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slc7a11 shrna sequence 1  (OriGene)


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    OriGene slc7a11 shrna sequence 1
    A) Quantitative real-time PCR analysis of <t>SLC7A11</t> and SLC3A2 expression in total RNA extracts from normal pancreatic fibroblasts (isolated from n=8 patients with benign pancreatic conditions) and CAFs (isolated from n=10 PDAC patients). Bars show mean+s.e.m., circles indicate independent replicates (*p≤0.05, student t-test). B) Western blot of SLC7A11 in total protein extracts from PDAC cells and human CAFs (Cell lines 1-6). α-tubulin was used as a loading control. C) Immunoflourescence for DAPI, αSMA (CAF marker) and SLC7A11 in a human PDAC tissue specimen obtained through the Australian Pancreatic Cancer Genome Initiative (APGI). (D-G) Human PDA tissue microarrays obtained through the APGI (International Cancer Genome Consortium cohort) were stained for SLC7A11 by immunohistochemistry. D) Samples selected as references for scoring (0,1,2,3) for tumour and stromal compartments are shown (insets show magnified view of cells). Scores of 0-1 were classified as low SLC7A11 expression (“Tumour low ” and “Stroma low ”), scores of 2-3 were classified as high SLC7A11 expression (“Tumour high ” and “Stroma high ”). E-G) Kaplan-Meier survival curves showing the correlation between SLC7A11 expression in tumour cells (E), stroma (F), or a combination of both (G) with overall patient survival (days survived post-diagnosis). Patients that were deceased due to other causes or that were still alive were censored (shown as black ticks on each line graph). Total patient numbers for each group are indicated in the graph keys. Asterisks indicate significance based on (F) multivariate analysis (G) univariate Log-Rank test (*p≤0.05). H) Representative photos of Tumour low Stroma low , Tumour high Stroma low , Tumour low Stroma high , and Tumour high Stroma high groups. Scale bars in all photos = 100μm.
    Slc7a11 Shrna Sequence 1, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/slc7a11 shrna sequence 1/product/OriGene
    Average 91 stars, based on 2 article reviews
    slc7a11 shrna sequence 1 - by Bioz Stars, 2026-03
    91/100 stars

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    1) Product Images from "Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition"

    Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

    Journal: bioRxiv

    doi: 10.1101/2020.07.12.199638

    A) Quantitative real-time PCR analysis of SLC7A11 and SLC3A2 expression in total RNA extracts from normal pancreatic fibroblasts (isolated from n=8 patients with benign pancreatic conditions) and CAFs (isolated from n=10 PDAC patients). Bars show mean+s.e.m., circles indicate independent replicates (*p≤0.05, student t-test). B) Western blot of SLC7A11 in total protein extracts from PDAC cells and human CAFs (Cell lines 1-6). α-tubulin was used as a loading control. C) Immunoflourescence for DAPI, αSMA (CAF marker) and SLC7A11 in a human PDAC tissue specimen obtained through the Australian Pancreatic Cancer Genome Initiative (APGI). (D-G) Human PDA tissue microarrays obtained through the APGI (International Cancer Genome Consortium cohort) were stained for SLC7A11 by immunohistochemistry. D) Samples selected as references for scoring (0,1,2,3) for tumour and stromal compartments are shown (insets show magnified view of cells). Scores of 0-1 were classified as low SLC7A11 expression (“Tumour low ” and “Stroma low ”), scores of 2-3 were classified as high SLC7A11 expression (“Tumour high ” and “Stroma high ”). E-G) Kaplan-Meier survival curves showing the correlation between SLC7A11 expression in tumour cells (E), stroma (F), or a combination of both (G) with overall patient survival (days survived post-diagnosis). Patients that were deceased due to other causes or that were still alive were censored (shown as black ticks on each line graph). Total patient numbers for each group are indicated in the graph keys. Asterisks indicate significance based on (F) multivariate analysis (G) univariate Log-Rank test (*p≤0.05). H) Representative photos of Tumour low Stroma low , Tumour high Stroma low , Tumour low Stroma high , and Tumour high Stroma high groups. Scale bars in all photos = 100μm.
    Figure Legend Snippet: A) Quantitative real-time PCR analysis of SLC7A11 and SLC3A2 expression in total RNA extracts from normal pancreatic fibroblasts (isolated from n=8 patients with benign pancreatic conditions) and CAFs (isolated from n=10 PDAC patients). Bars show mean+s.e.m., circles indicate independent replicates (*p≤0.05, student t-test). B) Western blot of SLC7A11 in total protein extracts from PDAC cells and human CAFs (Cell lines 1-6). α-tubulin was used as a loading control. C) Immunoflourescence for DAPI, αSMA (CAF marker) and SLC7A11 in a human PDAC tissue specimen obtained through the Australian Pancreatic Cancer Genome Initiative (APGI). (D-G) Human PDA tissue microarrays obtained through the APGI (International Cancer Genome Consortium cohort) were stained for SLC7A11 by immunohistochemistry. D) Samples selected as references for scoring (0,1,2,3) for tumour and stromal compartments are shown (insets show magnified view of cells). Scores of 0-1 were classified as low SLC7A11 expression (“Tumour low ” and “Stroma low ”), scores of 2-3 were classified as high SLC7A11 expression (“Tumour high ” and “Stroma high ”). E-G) Kaplan-Meier survival curves showing the correlation between SLC7A11 expression in tumour cells (E), stroma (F), or a combination of both (G) with overall patient survival (days survived post-diagnosis). Patients that were deceased due to other causes or that were still alive were censored (shown as black ticks on each line graph). Total patient numbers for each group are indicated in the graph keys. Asterisks indicate significance based on (F) multivariate analysis (G) univariate Log-Rank test (*p≤0.05). H) Representative photos of Tumour low Stroma low , Tumour high Stroma low , Tumour low Stroma high , and Tumour high Stroma high groups. Scale bars in all photos = 100μm.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Isolation, Western Blot, Marker, Staining, Immunohistochemistry

