured with activated Jurkat T cells, after treatment of PD-1/PD-L1 inhibitor for 24 h.
    Figure Legend Snippet: PD-L1 is an unpredictable biomarker of response to PD-1/PD-L1 Inhibition. (A) PD-L1 mRNA and protein expression levels and (B and C) surface PD-L1 expression in MCF7, BT20, and MDA-MB-231 cells; (D) western blot analysis for PD-1 expression and ELISA assay for IL-2 and IFNγ levels in activated Jurkat T cells after PMA (50 ng/mL) and Ionomycin (1 µg/mL) treatment; (E) Caspase 3/7 activity of PD-L1(-) MCF7 cells, co-cultured with activated Jurkat T cells; Caspase 3/7 activity of PD-L1(+) (F) BT20 and (G) MDA-MB-231 cells, co-cultured with activated Jurkat T cells, after treatment of PD-1/PD-L1 inhibitor for 24 h.

    Techniques Used: Biomarker Assay, Inhibition, Expressing, Multiple Displacement Amplification, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Cell Culture

    TG2 induces PD-L1 expression by blocking PTEN and activating NF-κB in TNBC cells. (A) Fold change in TG2 in MDA-MB-231 and BT20 cells, and western blot analysis of TG2, PD-L1, PTEN, pAKT, and IκBα in MCF7 and MDA-MB-231 cells, with β-Actin as a loading control. (B) Western blot analysis of TG2, PD-L1, PTEN, pAKT, and IκBα in MCF7/Mock, MCF7/TG2, MDA-MB-231, and MDA-MB-231/TG2 siRNA cells, with β-Actin as a loading control. (C) Cell Immunohistochemistry and (D) FACS analysis for surface PD-L1 expression level in MCF7/Mock, MCF7/TG2, MDA-MB-231 and MDA-MB-231/TG2 siRNA. (E) Incidence of TG2 and PD-L1 expression in Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples of 648 TNBC patients.
    Figure Legend Snippet: TG2 induces PD-L1 expression by blocking PTEN and activating NF-κB in TNBC cells. (A) Fold change in TG2 in MDA-MB-231 and BT20 cells, and western blot analysis of TG2, PD-L1, PTEN, pAKT, and IκBα in MCF7 and MDA-MB-231 cells, with β-Actin as a loading control. (B) Western blot analysis of TG2, PD-L1, PTEN, pAKT, and IκBα in MCF7/Mock, MCF7/TG2, MDA-MB-231, and MDA-MB-231/TG2 siRNA cells, with β-Actin as a loading control. (C) Cell Immunohistochemistry and (D) FACS analysis for surface PD-L1 expression level in MCF7/Mock, MCF7/TG2, MDA-MB-231 and MDA-MB-231/TG2 siRNA. (E) Incidence of TG2 and PD-L1 expression in Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples of 648 TNBC patients.

    Techniques Used: Expressing, Blocking Assay, Multiple Displacement Amplification, Western Blot, Immunohistochemistry, FACS, Formalin-fixed Paraffin-Embedded

    CCL2 induction by TG2 contributes to PD-L1 inhibitor-resistance via regulation of negatively cytotoxic T cells in TNBC cells. The level of (A) PD-L1 and (B) CCL2 mRNA expression in MCF7, BT20, MCF7/TG2, and MDA-MB-231 cells measured by qRT-PCR analysis; (C) western blot analysis to check expression levels of TG2, P-AKT, IκBα, PD-L1, CCL2, and β-Actin in MCF7, BT20, MCF7/TG2, and MDA-MB-231 cells; (D) the expression of TG2, PD-L1, and CCL2 in MCF7/TG2 and MDA-MB-231 cells tested by western blotting analysis after TG2 siRNA transfection; (E) expression of PD-1 and CCR2 in Jurkat T cells with or without treatment of PMA (50 ng/mL) and Ionomycin (1 µg/mL) for 24 h measured by western blotting; (F and G) the protein levels of cPARP, IκBα, PD-L1, and CCL2 in MCF7/TG and MDA-MB-231 cells with or without activated Jurkat T cell co-culture for 24 h measured by western blotting after CCL2 siRNA transfection, Avelumab (20 µg/mL) treatment, or dual combination treatment and Caspase 3/7 activity measured by Caspase 3/7 assay using proteins from MCF7/TG and MDA-MB-231 cells.
    Figure Legend Snippet: CCL2 induction by TG2 contributes to PD-L1 inhibitor-resistance via regulation of negatively cytotoxic T cells in TNBC cells. The level of (A) PD-L1 and (B) CCL2 mRNA expression in MCF7, BT20, MCF7/TG2, and MDA-MB-231 cells measured by qRT-PCR analysis; (C) western blot analysis to check expression levels of TG2, P-AKT, IκBα, PD-L1, CCL2, and β-Actin in MCF7, BT20, MCF7/TG2, and MDA-MB-231 cells; (D) the expression of TG2, PD-L1, and CCL2 in MCF7/TG2 and MDA-MB-231 cells tested by western blotting analysis after TG2 siRNA transfection; (E) expression of PD-1 and CCR2 in Jurkat T cells with or without treatment of PMA (50 ng/mL) and Ionomycin (1 µg/mL) for 24 h measured by western blotting; (F and G) the protein levels of cPARP, IκBα, PD-L1, and CCL2 in MCF7/TG and MDA-MB-231 cells with or without activated Jurkat T cell co-culture for 24 h measured by western blotting after CCL2 siRNA transfection, Avelumab (20 µg/mL) treatment, or dual combination treatment and Caspase 3/7 activity measured by Caspase 3/7 assay using proteins from MCF7/TG and MDA-MB-231 cells.

    Techniques Used: Expressing, Multiple Displacement Amplification, Quantitative RT-PCR, Western Blot, Transfection, Co-Culture Assay, Activity Assay

    6) Product Images from "Blockade of CCL2 expression overcomes intrinsic PD-1/PD-L1 inhibitor-resistance in transglutaminase 2-induced PD-L1 positive triple negative breast cancer"

    Article Title: Blockade of CCL2 expression overcomes intrinsic PD-1/PD-L1 inhibitor-resistance in transglutaminase 2-induced PD-L1 positive triple negative breast cancer

    Journal: American Journal of Cancer Research

    doi:

    PD-L1 is an unpredictable biomarker of response to PD-1/PD-L1 Inhibition. (A) PD-L1 mRNA and protein expression levels and (B and C) surface PD-L1 expression in MCF7, BT20, and MDA-MB-231 cells; (D) western blot analysis for PD-1 expression and ELISA assay for IL-2 and IFNγ levels in activated Jurkat T cells after PMA (50 ng/mL) and Ionomycin (1 µg/mL) treatment; (E) Caspase 3/7 activity of PD-L1(-) MCF7 cells, co-cultured with activated Jurkat T cells; Caspase 3/7 activity of PD-L1(+) (F) BT20 and (G) MDA-MB-231 cells, co-cultured with activated Jurkat T cells, after treatment of PD-1/PD-L1 inhibitor for 24 h.
    Figure Legend Snippet: PD-L1 is an unpredictable biomarker of response to PD-1/PD-L1 Inhibition. (A) PD-L1 mRNA and protein expression levels and (B and C) surface PD-L1 expression in MCF7, BT20, and MDA-MB-231 cells; (D) western blot analysis for PD-1 expression and ELISA assay for IL-2 and IFNγ levels in activated Jurkat T cells after PMA (50 ng/mL) and Ionomycin (1 µg/mL) treatment; (E) Caspase 3/7 activity of PD-L1(-) MCF7 cells, co-cultured with activated Jurkat T cells; Caspase 3/7 activity of PD-L1(+) (F) BT20 and (G) MDA-MB-231 cells, co-cultured with activated Jurkat T cells, after treatment of PD-1/PD-L1 inhibitor for 24 h.

    Techniques Used: Biomarker Assay, Inhibition, Expressing, Multiple Displacement Amplification, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Cell Culture

    TG2 induces PD-L1 expression by blocking PTEN and activating NF-κB in TNBC cells. (A) Fold change in TG2 in MDA-MB-231 and BT20 cells, and western blot analysis of TG2, PD-L1, PTEN, pAKT, and IκBα in MCF7 and MDA-MB-231 cells, with β-Actin as a loading control. (B) Western blot analysis of TG2, PD-L1, PTEN, pAKT, and IκBα in MCF7/Mock, MCF7/TG2, MDA-MB-231, and MDA-MB-231/TG2 siRNA cells, with β-Actin as a loading control. (C) Cell Immunohistochemistry and (D) FACS analysis for surface PD-L1 expression level in MCF7/Mock, MCF7/TG2, MDA-MB-231 and MDA-MB-231/TG2 siRNA. (E) Incidence of TG2 and PD-L1 expression in Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples of 648 TNBC patients.
    Figure Legend Snippet: TG2 induces PD-L1 expression by blocking PTEN and activating NF-κB in TNBC cells. (A) Fold change in TG2 in MDA-MB-231 and BT20 cells, and western blot analysis of TG2, PD-L1, PTEN, pAKT, and IκBα in MCF7 and MDA-MB-231 cells, with β-Actin as a loading control. (B) Western blot analysis of TG2, PD-L1, PTEN, pAKT, and IκBα in MCF7/Mock, MCF7/TG2, MDA-MB-231, and MDA-MB-231/TG2 siRNA cells, with β-Actin as a loading control. (C) Cell Immunohistochemistry and (D) FACS analysis for surface PD-L1 expression level in MCF7/Mock, MCF7/TG2, MDA-MB-231 and MDA-MB-231/TG2 siRNA. (E) Incidence of TG2 and PD-L1 expression in Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples of 648 TNBC patients.