    A) Cell proliferation (based on cell counting kit 8 absorbance) of CAFs 72h post-transfection with non-silencing siRNA (ns-siRNA) or SLC7A11-siRNA pool (pool of 4 siRNA sequences). Asterisks indicate significance (****p≤0.0001; n=6, student t-test). B) Cell proliferation (cell counting kit 8 absorbance) of CAFs treated with sulfasalazine (SSZ) ± 66μM 2-mercaptoethanol (2-ME), as a % of controls. Circles indicate replicates, asterisks indicate significance (***p≤0.001, ****p≤0.0001; One-way ANOVA). C) Live cell counts of CAFs (as a fraction of controls) treated with erastin for 48h. Asterisks indicate significance (****p≤0.0001, n=4; student t-test). D-E) Radiolabelled cystine uptake as a fraction of ns-siRNA (72h post-transfection) or untreated control cells (48h post-treatment with SSZ). Asterisks indicate significance (*p≤0.05; student t-test). F-G) Intracellular glutathione levels as assessed by colorimetric assay, and as a fraction of ns-siRNA (72h post-transfection) or untreated control cells [16h post-treatment with SSZ±N-acetyl-cysteine (NAC)]. Asterisks indicate significance (*p≤0.05, **p≤0.01, n=6; One-way ANOVA). H-I) Intracellular oxidative stress in the presence or absence of tert-butyl hydroperoxide (tBHP; oxidative stress), as measured by CellROX staining and flow cytometry (as a fraction of ns-siRNA + 0 μM tBHP). Asterisks indicate significance (ns=not significant, **p≤0.01, n=3; One-way ANOVA). Circles in all graphs indicate replicates, lines and bars in all graphs represent mean±s.e.m.
    Figure Legend Snippet: A) Cell proliferation (based on cell counting kit 8 absorbance) of CAFs 72h post-transfection with non-silencing siRNA (ns-siRNA) or SLC7A11-siRNA pool (pool of 4 siRNA sequences). Asterisks indicate significance (****p≤0.0001; n=6, student t-test). B) Cell proliferation (cell counting kit 8 absorbance) of CAFs treated with sulfasalazine (SSZ) ± 66μM 2-mercaptoethanol (2-ME), as a % of controls. Circles indicate replicates, asterisks indicate significance (***p≤0.001, ****p≤0.0001; One-way ANOVA). C) Live cell counts of CAFs (as a fraction of controls) treated with erastin for 48h. Asterisks indicate significance (****p≤0.0001, n=4; student t-test). D-E) Radiolabelled cystine uptake as a fraction of ns-siRNA (72h post-transfection) or untreated control cells (48h post-treatment with SSZ). Asterisks indicate significance (*p≤0.05; student t-test). F-G) Intracellular glutathione levels as assessed by colorimetric assay, and as a fraction of ns-siRNA (72h post-transfection) or untreated control cells [16h post-treatment with SSZ±N-acetyl-cysteine (NAC)]. Asterisks indicate significance (*p≤0.05, **p≤0.01, n=6; One-way ANOVA). H-I) Intracellular oxidative stress in the presence or absence of tert-butyl hydroperoxide (tBHP; oxidative stress), as measured by CellROX staining and flow cytometry (as a fraction of ns-siRNA + 0 μM tBHP). Asterisks indicate significance (ns=not significant, **p≤0.01, n=3; One-way ANOVA). Circles in all graphs indicate replicates, lines and bars in all graphs represent mean±s.e.m.