    Techniques Used: Expressing, Blocking Assay, Multiple Displacement Amplification, Western Blot, Immunohistochemistry, FACS, Formalin-fixed Paraffin-Embedded

    CCL2 induction by TG2 contributes to PD-L1 inhibitor-resistance via regulation of negatively cytotoxic T cells in TNBC cells. The level of (A) PD-L1 and (B) CCL2 mRNA expression in MCF7, BT20, MCF7/TG2, and MDA-MB-231 cells measured by qRT-PCR analysis; (C) western blot analysis to check expression levels of TG2, P-AKT, IκBα, PD-L1, CCL2, and β-Actin in MCF7, BT20, MCF7/TG2, and MDA-MB-231 cells; (D) the expression of TG2, PD-L1, and CCL2 in MCF7/TG2 and MDA-MB-231 cells tested by western blotting analysis after TG2 siRNA transfection; (E) expression of PD-1 and CCR2 in Jurkat T cells with or without treatment of PMA (50 ng/mL) and Ionomycin (1 µg/mL) for 24 h measured by western blotting; (F and G) the protein levels of cPARP, IκBα, PD-L1, and CCL2 in MCF7/TG and MDA-MB-231 cells with or without activated Jurkat T cell co-culture for 24 h measured by western blotting after CCL2 siRNA transfection, Avelumab (20 µg/mL) treatment, or dual combination treatment and Caspase 3/7 activity measured by Caspase 3/7 assay using proteins from MCF7/TG and MDA-MB-231 cells.
    Figure Legend Snippet: CCL2 induction by TG2 contributes to PD-L1 inhibitor-resistance via regulation of negatively cytotoxic T cells in TNBC cells. The level of (A) PD-L1 and (B) CCL2 mRNA expression in MCF7, BT20, MCF7/TG2, and MDA-MB-231 cells measured by qRT-PCR analysis; (C) western blot analysis to check expression levels of TG2, P-AKT, IκBα, PD-L1, CCL2, and β-Actin in MCF7, BT20, MCF7/TG2, and MDA-MB-231 cells; (D) the expression of TG2, PD-L1, and CCL2 in MCF7/TG2 and MDA-MB-231 cells tested by western blotting analysis after TG2 siRNA transfection; (E) expression of PD-1 and CCR2 in Jurkat T cells with or without treatment of PMA (50 ng/mL) and Ionomycin (1 µg/mL) for 24 h measured by western blotting; (F and G) the protein levels of cPARP, IκBα, PD-L1, and CCL2 in MCF7/TG and MDA-MB-231 cells with or without activated Jurkat T cell co-culture for 24 h measured by western blotting after CCL2 siRNA transfection, Avelumab (20 µg/mL) treatment, or dual combination treatment and Caspase 3/7 activity measured by Caspase 3/7 assay using proteins from MCF7/TG and MDA-MB-231 cells.

    Techniques Used: Expressing, Multiple Displacement Amplification, Quantitative RT-PCR, Western Blot, Transfection, Co-Culture Assay, Activity Assay

    7) Product Images from "TGLI1 transcription factor mediates breast cancer brain metastasis via activating metastasis-initiating cancer stem cells and astrocytes in the tumor microenvironment"

    Article Title: TGLI1 transcription factor mediates breast cancer brain metastasis via activating metastasis-initiating cancer stem cells and astrocytes in the tumor microenvironment

    Journal: Oncogene

    doi: 10.1038/s41388-019-0959-3

    TGLI1 promotes breast cancer stem cells and TGLI1-positive stem cells activate human astrocytes. A) Expression of GLI1 and TGLI1 mRNA in a two breast cancer cell lines grown in monolayer or mammospheres. Regular RT-PCR was conducted. MS, mammosphere. ML, monolayer. B) GLI1 and TGLI1 mRNA expression in SKBRM MS relative to ML, as determined by qRT-PCR. C) TGLI1 overexpression in MDA-MB-231 cells led to increased stem cell population defined by CD44 high /CD24 low as determined by FACS. D-F) TGLI1 ectopic expression enhanced the mammosphere-forming ability of BT20 cells. Panel D, average number of mammospheres. Panel E, average size of mammospheres. Panel F, representative images of mammospheres. G-I) TGLI1 ectopic expression enhanced the mammosphere-forming ability of SKBRM cells. Panel G, average number of mammospheres. Panel H, average size of mammospheres. Panel I, representative images of mammospheres. J-K) GFAP expression is increased in human astrocytes upon stimulation with conditioned medium (CM) from TGLI1-positive mammospheres. Isogenic SKBRM cells grown in monolayer or mammospheres were used. Panel J, representative images. Panel K, percent GFAP positive cells. L) Co-culture of human astrocytes (GFP-tagged) with isogenic SKBRM cells (RFP-tagged) under the sphere-forming condition. M-N) Analysis of isogenic MDA-MB-231 intracranial tumors for activated astrocytes (GFAP IHC). M) Representative images. N) Quantified number of astrocytes per area of tumor. Student’s t -test was used to determine p-values.
    Figure Legend Snippet: TGLI1 promotes breast cancer stem cells and TGLI1-positive stem cells activate human astrocytes. A) Expression of GLI1 and TGLI1 mRNA in a two breast cancer cell lines grown in monolayer or mammospheres. Regular RT-PCR was conducted. MS, mammosphere. ML, monolayer. B) GLI1 and TGLI1 mRNA expression in SKBRM MS relative to ML, as determined by qRT-PCR. C) TGLI1 overexpression in MDA-MB-231 cells led to increased stem cell population defined by CD44 high /CD24 low as determined by FACS. D-F) TGLI1 ectopic expression enhanced the mammosphere-forming ability of BT20 cells. Panel D, average number of mammospheres. Panel E, average size of mammospheres. Panel F, representative images of mammospheres. G-I) TGLI1 ectopic expression enhanced the mammosphere-forming ability of SKBRM cells. Panel G, average number of mammospheres. Panel H, average size of mammospheres. Panel I, representative images of mammospheres. J-K) GFAP expression is increased in human astrocytes upon stimulation with conditioned medium (CM) from TGLI1-positive mammospheres. Isogenic SKBRM cells grown in monolayer or mammospheres were used. Panel J, representative images. Panel K, percent GFAP positive cells. L) Co-culture of human astrocytes (GFP-tagged) with isogenic SKBRM cells (RFP-tagged) under the sphere-forming condition. M-N) Analysis of isogenic MDA-MB-231 intracranial tumors for activated astrocytes (GFAP IHC). M) Representative images. N) Quantified number of astrocytes per area of tumor. Student’s t -test was used to determine p-values.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Over Expression, Multiple Displacement Amplification, FACS, Co-Culture Assay, Immunohistochemistry

    8) Product Images from "Identification of CP77 as the Third Orthopoxvirus SAMD9 and SAMD9L Inhibitor with Unique Specificity for a Rodent SAMD9L"

    Article Title: Identification of CP77 as the Third Orthopoxvirus SAMD9 and SAMD9L Inhibitor with Unique Specificity for a Rodent SAMD9L

    Journal: Journal of Virology

    doi: 10.1128/JVI.00225-19

    Expression of chSAMD9L in human cells is sufficient for recapitulating the poxvirus restriction property of CHO cells. (A) A stable human BT20 cell line with inducible expression of chSAMD9L was established via lentiviral transduction. The cell line (i-chSAMD9L) was cultured with medium containing the indicated concentration of doxycycline (Dox). The levels of chSAMD9L and the control HSP70 protein in the cell lysates were determined by Western blotting. (B) The i-chSAMD9L cells were untreated or treated with 1 μg/ml Dox. The cells were then infected with the panel of vK1 − C7 − -derived VACVs. Viral titers at 0 and 24 hpi were measured by plaque assay in Vero cells.
    Figure Legend Snippet: Expression of chSAMD9L in human cells is sufficient for recapitulating the poxvirus restriction property of CHO cells. (A) A stable human BT20 cell line with inducible expression of chSAMD9L was established via lentiviral transduction. The cell line (i-chSAMD9L) was cultured with medium containing the indicated concentration of doxycycline (Dox). The levels of chSAMD9L and the control HSP70 protein in the cell lysates were determined by Western blotting. (B) The i-chSAMD9L cells were untreated or treated with 1 μg/ml Dox. The cells were then infected with the panel of vK1 − C7 − -derived VACVs. Viral titers at 0 and 24 hpi were measured by plaque assay in Vero cells.

    Techniques Used: Expressing, Transduction, Cell Culture, Concentration Assay, Western Blot, Infection, Derivative Assay, Plaque Assay

    9) Product Images from "Identification of CP77 as the Third Orthopoxvirus SAMD9 and SAMD9L Inhibitor with Unique Specificity for a Rodent SAMD9L"

    Article Title: Identification of CP77 as the Third Orthopoxvirus SAMD9 and SAMD9L Inhibitor with Unique Specificity for a Rodent SAMD9L

    Journal: Journal of Virology

    doi: 10.1128/JVI.00225-19

    Expression of chSAMD9L in human cells is sufficient for recapitulating the poxvirus restriction property of CHO cells. (A) A stable human BT20 cell line with inducible expression of chSAMD9L was established via lentiviral transduction. The cell line (i-chSAMD9L) was cultured with medium containing the indicated concentration of doxycycline (Dox). The levels of chSAMD9L and the control HSP70 protein in the cell lysates were determined by Western blotting. (B) The i-chSAMD9L cells were untreated or treated with 1 μg/ml Dox. The cells were then infected with the panel of vK1 − C7 − -derived VACVs. Viral titers at 0 and 24 hpi were measured by plaque assay in Vero cells.
    Figure Legend Snippet: Expression of chSAMD9L in human cells is sufficient for recapitulating the poxvirus restriction property of CHO cells. (A) A stable human BT20 cell line with inducible expression of chSAMD9L was established via lentiviral transduction. The cell line (i-chSAMD9L) was cultured with medium containing the indicated concentration of doxycycline (Dox). The levels of chSAMD9L and the control HSP70 protein in the cell lysates were determined by Western blotting. (B) The i-chSAMD9L cells were untreated or treated with 1 μg/ml Dox. The cells were then infected with the panel of vK1 − C7 − -derived VACVs. Viral titers at 0 and 24 hpi were measured by plaque assay in Vero cells.

    Techniques Used: Expressing, Transduction, Cell Culture, Concentration Assay, Western Blot, Infection, Derivative Assay, Plaque Assay

    10) Product Images from "Leptin produced by obesity-altered adipose stem cells promotes metastasis but not tumorigenesis of triple-negative breast cancer in orthotopic xenograft and patient-derived xenograft models"

    Article Title: Leptin produced by obesity-altered adipose stem cells promotes metastasis but not tumorigenesis of triple-negative breast cancer in orthotopic xenograft and patient-derived xenograft models

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/s13058-019-1153-9

    Transwell co-culture of breast cancer cells with obASCs increases expression of metastatic genes. a TNBC cell line BT20 gene expression was screened for changes in epithelial-to-mesenchymal transition (EMT), inflammatory, and cancer stem cell (CSC) genes after 96-h of transwell co-culture (shown in heat map). b Serpine1, TWIST1, and SNAI2 were evaluated across four cell lines: BT20, HCC1806, MCF7, and TU-BcX-2 K1 PDX-derived cells. Values reported are the mean of three independent experiments each performed in triplicate. Bars, ± SEM. * p
    Figure Legend Snippet: Transwell co-culture of breast cancer cells with obASCs increases expression of metastatic genes. a TNBC cell line BT20 gene expression was screened for changes in epithelial-to-mesenchymal transition (EMT), inflammatory, and cancer stem cell (CSC) genes after 96-h of transwell co-culture (shown in heat map). b Serpine1, TWIST1, and SNAI2 were evaluated across four cell lines: BT20, HCC1806, MCF7, and TU-BcX-2 K1 PDX-derived cells. Values reported are the mean of three independent experiments each performed in triplicate. Bars, ± SEM. * p

    Techniques Used: Co-Culture Assay, Expressing, Derivative Assay

    Knockdown of leptin expression in obASCs decreases promotion of metastatic phenotype. a Western blots of signaling pathways activated in BCCS after Transwell co-culture with control shRNA obASCs compared to leptin shRNA obASCs. b RT-qPCR after transwell co-culture with obASC or obASC with leptin shRNA demonstrates decreased mRNA levels of Serpine1, TWIST1, and SNAI2. c Leptin shRNA obASCs decreased migration of BT20, HCC1806, and MCF7 breast cancer cell lines through a 0.4-um transwell membrane. Values reported are the mean of three independent experiments each performed in triplicate. Bars, ± SEM. * p
    Figure Legend Snippet: Knockdown of leptin expression in obASCs decreases promotion of metastatic phenotype. a Western blots of signaling pathways activated in BCCS after Transwell co-culture with control shRNA obASCs compared to leptin shRNA obASCs. b RT-qPCR after transwell co-culture with obASC or obASC with leptin shRNA demonstrates decreased mRNA levels of Serpine1, TWIST1, and SNAI2. c Leptin shRNA obASCs decreased migration of BT20, HCC1806, and MCF7 breast cancer cell lines through a 0.4-um transwell membrane. Values reported are the mean of three independent experiments each performed in triplicate. Bars, ± SEM. * p

    Techniques Used: Expressing, Western Blot, Co-Culture Assay, shRNA, Quantitative RT-PCR, Migration

    Adipose stem cells do not affect tumor growth of TNBC xenogafts, but obASCs increase metastases. a Average tumor volume of TNBC cell line BT20 xenograft was consistent for all groups across the time course of the experiment. b Evaluation of metastasis revealed increased area of lung metastases from BT20 tumors grown with obASCs. c Average tumor volume of TNBC PDX TU-BcX-2 K1 was consistent for all groups across the time course of the experiment. d Evaluation of metastasis revealed increased area of metastases in lungs of mice with PDX tumors grown with obASCs in comparison to lnASCs or PDX only. e Flow cytometric analysis of the blood demonstrates increased circulating HLA1 + “human cells” in the blood of the obASC group. There is an increase in cancer stem cell marker CD44 + CD24 − in the CTCs from the obASC group. There is an increase in circulating CD326 + (epithelial cell adhesion molecule) cells in the obASC group. Caliper measurements were taken every 3 to 4 days until tumor volume reached 750–1000 mm 3 . Values reported are the mean ( n = 5 mice/group). Bars, ± SEM. * p
    Figure Legend Snippet: Adipose stem cells do not affect tumor growth of TNBC xenogafts, but obASCs increase metastases. a Average tumor volume of TNBC cell line BT20 xenograft was consistent for all groups across the time course of the experiment. b Evaluation of metastasis revealed increased area of lung metastases from BT20 tumors grown with obASCs. c Average tumor volume of TNBC PDX TU-BcX-2 K1 was consistent for all groups across the time course of the experiment. d Evaluation of metastasis revealed increased area of metastases in lungs of mice with PDX tumors grown with obASCs in comparison to lnASCs or PDX only. e Flow cytometric analysis of the blood demonstrates increased circulating HLA1 + “human cells” in the blood of the obASC group. There is an increase in cancer stem cell marker CD44 + CD24 − in the CTCs from the obASC group. There is an increase in circulating CD326 + (epithelial cell adhesion molecule) cells in the obASC group. Caliper measurements were taken every 3 to 4 days until tumor volume reached 750–1000 mm 3 . Values reported are the mean ( n = 5 mice/group). Bars, ± SEM. * p

    Techniques Used: Mouse Assay, Marker

    Secreted factors from adipose stem cells promotes migration of breast cancer. obASCs promoted increased migration of BT20, MCC1806, and MCF7, patient-derived xenograft TU-BcX-2 K1-derived cells through a 0.4-um membrane. All images were acquired at the same magnification. Scale bar represents 100 μm. Values reported are the mean of three independent experiments each performed in triplicate. Bars, ± SEM. * p
    Figure Legend Snippet: Secreted factors from adipose stem cells promotes migration of breast cancer. obASCs promoted increased migration of BT20, MCC1806, and MCF7, patient-derived xenograft TU-BcX-2 K1-derived cells through a 0.4-um membrane. All images were acquired at the same magnification. Scale bar represents 100 μm. Values reported are the mean of three independent experiments each performed in triplicate. Bars, ± SEM. * p

    Techniques Used: Migration, Derivative Assay

    11) Product Images from "ITGA2 is a target of miR-206 promoting cancer stemness and lung metastasis through enhanced ACLY and CCND1 expression in triple negative breast cancer"

    Article Title: ITGA2 is a target of miR-206 promoting cancer stemness and lung metastasis through enhanced ACLY and CCND1 expression in triple negative breast cancer

    Journal: bioRxiv

    doi: 10.1101/583062

    ITGA2 knockdown inhibits cell cycle. A B . Images (A) and cell counts (B) of MDA-MB-231 cells 48 hours after transfections with si ITGA2 ( ITGA2 -KD) or miR-206 mimic. Cell number was measured via hemocytometer counting (B), n=12, p=0.002 ( ITGA2 -KD), and n=3, p=0.001231 (miR- 206). C. Representative flow cytometry analyses of cell cycle with phases of subG1, G1, S, and G2M using propidium iodide upon ITGA2 -KD and miR-206 overexpression in MDA- MB-231 cells. D. Quantified percentage of MDA-MB-231 cells in each cell cycle phase as shown in C. Top panel: si ITGA2 mediated ITGA2- KD and control, n=4 biological replicates; p=0.03 (G1); p=0.004 (G2M). Bottom panel: miR-206 overexpression and control, n=3 biological replicates; p=0.01 (G1), 0.02 (S), 0.04 (G2M). E. Percentage of BT20 cells in each cell cycle phase upon ITGA2 -KD. F. Percentage of MDA-MB-231 cells in each cell cycle phase upon ITGA2 cDNA- mediated overexpression (gated GFP + cells with ITGA2 overexpression).
    Figure Legend Snippet: ITGA2 knockdown inhibits cell cycle. A B . Images (A) and cell counts (B) of MDA-MB-231 cells 48 hours after transfections with si ITGA2 ( ITGA2 -KD) or miR-206 mimic. Cell number was measured via hemocytometer counting (B), n=12, p=0.002 ( ITGA2 -KD), and n=3, p=0.001231 (miR- 206). C. Representative flow cytometry analyses of cell cycle with phases of subG1, G1, S, and G2M using propidium iodide upon ITGA2 -KD and miR-206 overexpression in MDA- MB-231 cells. D. Quantified percentage of MDA-MB-231 cells in each cell cycle phase as shown in C. Top panel: si ITGA2 mediated ITGA2- KD and control, n=4 biological replicates; p=0.03 (G1); p=0.004 (G2M). Bottom panel: miR-206 overexpression and control, n=3 biological replicates; p=0.01 (G1), 0.02 (S), 0.04 (G2M). E. Percentage of BT20 cells in each cell cycle phase upon ITGA2 -KD. F. Percentage of MDA-MB-231 cells in each cell cycle phase upon ITGA2 cDNA- mediated overexpression (gated GFP + cells with ITGA2 overexpression).

    Techniques Used: Multiple Displacement Amplification, Transfection, Flow Cytometry, Over Expression

    12) Product Images from "MFAP5 promotes basal-like breast cancer progression by activating the EMT program"

    Article Title: MFAP5 promotes basal-like breast cancer progression by activating the EMT program

    Journal: Cell & Bioscience

    doi: 10.1186/s13578-019-0284-0

    GO/KEGG pathway enrichment analysis of differentially expressed genes between BT20-LV-vehicle and BT20-LV-MFAP5 cells using RNA-sequencing. a M (log ratio) and A (mean average) scales indicated the mRNA differentially expressed (red spots) in BT20-LV-vehicle and BT20-LV-MFAP5 cells. b The mRNA differentially expressed volcano plots in BT20-LV-vehicle and BT20-LV-MFAP5 cells. c Heatmap of differential expression levels of mRNA (log2RPKM) in these two cells. d Differentially expressed genes enriched and identified by GO analysis in these two cells. e Differentially expressed mRNA in 19 pathways including TGF-β and Notch pathway were figured out by KEGG analysis
    Figure Legend Snippet: GO/KEGG pathway enrichment analysis of differentially expressed genes between BT20-LV-vehicle and BT20-LV-MFAP5 cells using RNA-sequencing. a M (log ratio) and A (mean average) scales indicated the mRNA differentially expressed (red spots) in BT20-LV-vehicle and BT20-LV-MFAP5 cells. b The mRNA differentially expressed volcano plots in BT20-LV-vehicle and BT20-LV-MFAP5 cells. c Heatmap of differential expression levels of mRNA (log2RPKM) in these two cells. d Differentially expressed genes enriched and identified by GO analysis in these two cells. e Differentially expressed mRNA in 19 pathways including TGF-β and Notch pathway were figured out by KEGG analysis

    Techniques Used: RNA Sequencing Assay, Expressing

    TGF-β and Notch pathways mediated the promoting effects of MPAP5 on BLBC cells motility. a BT20-LV-vehicle and BT20-LV-MFAP5 cells were treated with SB431542 or ly-411575 as indicated for 0, 24, 48 and 72 h. Then the OD value of cells was measured. Overexpression of MFAP5 time dependently improved the OD value compared to LV-vehicle group. But OD value elevated by MFAP5 was strikingly weakened by SB431542 and ly-411575. **P
    Figure Legend Snippet: TGF-β and Notch pathways mediated the promoting effects of MPAP5 on BLBC cells motility. a BT20-LV-vehicle and BT20-LV-MFAP5 cells were treated with SB431542 or ly-411575 as indicated for 0, 24, 48 and 72 h. Then the OD value of cells was measured. Overexpression of MFAP5 time dependently improved the OD value compared to LV-vehicle group. But OD value elevated by MFAP5 was strikingly weakened by SB431542 and ly-411575. **P

    Techniques Used: Over Expression

    MFAP5 regulated the activation of TGF-β and Notch pathways in BT20 cells. BT20-LV-vehicle and BT20-LV-MFAP5 cells were treated with SB431542 or ly-411575 as indicated for 24 h and then collected to detect the protein level of p-Smad2/Smad3 by western blot and the mRNA levels of Hes1 and Hes5 by RT-PCR. a , b Compared with LV-vehicle group, overexpression of MFAP5 significantly improved the overexpression of p-Smad2/Smad3 and the mRNA levels of Hes1, Hes5 which were strikingly attenuated by SB431542 and ly-411575. ** P
    Figure Legend Snippet: MFAP5 regulated the activation of TGF-β and Notch pathways in BT20 cells. BT20-LV-vehicle and BT20-LV-MFAP5 cells were treated with SB431542 or ly-411575 as indicated for 24 h and then collected to detect the protein level of p-Smad2/Smad3 by western blot and the mRNA levels of Hes1 and Hes5 by RT-PCR. a , b Compared with LV-vehicle group, overexpression of MFAP5 significantly improved the overexpression of p-Smad2/Smad3 and the mRNA levels of Hes1, Hes5 which were strikingly attenuated by SB431542 and ly-411575. ** P

    Techniques Used: Activation Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Over Expression

    MPAP5 regulated epithelial–mesenchymal transition of BLBC cells through TGF-β and Notch pathways. BT20-LV-vehicle and BT20-LV-MFAP5 cells were treated with SB431542 or ly-411575 as indicated for 24 h and then collected to test the protein levels of E-cadherin, N-cadherin and Type I Collagen by Western blotting and the mRNA levels of Slug, Snail, Zeb1, Zeb2 and Twist by RT-PCR. a Overexpression of MFAP5 reduced the protein level of E-cadherin and increased N-cadherin and Collagen I expression in BT20 cells. SB431542 and ly-411575 partly abolished the inhibitory effect of MFAP5 on E-cadherin and the promoting role on N-cadherin and collagen I. ** P
    Figure Legend Snippet: MPAP5 regulated epithelial–mesenchymal transition of BLBC cells through TGF-β and Notch pathways. BT20-LV-vehicle and BT20-LV-MFAP5 cells were treated with SB431542 or ly-411575 as indicated for 24 h and then collected to test the protein levels of E-cadherin, N-cadherin and Type I Collagen by Western blotting and the mRNA levels of Slug, Snail, Zeb1, Zeb2 and Twist by RT-PCR. a Overexpression of MFAP5 reduced the protein level of E-cadherin and increased N-cadherin and Collagen I expression in BT20 cells. SB431542 and ly-411575 partly abolished the inhibitory effect of MFAP5 on E-cadherin and the promoting role on N-cadherin and collagen I. ** P

    Techniques Used: Western Blot, Reverse Transcription Polymerase Chain Reaction, Over Expression, Expressing

    MFAP5 regulated the proliferation, adhesion and migration of BT20 and HS578T cells. BT20 cells in mock, LV-vehicle, LV-MFAP5 groups and HS578T cells in mock, control shRNA, MFAP5 shRNA groups were cultured at different time as indicated and then the proliferation, adhesion and migration of cells were examined. a Overexpressed MFAP5 time-dependently increased the proliferation of BT20 cells. ** P
    Figure Legend Snippet: MFAP5 regulated the proliferation, adhesion and migration of BT20 and HS578T cells. BT20 cells in mock, LV-vehicle, LV-MFAP5 groups and HS578T cells in mock, control shRNA, MFAP5 shRNA groups were cultured at different time as indicated and then the proliferation, adhesion and migration of cells were examined. a Overexpressed MFAP5 time-dependently increased the proliferation of BT20 cells. ** P

    Techniques Used: Migration, shRNA, Cell Culture

    MFAP5 regulates the invasion of BT20 and HS578T cells. Cell invasion was measured by transwell migration assay. a Compared to BT20-LV-vehicle cells, the invasion was increased of BT20-LV-MFAP5 cells. ** P
    Figure Legend Snippet: MFAP5 regulates the invasion of BT20 and HS578T cells. Cell invasion was measured by transwell migration assay. a Compared to BT20-LV-vehicle cells, the invasion was increased of BT20-LV-MFAP5 cells. ** P

    Techniques Used: Transwell Migration Assay

    MFAP5 regulated tumor proliferation and BT20 cell metastasis. a The volume of tumors in BT20-LV-MFAP5 nude mice was bigger than those in BT20-LV-vehicle group. b Compared with nude mice in BT20-LV-vehicle group, the weight of tumors in BT20-LV-MFAP5 group was significantly increased. ** P
    Figure Legend Snippet: MFAP5 regulated tumor proliferation and BT20 cell metastasis. a The volume of tumors in BT20-LV-MFAP5 nude mice was bigger than those in BT20-LV-vehicle group. b Compared with nude mice in BT20-LV-vehicle group, the weight of tumors in BT20-LV-MFAP5 group was significantly increased. ** P

    Techniques Used: Mouse Assay

    MFAP5 regulated angiogenesis of HUVEC cells. a The length of blood vessels of HUVEC cells in the supernatant of BT20-LV-MFAP5 cells was increased compared with those in BT20-LV-vehicle cell supernatant. ** P
    Figure Legend Snippet: MFAP5 regulated angiogenesis of HUVEC cells. a The length of blood vessels of HUVEC cells in the supernatant of BT20-LV-MFAP5 cells was increased compared with those in BT20-LV-vehicle cell supernatant. ** P

    Techniques Used:

    13) Product Images from "SRC inhibition prevents P-cadherin mediated signaling and function in basal-like breast cancer cells"

    Article Title: SRC inhibition prevents P-cadherin mediated signaling and function in basal-like breast cancer cells

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-018-0286-2

    Dasatinib treatment inhibits the in vitro functional activity induced by P-cadherin expression. a Western blotting for pSRC(Tyr416), total Src, P-cadherin, E-cadherin and p120ctn in P-cadherin overexpressing cells after dasatinib treatment (100 nM) for 48 h. Protein levels of β-actin were analyzed and used as the loading control. b Representative experiment from a wound healing migration assay, in both P-cadherin overexpressing BCC (MCF-7/AZ.Pcad and BT20), treated with 100 nM of dasatinib or DMSO for 24 h. c Fold change in the number of invasive cells, evaluated by the matrigel invasion assay for both MCF-7/AZ.Pcad and BT20 treated with DMSO or 100 nM of dasatinib. d Zymography for MMP2 activity and Western blotting for sP-cad, using the conditioned medium from cells treated with DMSO or 100 nM of dasatinib for 48 h, in both MCF-7/AZ.Pcad and BT20 BCC models. e Mammosphere forming assay was performed for both cell models. The P -values indicate the statistically significant difference between DMSO and dasatinib treated cells, in both P-cadherin BCC models. f Average values of Work (J), representing the cell-cell adhesion strength, of both BCC cancer cell models treated with DMSO and dasatinib, using AFM Force Spectroscopy analysis. g Box-plot quantification of the length (μm) and h number of invasive protrusions of MCF-7/AZ.Pcad spheroids, treated with DMSO or 100 nM dasatinib, for 24 h. P -values
    Figure Legend Snippet: Dasatinib treatment inhibits the in vitro functional activity induced by P-cadherin expression. a Western blotting for pSRC(Tyr416), total Src, P-cadherin, E-cadherin and p120ctn in P-cadherin overexpressing cells after dasatinib treatment (100 nM) for 48 h. Protein levels of β-actin were analyzed and used as the loading control. b Representative experiment from a wound healing migration assay, in both P-cadherin overexpressing BCC (MCF-7/AZ.Pcad and BT20), treated with 100 nM of dasatinib or DMSO for 24 h. c Fold change in the number of invasive cells, evaluated by the matrigel invasion assay for both MCF-7/AZ.Pcad and BT20 treated with DMSO or 100 nM of dasatinib. d Zymography for MMP2 activity and Western blotting for sP-cad, using the conditioned medium from cells treated with DMSO or 100 nM of dasatinib for 48 h, in both MCF-7/AZ.Pcad and BT20 BCC models. e Mammosphere forming assay was performed for both cell models. The P -values indicate the statistically significant difference between DMSO and dasatinib treated cells, in both P-cadherin BCC models. f Average values of Work (J), representing the cell-cell adhesion strength, of both BCC cancer cell models treated with DMSO and dasatinib, using AFM Force Spectroscopy analysis. g Box-plot quantification of the length (μm) and h number of invasive protrusions of MCF-7/AZ.Pcad spheroids, treated with DMSO or 100 nM dasatinib, for 24 h. P -values

    Techniques Used: In Vitro, Functional Assay, Activity Assay, Expressing, Western Blot, Migration, Invasion Assay, Zymography, Spectroscopy

    Dasatinib treatment affects the in vivo tumorigenic and metastatic capacity of P-cadherin overexpressing tumors. a Kaplan-Meyer survival curve for the overall survival of mice treated with DMSO and dasatinib 10 mg/kg, for a maximum period of 210 days. P-value was calculated using a log-rank test to assess significant differences for mice overall survival. b Percentage of tumours with vascular invasion, observed by HE staining. Presence or absence of clusters of tumour cells inside blood vessels surrounding the primary tumour was quantified in control versus dasatinib treated mice (10 mg/kg). c Tumour volume measured at week 5, post inoculation of cancer cells, from control and dasatinib treated groups, in all BCC models (SUM149, BT20 and MDA-MB-468). d Immunofluorescence for p120ctn (red) and DAPI (blue in the primary tumours from control and dasatinib treated animals. Scale bar = 50 μm. e Immunohistochemistry for p120ctn and pSRC(Tyr416) in the primary tumours from control and dasatinib treated animals. The images shown are representative ones. f Quantification of the p120ctn cytoplasmic intensity expression in primary tumours from control and dasatinib treated animals. g Quantification of the p120ctn membrane intensity. Each dot represents the mean of p120ctn membrane intensity of 100 pairs of cells from a single biological replicate. Student’s t-tests were used to determine statistically significant differences, and P-values are indicated in the figure. Scale bar = 50 μm. h Proximity ligation assay for E-cadherin and p120ctn, from tumours recovered from different mice from control and dasatinib treated groups. i Box-plot for the average number of blobs/cells, quantified using the images of the proximity ligation assay for E-cadherin and p120ctn, between tumours from control and dasatinib treated mice. Student’s t-tests were used to determine statistically significant differences, and the P-value are indicated in the figure. Scale bar = 50 μm
    Figure Legend Snippet: Dasatinib treatment affects the in vivo tumorigenic and metastatic capacity of P-cadherin overexpressing tumors. a Kaplan-Meyer survival curve for the overall survival of mice treated with DMSO and dasatinib 10 mg/kg, for a maximum period of 210 days. P-value was calculated using a log-rank test to assess significant differences for mice overall survival. b Percentage of tumours with vascular invasion, observed by HE staining. Presence or absence of clusters of tumour cells inside blood vessels surrounding the primary tumour was quantified in control versus dasatinib treated mice (10 mg/kg). c Tumour volume measured at week 5, post inoculation of cancer cells, from control and dasatinib treated groups, in all BCC models (SUM149, BT20 and MDA-MB-468). d Immunofluorescence for p120ctn (red) and DAPI (blue in the primary tumours from control and dasatinib treated animals. Scale bar = 50 μm. e Immunohistochemistry for p120ctn and pSRC(Tyr416) in the primary tumours from control and dasatinib treated animals. The images shown are representative ones. f Quantification of the p120ctn cytoplasmic intensity expression in primary tumours from control and dasatinib treated animals. g Quantification of the p120ctn membrane intensity. Each dot represents the mean of p120ctn membrane intensity of 100 pairs of cells from a single biological replicate. Student’s t-tests were used to determine statistically significant differences, and P-values are indicated in the figure. Scale bar = 50 μm. h Proximity ligation assay for E-cadherin and p120ctn, from tumours recovered from different mice from control and dasatinib treated groups. i Box-plot for the average number of blobs/cells, quantified using the images of the proximity ligation assay for E-cadherin and p120ctn, between tumours from control and dasatinib treated mice. Student’s t-tests were used to determine statistically significant differences, and the P-value are indicated in the figure. Scale bar = 50 μm

    Techniques Used: In Vivo, Mouse Assay, Staining, Multiple Displacement Amplification, Immunofluorescence, Immunohistochemistry, Expressing, Proximity Ligation Assay

    Dasatinib treatment promotes the recovery of E-cadherin function with stabilization of the E-cad/p120ctn complex to the cell membrane. a Dual Immunofluorescence for anti-p120ctn antibody (green), F-actin (red), E-cadherin (green) and DAPI (blue) in MCF-7/AZ.Pcad cells after 100 nM dasatinib treatment for 48 h. Scale bar = 50 μm. b Internuclear profile for p120ctn and E-cadherin, obtained through the computational analysis of confocal immunofluorescent images. P-values indicate significant differences observed in the membrane mean fluorescent intensity for p120ctn between MCF-7/AZ.Pcad treated with DMSO versus 100 nM dasatinib, 48 h. c Quantification of the active Rac1-GTP, measured by a G-Lisa assay, was performed to measure Rac1 activity in P-cadherin overexpressing cells (MCF-7/AZ.Pcad and BT20) treated with DMSO and dasatinib. d Proximity ligation assay for E-cadherin and p120ctn, for MCF-7/AZ.Pcad treated with DMSO versus 100 nM dasatinib during 48 h. e 72 h slow aggregation assay images for dasatinib treated MCF-7/AZ.Pcad cells with inhibition of E-cadherin function by MB2 antibody. The images shown are representative ones. P-values are indicated in the figure and considered statistically significant when
    Figure Legend Snippet: Dasatinib treatment promotes the recovery of E-cadherin function with stabilization of the E-cad/p120ctn complex to the cell membrane. a Dual Immunofluorescence for anti-p120ctn antibody (green), F-actin (red), E-cadherin (green) and DAPI (blue) in MCF-7/AZ.Pcad cells after 100 nM dasatinib treatment for 48 h. Scale bar = 50 μm. b Internuclear profile for p120ctn and E-cadherin, obtained through the computational analysis of confocal immunofluorescent images. P-values indicate significant differences observed in the membrane mean fluorescent intensity for p120ctn between MCF-7/AZ.Pcad treated with DMSO versus 100 nM dasatinib, 48 h. c Quantification of the active Rac1-GTP, measured by a G-Lisa assay, was performed to measure Rac1 activity in P-cadherin overexpressing cells (MCF-7/AZ.Pcad and BT20) treated with DMSO and dasatinib. d Proximity ligation assay for E-cadherin and p120ctn, for MCF-7/AZ.Pcad treated with DMSO versus 100 nM dasatinib during 48 h. e 72 h slow aggregation assay images for dasatinib treated MCF-7/AZ.Pcad cells with inhibition of E-cadherin function by MB2 antibody. The images shown are representative ones. P-values are indicated in the figure and considered statistically significant when

    Techniques Used: Immunofluorescence, Activity Assay, Proximity Ligation Assay, Inhibition

    14) Product Images from "SRC inhibition prevents P-cadherin mediated signaling and function in basal-like breast cancer cells"

    Article Title: SRC inhibition prevents P-cadherin mediated signaling and function in basal-like breast cancer cells

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-018-0286-2

    Dasatinib treatment inhibits the in vitro functional activity induced by P-cadherin expression. a Western blotting for pSRC(Tyr416), total Src, P-cadherin, E-cadherin and p120ctn in P-cadherin overexpressing cells after dasatinib treatment (100 nM) for 48 h. Protein levels of β-actin were analyzed and used as the loading control. b Representative experiment from a wound healing migration assay, in both P-cadherin overexpressing BCC (MCF-7/AZ.Pcad and BT20), treated with 100 nM of dasatinib or DMSO for 24 h. c Fold change in the number of invasive cells, evaluated by the matrigel invasion assay for both MCF-7/AZ.Pcad and BT20 treated with DMSO or 100 nM of dasatinib. d Zymography for MMP2 activity and Western blotting for sP-cad, using the conditioned medium from cells treated with DMSO or 100 nM of dasatinib for 48 h, in both MCF-7/AZ.Pcad and BT20 BCC models. e Mammosphere forming assay was performed for both cell models. The P -values indicate the statistically significant difference between DMSO and dasatinib treated cells, in both P-cadherin BCC models. f Average values of Work (J), representing the cell-cell adhesion strength, of both BCC cancer cell models treated with DMSO and dasatinib, using AFM Force Spectroscopy analysis. g Box-plot quantification of the length (μm) and h number of invasive protrusions of MCF-7/AZ.Pcad spheroids, treated with DMSO or 100 nM dasatinib, for 24 h. P -values
    Figure Legend Snippet: Dasatinib treatment inhibits the in vitro functional activity induced by P-cadherin expression. a Western blotting for pSRC(Tyr416), total Src, P-cadherin, E-cadherin and p120ctn in P-cadherin overexpressing cells after dasatinib treatment (100 nM) for 48 h. Protein levels of β-actin were analyzed and used as the loading control. b Representative experiment from a wound healing migration assay, in both P-cadherin overexpressing BCC (MCF-7/AZ.Pcad and BT20), treated with 100 nM of dasatinib or DMSO for 24 h. c Fold change in the number of invasive cells, evaluated by the matrigel invasion assay for both MCF-7/AZ.Pcad and BT20 treated with DMSO or 100 nM of dasatinib. d Zymography for MMP2 activity and Western blotting for sP-cad, using the conditioned medium from cells treated with DMSO or 100 nM of dasatinib for 48 h, in both MCF-7/AZ.Pcad and BT20 BCC models. e Mammosphere forming assay was performed for both cell models. The P -values indicate the statistically significant difference between DMSO and dasatinib treated cells, in both P-cadherin BCC models. f Average values of Work (J), representing the cell-cell adhesion strength, of both BCC cancer cell models treated with DMSO and dasatinib, using AFM Force Spectroscopy analysis. g Box-plot quantification of the length (μm) and h number of invasive protrusions of MCF-7/AZ.Pcad spheroids, treated with DMSO or 100 nM dasatinib, for 24 h. P -values

    Techniques Used: In Vitro, Functional Assay, Activity Assay, Expressing, Western Blot, Migration, Invasion Assay, Zymography, Spectroscopy

    Dasatinib treatment affects the in vivo tumorigenic and metastatic capacity of P-cadherin overexpressing tumors. a Kaplan-Meyer survival curve for the overall survival of mice treated with DMSO and dasatinib 10 mg/kg, for a maximum period of 210 days. P-value was calculated using a log-rank test to assess significant differences for mice overall survival. b Percentage of tumours with vascular invasion, observed by HE staining. Presence or absence of clusters of tumour cells inside blood vessels surrounding the primary tumour was quantified in control versus dasatinib treated mice (10 mg/kg). c Tumour volume measured at week 5, post inoculation of cancer cells, from control and dasatinib treated groups, in all BCC models (SUM149, BT20 and MDA-MB-468). d Immunofluorescence for p120ctn (red) and DAPI (blue in the primary tumours from control and dasatinib treated animals. Scale bar = 50 μm. e Immunohistochemistry for p120ctn and pSRC(Tyr416) in the primary tumours from control and dasatinib treated animals. The images shown are representative ones. f Quantification of the p120ctn cytoplasmic intensity expression in primary tumours from control and dasatinib treated animals. g Quantification of the p120ctn membrane intensity. Each dot represents the mean of p120ctn membrane intensity of 100 pairs of cells from a single biological replicate. Student’s t-tests were used to determine statistically significant differences, and P-values are indicated in the figure. Scale bar = 50 μm. h Proximity ligation assay for E-cadherin and p120ctn, from tumours recovered from different mice from control and dasatinib treated groups. i Box-plot for the average number of blobs/cells, quantified using the images of the proximity ligation assay for E-cadherin and p120ctn, between tumours from control and dasatinib treated mice. Student’s t-tests were used to determine statistically significant differences, and the P-value are indicated in the figure. Scale bar = 50 μm
    Figure Legend Snippet: Dasatinib treatment affects the in vivo tumorigenic and metastatic capacity of P-cadherin overexpressing tumors. a Kaplan-Meyer survival curve for the overall survival of mice treated with DMSO and dasatinib 10 mg/kg, for a maximum period of 210 days. P-value was calculated using a log-rank test to assess significant differences for mice overall survival. b Percentage of tumours with vascular invasion, observed by HE staining. Presence or absence of clusters of tumour cells inside blood vessels surrounding the primary tumour was quantified in control versus dasatinib treated mice (10 mg/kg). c Tumour volume measured at week 5, post inoculation of cancer cells, from control and dasatinib treated groups, in all BCC models (SUM149, BT20 and MDA-MB-468). d Immunofluorescence for p120ctn (red) and DAPI (blue in the primary tumours from control and dasatinib treated animals. Scale bar = 50 μm. e Immunohistochemistry for p120ctn and pSRC(Tyr416) in the primary tumours from control and dasatinib treated animals. The images shown are representative ones. f Quantification of the p120ctn cytoplasmic intensity expression in primary tumours from control and dasatinib treated animals. g Quantification of the p120ctn membrane intensity. Each dot represents the mean of p120ctn membrane intensity of 100 pairs of cells from a single biological replicate. Student’s t-tests were used to determine statistically significant differences, and P-values are indicated in the figure. Scale bar = 50 μm. h Proximity ligation assay for E-cadherin and p120ctn, from tumours recovered from different mice from control and dasatinib treated groups. i Box-plot for the average number of blobs/cells, quantified using the images of the proximity ligation assay for E-cadherin and p120ctn, between tumours from control and dasatinib treated mice. Student’s t-tests were used to determine statistically significant differences, and the P-value are indicated in the figure. Scale bar = 50 μm

    Techniques Used: In Vivo, Mouse Assay, Staining, Multiple Displacement Amplification, Immunofluorescence, Immunohistochemistry, Expressing, Proximity Ligation Assay

    15) Product Images from "SRC inhibition prevents P-cadherin mediated signaling and function in basal-like breast cancer cells"

    Article Title: SRC inhibition prevents P-cadherin mediated signaling and function in basal-like breast cancer cells

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-018-0286-2

    Dasatinib treatment inhibits the in vitro functional activity induced by P-cadherin expression. a Western blotting for pSRC(Tyr416), total Src, P-cadherin, E-cadherin and p120ctn in P-cadherin overexpressing cells after dasatinib treatment (100 nM) for 48 h. Protein levels of β-actin were analyzed and used as the loading control. b Representative experiment from a wound healing migration assay, in both P-cadherin overexpressing BCC (MCF-7/AZ.Pcad and BT20), treated with 100 nM of dasatinib or DMSO for 24 h. c Fold change in the number of invasive cells, evaluated by the matrigel invasion assay for both MCF-7/AZ.Pcad and BT20 treated with DMSO or 100 nM of dasatinib. d Zymography for MMP2 activity and Western blotting for sP-cad, using the conditioned medium from cells treated with DMSO or 100 nM of dasatinib for 48 h, in both MCF-7/AZ.Pcad and BT20 BCC models. e Mammosphere forming assay was performed for both cell models. The P -values indicate the statistically significant difference between DMSO and dasatinib treated cells, in both P-cadherin BCC models. f Average values of Work (J), representing the cell-cell adhesion strength, of both BCC cancer cell models treated with DMSO and dasatinib, using AFM Force Spectroscopy analysis. g Box-plot quantification of the length (μm) and h number of invasive protrusions of MCF-7/AZ.Pcad spheroids, treated with DMSO or 100 nM dasatinib, for 24 h. P -values
    Figure Legend Snippet: Dasatinib treatment inhibits the in vitro functional activity induced by P-cadherin expression. a Western blotting for pSRC(Tyr416), total Src, P-cadherin, E-cadherin and p120ctn in P-cadherin overexpressing cells after dasatinib treatment (100 nM) for 48 h. Protein levels of β-actin were analyzed and used as the loading control. b Representative experiment from a wound healing migration assay, in both P-cadherin overexpressing BCC (MCF-7/AZ.Pcad and BT20), treated with 100 nM of dasatinib or DMSO for 24 h. c Fold change in the number of invasive cells, evaluated by the matrigel invasion assay for both MCF-7/AZ.Pcad and BT20 treated with DMSO or 100 nM of dasatinib. d Zymography for MMP2 activity and Western blotting for sP-cad, using the conditioned medium from cells treated with DMSO or 100 nM of dasatinib for 48 h, in both MCF-7/AZ.Pcad and BT20 BCC models. e Mammosphere forming assay was performed for both cell models. The P -values indicate the statistically significant difference between DMSO and dasatinib treated cells, in both P-cadherin BCC models. f Average values of Work (J), representing the cell-cell adhesion strength, of both BCC cancer cell models treated with DMSO and dasatinib, using AFM Force Spectroscopy analysis. g Box-plot quantification of the length (μm) and h number of invasive protrusions of MCF-7/AZ.Pcad spheroids, treated with DMSO or 100 nM dasatinib, for 24 h. P -values

    Techniques Used: In Vitro, Functional Assay, Activity Assay, Expressing, Western Blot, Migration, Invasion Assay, Zymography, Spectroscopy

    Dasatinib treatment affects the in vivo tumorigenic and metastatic capacity of P-cadherin overexpressing tumors. a Kaplan-Meyer survival curve for the overall survival of mice treated with DMSO and dasatinib 10 mg/kg, for a maximum period of 210 days. P-value was calculated using a log-rank test to assess significant differences for mice overall survival. b Percentage of tumours with vascular invasion, observed by HE staining. Presence or absence of clusters of tumour cells inside blood vessels surrounding the primary tumour was quantified in control versus dasatinib treated mice (10 mg/kg). c Tumour volume measured at week 5, post inoculation of cancer cells, from control and dasatinib treated groups, in all BCC models (SUM149, BT20 and MDA-MB-468). d Immunofluorescence for p120ctn (red) and DAPI (blue in the primary tumours from control and dasatinib treated animals. Scale bar = 50 μm. e Immunohistochemistry for p120ctn and pSRC(Tyr416) in the primary tumours from control and dasatinib treated animals. The images shown are representative ones. f Quantification of the p120ctn cytoplasmic intensity expression in primary tumours from control and dasatinib treated animals. g Quantification of the p120ctn membrane intensity. Each dot represents the mean of p120ctn membrane intensity of 100 pairs of cells from a single biological replicate. Student’s t-tests were used to determine statistically significant differences, and P-values are indicated in the figure. Scale bar = 50 μm. h Proximity ligation assay for E-cadherin and p120ctn, from tumours recovered from different mice from control and dasatinib treated groups. i Box-plot for the average number of blobs/cells, quantified using the images of the proximity ligation assay for E-cadherin and p120ctn, between tumours from control and dasatinib treated mice. Student’s t-tests were used to determine statistically significant differences, and the P-value are indicated in the figure. Scale bar = 50 μm
    Figure Legend Snippet: Dasatinib treatment affects the in vivo tumorigenic and metastatic capacity of P-cadherin overexpressing tumors. a Kaplan-Meyer survival curve for the overall survival of mice treated with DMSO and dasatinib 10 mg/kg, for a maximum period of 210 days. P-value was calculated using a log-rank test to assess significant differences for mice overall survival. b Percentage of tumours with vascular invasion, observed by HE staining. Presence or absence of clusters of tumour cells inside blood vessels surrounding the primary tumour was quantified in control versus dasatinib treated mice (10 mg/kg). c Tumour volume measured at week 5, post inoculation of cancer cells, from control and dasatinib treated groups, in all BCC models (SUM149, BT20 and MDA-MB-468). d Immunofluorescence for p120ctn (red) and DAPI (blue in the primary tumours from control and dasatinib treated animals. Scale bar = 50 μm. e Immunohistochemistry for p120ctn and pSRC(Tyr416) in the primary tumours from control and dasatinib treated animals. The images shown are representative ones. f Quantification of the p120ctn cytoplasmic intensity expression in primary tumours from control and dasatinib treated animals. g Quantification of the p120ctn membrane intensity. Each dot represents the mean of p120ctn membrane intensity of 100 pairs of cells from a single biological replicate. Student’s t-tests were used to determine statistically significant differences, and P-values are indicated in the figure. Scale bar = 50 μm. h Proximity ligation assay for E-cadherin and p120ctn, from tumours recovered from different mice from control and dasatinib treated groups. i Box-plot for the average number of blobs/cells, quantified using the images of the proximity ligation assay for E-cadherin and p120ctn, between tumours from control and dasatinib treated mice. Student’s t-tests were used to determine statistically significant differences, and the P-value are indicated in the figure. Scale bar = 50 μm

    Techniques Used: In Vivo, Mouse Assay, Staining, Multiple Displacement Amplification, Immunofluorescence, Immunohistochemistry, Expressing, Proximity Ligation Assay

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    ATCC bt 20
    TLN1 exon 17b inclusion is associated with altered drug response and gene dependencies in cancer cell lines. (A) Differential splicing of TLN1 exon 17b is detected in cancer cell lines. Boxplots show distribution of percent spliced-in (PSI) values for TLN1 exon 17b in lung, colon and breast cancer cell lines. The individually labelled <t>BT20,</t> ZR751, MDA231 and BT549 breast cancer cell lines were used for RT-PCR validation in (B). (B) RT-PCR validation of TLN1 exon 17b expression in four representative breast cancer cell lines. RT-PCR was performed with primers flanking exon 17b (primer positions indicated by black arrows). Exon 17b spans 51 base pairs (bp) and exon 17b inclusion results in an amplicon size increase from 183 bp to 234 bp. BT20 and ZR751 cell lines show exon 17b inclusion whereas MDA231 and BT549 cell lines show exon 17b skipping. (C) Expression of TLN1 inclusion junctions (18, 20) and skipping junction (19) was correlated to cell survival after drug treatment using DepMap drug sensitivity data across all cancer cell lines. The top-ranked correlation coefficients (FDR
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    TLN1 exon 17b inclusion is associated with altered drug response and gene dependencies in cancer cell lines. (A) Differential splicing of TLN1 exon 17b is detected in cancer cell lines. Boxplots show distribution of percent spliced-in (PSI) values for TLN1 exon 17b in lung, colon and breast cancer cell lines. The individually labelled BT20, ZR751, MDA231 and BT549 breast cancer cell lines were used for RT-PCR validation in (B). (B) RT-PCR validation of TLN1 exon 17b expression in four representative breast cancer cell lines. RT-PCR was performed with primers flanking exon 17b (primer positions indicated by black arrows). Exon 17b spans 51 base pairs (bp) and exon 17b inclusion results in an amplicon size increase from 183 bp to 234 bp. BT20 and ZR751 cell lines show exon 17b inclusion whereas MDA231 and BT549 cell lines show exon 17b skipping. (C) Expression of TLN1 inclusion junctions (18, 20) and skipping junction (19) was correlated to cell survival after drug treatment using DepMap drug sensitivity data across all cancer cell lines. The top-ranked correlation coefficients (FDR

    Journal: bioRxiv

    Article Title: A novel cancer-associated cassette exon in TLN1 alters Talin 1 mechanosensitivity

    doi: 10.1101/2022.09.06.506802

    Figure Lengend Snippet: TLN1 exon 17b inclusion is associated with altered drug response and gene dependencies in cancer cell lines. (A) Differential splicing of TLN1 exon 17b is detected in cancer cell lines. Boxplots show distribution of percent spliced-in (PSI) values for TLN1 exon 17b in lung, colon and breast cancer cell lines. The individually labelled BT20, ZR751, MDA231 and BT549 breast cancer cell lines were used for RT-PCR validation in (B). (B) RT-PCR validation of TLN1 exon 17b expression in four representative breast cancer cell lines. RT-PCR was performed with primers flanking exon 17b (primer positions indicated by black arrows). Exon 17b spans 51 base pairs (bp) and exon 17b inclusion results in an amplicon size increase from 183 bp to 234 bp. BT20 and ZR751 cell lines show exon 17b inclusion whereas MDA231 and BT549 cell lines show exon 17b skipping. (C) Expression of TLN1 inclusion junctions (18, 20) and skipping junction (19) was correlated to cell survival after drug treatment using DepMap drug sensitivity data across all cancer cell lines. The top-ranked correlation coefficients (FDR

    Article Snippet: Cell line and organoid culture BT20, BT549, MCF7, MCF10a, MDA-MB231, MDA-MB453, SKBR3, SUM44PE, T47D and ZR751 cell lines were obtained from the American Type Culture Collection (ATCC), STR typed verified by PCR and cultured as described previously.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification

    CLSTN1 exon 11 expression is inversely correlated with TLN1 exon 17b expression in cancer patients and cell lines (A) Correlation analysis of percent spliced-in (PSI) values of TLN1 exon 17b and CLSTN1 exon 11 in tumor patient tissue and cancer cell lines. TLN1 exon 17b and CLSTN1 exon 11 splicing is inversely correlated suggesting nearly trans-mutually exclusive splicing regulation of these two events ( R = Pearson correlation coefficient, P = adjusted P -value). (B) RT-PCR validation of CLSTN1 exon 11 expression in four representative breast cancer cell lines. RT-PCR was performed with primers flanking exon 11 (primer positions indicated by black arrows). Exon 11 skipping results in an amplicon size reduction from 278 bp to 221 base pairs. BT20 and ZR751 cell lines show exon 11 skipping whereas MDA231 and BT549 cell lines show exon 11 inclusion. The RT-PCR analysis of TLN1 exon 17b expression in cancer cell lines shown in Figure 2B was repurposed here to illustrate the inverse splicing pattern with CLSTN1 exon 11. (C) Analogous analysis to Figure 2E , that reveals dynamic CLSTN1 exon 11 splicing in response to combined TGF-β/EGF treatment. Gene-wise splice plots of CLSTN1 junction expression in HeLa cells, which show baseline skipping of exon 11. The analysis of the TLN1 exon 17b expression in HeLa cells shown in Figure 2E was repurposed here to illustrate the inverse splicing pattern with CLSTN1 exon 11 (The plots shown in this figure were generated by DJExpress -based re-analysis of RNA-Seq data from GSE72419; Grey area indicates the log-fold change cut-off (|logFC| > 0.5). Inclusion junctions are shown in red, skipping junctions are shown in blue. Junctions with FDR > 0.05 for absolute or relative logFC (or both) are shown in black. Black arrow indicates the direction of transcription on the reverse strand).

    Journal: bioRxiv

    Article Title: A novel cancer-associated cassette exon in TLN1 alters Talin 1 mechanosensitivity

    doi: 10.1101/2022.09.06.506802

    Figure Lengend Snippet: CLSTN1 exon 11 expression is inversely correlated with TLN1 exon 17b expression in cancer patients and cell lines (A) Correlation analysis of percent spliced-in (PSI) values of TLN1 exon 17b and CLSTN1 exon 11 in tumor patient tissue and cancer cell lines. TLN1 exon 17b and CLSTN1 exon 11 splicing is inversely correlated suggesting nearly trans-mutually exclusive splicing regulation of these two events ( R = Pearson correlation coefficient, P = adjusted P -value). (B) RT-PCR validation of CLSTN1 exon 11 expression in four representative breast cancer cell lines. RT-PCR was performed with primers flanking exon 11 (primer positions indicated by black arrows). Exon 11 skipping results in an amplicon size reduction from 278 bp to 221 base pairs. BT20 and ZR751 cell lines show exon 11 skipping whereas MDA231 and BT549 cell lines show exon 11 inclusion. The RT-PCR analysis of TLN1 exon 17b expression in cancer cell lines shown in Figure 2B was repurposed here to illustrate the inverse splicing pattern with CLSTN1 exon 11. (C) Analogous analysis to Figure 2E , that reveals dynamic CLSTN1 exon 11 splicing in response to combined TGF-β/EGF treatment. Gene-wise splice plots of CLSTN1 junction expression in HeLa cells, which show baseline skipping of exon 11. The analysis of the TLN1 exon 17b expression in HeLa cells shown in Figure 2E was repurposed here to illustrate the inverse splicing pattern with CLSTN1 exon 11 (The plots shown in this figure were generated by DJExpress -based re-analysis of RNA-Seq data from GSE72419; Grey area indicates the log-fold change cut-off (|logFC| > 0.5). Inclusion junctions are shown in red, skipping junctions are shown in blue. Junctions with FDR > 0.05 for absolute or relative logFC (or both) are shown in black. Black arrow indicates the direction of transcription on the reverse strand).

    Article Snippet: Cell line and organoid culture BT20, BT549, MCF7, MCF10a, MDA-MB231, MDA-MB453, SKBR3, SUM44PE, T47D and ZR751 cell lines were obtained from the American Type Culture Collection (ATCC), STR typed verified by PCR and cultured as described previously.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Generated, RNA Sequencing Assay

    ER + and TN BC cells show distinct growth and metastatic behavior. a Scheme illustrating the intraductal xenografting approach used in this study. Η E: Haematoxylin Eosin, IF: Immunofluorescence. b Bar graph showing take rates for TN (blue) and ER + (red) BC cells injected intraductally, 13–19 mammary glands of 5–9 mice injected in each group. The vertical dashed line indicates 90%. c Graph showing the fold-change of bioluminescence over time for all intraductal xenografts. Data represent mean ± SEM of 13-19 mammary glands from 5–9 mice in each group. The dashed line indicates the experimental end-point for TNBC xenografts. d Fold-change of bioluminescence at 5 weeks after intraductal injection. Data represent mean ± SEM of 13–19 mammary glands from 5 to 9 mice in each group. Blue and red dashed lines represent the average change bioluminescence of TN (1148 fold) and ER + BC (32 fold), respectively. One-way ANOVA, Kruskal-Wallis test relative to MCF-7 (control). e Box plot showing the fold-change of bioluminescence at endpoint for TN (5 weeks) and ER + (5-6 months) BC cells from Fig. 1c. Boxes span the 25th to 75th percentile, whiskers 1.5 times the interquartile range. Boxplot whiskers show minimum and maximum values. Two-tailed Mann-Whitney test. f , g Bar plot showing ex vivo bioluminescence of resected organs from 9, 7, and 6 mice bearing BT20, HCC1806, and T70 xenografts, respectively, f and 20, 9, 10, and 14 mice bearing MCF-7, T47D, T99, METS15 xenografts, respectively, g Data represent mean ± SEM. h Dot plot showing the ratio of bioluminescence in lungs over primary tumor. Data represent mean ± SEM of n = 9 (BT20), 7 (HCC1806), 12 (MCF-7), 8 (T47D), 3 (T99) and 12 (METS15) mice. Blue and red dashed lines represent the average ratio for TN and ER + BC cells, respectively.

    Journal: Nature Communications

    Article Title: Epithelial-mesenchymal plasticity determines estrogen receptor positive breast cancer dormancy and epithelial reconversion drives recurrence

    doi: 10.1038/s41467-022-32523-6

    Figure Lengend Snippet: ER + and TN BC cells show distinct growth and metastatic behavior. a Scheme illustrating the intraductal xenografting approach used in this study. Η E: Haematoxylin Eosin, IF: Immunofluorescence. b Bar graph showing take rates for TN (blue) and ER + (red) BC cells injected intraductally, 13–19 mammary glands of 5–9 mice injected in each group. The vertical dashed line indicates 90%. c Graph showing the fold-change of bioluminescence over time for all intraductal xenografts. Data represent mean ± SEM of 13-19 mammary glands from 5–9 mice in each group. The dashed line indicates the experimental end-point for TNBC xenografts. d Fold-change of bioluminescence at 5 weeks after intraductal injection. Data represent mean ± SEM of 13–19 mammary glands from 5 to 9 mice in each group. Blue and red dashed lines represent the average change bioluminescence of TN (1148 fold) and ER + BC (32 fold), respectively. One-way ANOVA, Kruskal-Wallis test relative to MCF-7 (control). e Box plot showing the fold-change of bioluminescence at endpoint for TN (5 weeks) and ER + (5-6 months) BC cells from Fig. 1c. Boxes span the 25th to 75th percentile, whiskers 1.5 times the interquartile range. Boxplot whiskers show minimum and maximum values. Two-tailed Mann-Whitney test. f , g Bar plot showing ex vivo bioluminescence of resected organs from 9, 7, and 6 mice bearing BT20, HCC1806, and T70 xenografts, respectively, f and 20, 9, 10, and 14 mice bearing MCF-7, T47D, T99, METS15 xenografts, respectively, g Data represent mean ± SEM. h Dot plot showing the ratio of bioluminescence in lungs over primary tumor. Data represent mean ± SEM of n = 9 (BT20), 7 (HCC1806), 12 (MCF-7), 8 (T47D), 3 (T99) and 12 (METS15) mice. Blue and red dashed lines represent the average ratio for TN and ER + BC cells, respectively.

    Article Snippet: ER+ BC cell lines MCF-7 and T47D as well as TN BC cell lines BT20 and HCC1806 were purchased from American Type Culture Collection (ATCC).

    Techniques: Immunofluorescence, Injection, Mouse Assay, Two Tailed Test, MANN-WHITNEY, Ex Vivo

    ER + metastatic lesions are dormant. a Representative fluorescence stereo micrographs of lungs from ≥ 3 mice with BT20, HCC1806, MCF-7, or T47D intraductal xenografts, arrows point to DTCs. Scale bar, 1 mm. b , c Representative micrographs of H E stained lung sections from 3 mice bearing BT20 and HCC1806 ( b ) or MCF-7 and T47D ( c) intraductal xenografts. Scale bar, 50 µm. d , e Representative fluorescence stereo micrographs of the liver ( d ) and lungs ( e ) from ≥3 mice bearing T70 and METS15 MIND xenografts. Scale bars, 1 mm. f Representative micrographs of H E stained lung sections from 3 mice bearing METS15 intraductal xenografts. Scale bars, 50 µm. g Percentage of Ki67 + cells in matched primary and lung sections from mice 5 weeks after intraductal injection of BT20 and HCC1806 cells, n ≥ 14 sections. h Bar graph showing Ki67 index of BT20 and HCC1806 lung lesions of different sizes. i Ki67 + index in matched primary tumor and lung sections from mice 5 months after intraductal injection of MCF-7, T47D, and METS15 cells as indicated. In g and i each data point represents ≥ 1,000 and 100 cells analyzed, mean ± SD from ≥3 mice/condition, respectively. g – h Data represent mean ± SD from 3 different hosts. Student’s unpaired t-test, two-tailed. j Scheme of FUCCI reporter. k Bar plot showing percentage of cycling and non-cycling cells in primary tumors and in lung micro-metastases in MCF-7 intraductal xenografts-bearing mice. Data represent mean ± SD from 3 host mice. Paired t-test. l Representative fluorescence micrographs of MCF-7:FUCCI cells in matched primary tumor (left) and lung (right). Scale bars, 50 µm. m Bar plot showing the percentage of p27 + Ki67- cells over total human cells in the lung. Data represent mean ± SD from n ≥ 3 mice, and dots represent ≥40 cells analyzed. One-way ANOVA. n Representative immunofluorescence micrographs for CK8 (blue), p27 (magenta) and Ki67 (cyan), counterstained with DAPI (gray) on lung section from MCF-7-bearing mice. Scale bar, 50 µm; inlet, 20 µm. *, ***, ****, and n.s represent P

    Journal: Nature Communications

    Article Title: Epithelial-mesenchymal plasticity determines estrogen receptor positive breast cancer dormancy and epithelial reconversion drives recurrence

    doi: 10.1038/s41467-022-32523-6

    Figure Lengend Snippet: ER + metastatic lesions are dormant. a Representative fluorescence stereo micrographs of lungs from ≥ 3 mice with BT20, HCC1806, MCF-7, or T47D intraductal xenografts, arrows point to DTCs. Scale bar, 1 mm. b , c Representative micrographs of H E stained lung sections from 3 mice bearing BT20 and HCC1806 ( b ) or MCF-7 and T47D ( c) intraductal xenografts. Scale bar, 50 µm. d , e Representative fluorescence stereo micrographs of the liver ( d ) and lungs ( e ) from ≥3 mice bearing T70 and METS15 MIND xenografts. Scale bars, 1 mm. f Representative micrographs of H E stained lung sections from 3 mice bearing METS15 intraductal xenografts. Scale bars, 50 µm. g Percentage of Ki67 + cells in matched primary and lung sections from mice 5 weeks after intraductal injection of BT20 and HCC1806 cells, n ≥ 14 sections. h Bar graph showing Ki67 index of BT20 and HCC1806 lung lesions of different sizes. i Ki67 + index in matched primary tumor and lung sections from mice 5 months after intraductal injection of MCF-7, T47D, and METS15 cells as indicated. In g and i each data point represents ≥ 1,000 and 100 cells analyzed, mean ± SD from ≥3 mice/condition, respectively. g – h Data represent mean ± SD from 3 different hosts. Student’s unpaired t-test, two-tailed. j Scheme of FUCCI reporter. k Bar plot showing percentage of cycling and non-cycling cells in primary tumors and in lung micro-metastases in MCF-7 intraductal xenografts-bearing mice. Data represent mean ± SD from 3 host mice. Paired t-test. l Representative fluorescence micrographs of MCF-7:FUCCI cells in matched primary tumor (left) and lung (right). Scale bars, 50 µm. m Bar plot showing the percentage of p27 + Ki67- cells over total human cells in the lung. Data represent mean ± SD from n ≥ 3 mice, and dots represent ≥40 cells analyzed. One-way ANOVA. n Representative immunofluorescence micrographs for CK8 (blue), p27 (magenta) and Ki67 (cyan), counterstained with DAPI (gray) on lung section from MCF-7-bearing mice. Scale bar, 50 µm; inlet, 20 µm. *, ***, ****, and n.s represent P

    Article Snippet: ER+ BC cell lines MCF-7 and T47D as well as TN BC cell lines BT20 and HCC1806 were purchased from American Type Culture Collection (ATCC).

    Techniques: Fluorescence, Mouse Assay, Staining, Injection, Two Tailed Test, Immunofluorescence

    Dormant DTCs from ER + intraductal xenografts have an EMP signature. a CK8 staining on optically-cleared sections of METS15 cells in host’s lungs. Scale bars, 10 µm. b Representative masks representing different cellular aspect-ratio (CAR) with respect to the morphology c Percentage of cells with CAR > 1.7 in matched primary tumors and in the lung from intraductal ER + BC xenografts-bearing mice. Data represent mean ± SD, each data point represents at least 100 primary cells and 10 lung DTCs from 3 mice. Paired Student’s t -test. d Relative E-cad intensity (Int.) in matched primary tumor cells and lung DTCs in mice bearing indicated intraductal xenografts. Data represent n≥8 images, mean ± SD from 3 mice. Students t-test e , f Relative MKI67 ( e ) and CDH1 ( f ) mRNA levels in MCF-7 cells in the primary tumor (8 mice) or in the lung ( n = 8), brain ( n = 4), and liver ( n = 3) DTCs. Data represent mean ± SD. One-way ANOVA. g–i Relative levels of indicated mRNAs in matched MCF-7 primary tumors and lung ( g n = 8), brain ( h n = 4), and liver ( i n = 3) DTCs. Wilcoxon test. j Relative levels of indicated mRNAs in matched BT20 or HCC1806 primary tumors and lung metastases from 3 mice. Paired t-test. k Representative fluorescence stereo micrographs of lungs from at least 3 mice 3 weeks after intraductal injection of BT20 or HCC1806 cells. Arrows point to micro-metastases. Scale bar, 1 mm. l . Relative mRNA levels of the selected genes in 3 versus 5-6 weeks lung DTCs retrieved from at least 4 mice bearing BT20 (left) or HCC1806 (right), n ≥ 4. Data represent mean ± SD, Wilcoxon test. Gene expression was normalized to the geometric mean of GAPDH and HPRT in panels e – j , and l . *, **, ***, ****, and n.s. represent P

    Journal: Nature Communications

    Article Title: Epithelial-mesenchymal plasticity determines estrogen receptor positive breast cancer dormancy and epithelial reconversion drives recurrence

    doi: 10.1038/s41467-022-32523-6

    Figure Lengend Snippet: Dormant DTCs from ER + intraductal xenografts have an EMP signature. a CK8 staining on optically-cleared sections of METS15 cells in host’s lungs. Scale bars, 10 µm. b Representative masks representing different cellular aspect-ratio (CAR) with respect to the morphology c Percentage of cells with CAR > 1.7 in matched primary tumors and in the lung from intraductal ER + BC xenografts-bearing mice. Data represent mean ± SD, each data point represents at least 100 primary cells and 10 lung DTCs from 3 mice. Paired Student’s t -test. d Relative E-cad intensity (Int.) in matched primary tumor cells and lung DTCs in mice bearing indicated intraductal xenografts. Data represent n≥8 images, mean ± SD from 3 mice. Students t-test e , f Relative MKI67 ( e ) and CDH1 ( f ) mRNA levels in MCF-7 cells in the primary tumor (8 mice) or in the lung ( n = 8), brain ( n = 4), and liver ( n = 3) DTCs. Data represent mean ± SD. One-way ANOVA. g–i Relative levels of indicated mRNAs in matched MCF-7 primary tumors and lung ( g n = 8), brain ( h n = 4), and liver ( i n = 3) DTCs. Wilcoxon test. j Relative levels of indicated mRNAs in matched BT20 or HCC1806 primary tumors and lung metastases from 3 mice. Paired t-test. k Representative fluorescence stereo micrographs of lungs from at least 3 mice 3 weeks after intraductal injection of BT20 or HCC1806 cells. Arrows point to micro-metastases. Scale bar, 1 mm. l . Relative mRNA levels of the selected genes in 3 versus 5-6 weeks lung DTCs retrieved from at least 4 mice bearing BT20 (left) or HCC1806 (right), n ≥ 4. Data represent mean ± SD, Wilcoxon test. Gene expression was normalized to the geometric mean of GAPDH and HPRT in panels e – j , and l . *, **, ***, ****, and n.s. represent P

    Article Snippet: ER+ BC cell lines MCF-7 and T47D as well as TN BC cell lines BT20 and HCC1806 were purchased from American Type Culture Collection (ATCC).

    Techniques: Staining, Mouse Assay, Fluorescence, Injection, Expressing