    Techniques Used: Cell Counting, Transfection, Colorimetric Assay, Staining, Flow Cytometry

    A) Live cell counts of CAFs 72h post-transfection with non-silencing-siRNA (ns-siRNA), SLC7A11-siRNA pool or SLC7A11-siRNA single sequence (SLC7A11-siRNA single seq) and 24h post-treatment with tert-butyl hydroperoxide (tBHP). Circles indicate replicates, asterisks and hashes indicate significance (*p≤0.05, ** p≤0.01, **** p≤0.0001; # p≤0.05, ## p≤0.01, relative to ns-siRNA of the same tBHP concentration; n=4; One-way ANOVA). B) Frequency of AnnexinV+DAPI positive (apoptotic) cells, as a fraction of ns-siRNA+0μM tBHP controls, 72h post-transfection and 24h post-tBHP treatment. Circles indicate replicates, asterisks indicate significance (ns=not significant, **p≤0.01, ***p≤0.001; One-way ANOVA). C) Glutathione peroxidase activity of CAFs treated with erastin (9h) as a % of controls. Circles indicate replicates, asterisks indicate significance (*p≤0.05; n=4; student t-test). D) Live cell counts of CAFs 24h post-treatment with 40 μM erastin ± 2μM ferrostatin. Circles indicate replicates, asterisks indicate significance (*p≤0.05, n=4; One-way ANOVA). E) Live cell counts (trypan blue exclusion) of CAFs stably expressing scramble-shRNA or SLC7A11-shRNA seq 1, 72h post-seeding and 24h post-treatment with tBHP. Circles indicate replicate experiments. Asterisks represent significance (**p≤0.01, ***p≤0.001, ****p≤0.0001; n=3; One-way ANOVA). F) As per C, except CAFs stably expressed scramble-shRNA or SLC7A11-shRNA sequence 1 and were treated with 40uM tBHP for 9h, instead of erastin (**p≤0.01; n=3; One-way ANOVA). G-H) β-galactosidase positive cells (senescent cells) as a fraction of total cells (mean+s.e.m.): (G) 72h after transfection with control siRNA (ns-siRNA) or SLC7A11-siRNA or (H) 48h post-treatment with SSZ. Circles indicate replicates, asterisks indicate significance (**p≤0.01; G: n=4, student t-test; H: n=3, One-way ANOVA). Bars and lines in all graphs are mean±s.e.m. Replicate numbers in all panels refer to experiments performed using independent CAF cells isolated from different PDAC patients.
    Figure Legend Snippet: A) Live cell counts of CAFs 72h post-transfection with non-silencing-siRNA (ns-siRNA), SLC7A11-siRNA pool or SLC7A11-siRNA single sequence (SLC7A11-siRNA single seq) and 24h post-treatment with tert-butyl hydroperoxide (tBHP). Circles indicate replicates, asterisks and hashes indicate significance (*p≤0.05, ** p≤0.01, **** p≤0.0001; # p≤0.05, ## p≤0.01, relative to ns-siRNA of the same tBHP concentration; n=4; One-way ANOVA). B) Frequency of AnnexinV+DAPI positive (apoptotic) cells, as a fraction of ns-siRNA+0μM tBHP controls, 72h post-transfection and 24h post-tBHP treatment. Circles indicate replicates, asterisks indicate significance (ns=not significant, **p≤0.01, ***p≤0.001; One-way ANOVA). C) Glutathione peroxidase activity of CAFs treated with erastin (9h) as a % of controls. Circles indicate replicates, asterisks indicate significance (*p≤0.05; n=4; student t-test). D) Live cell counts of CAFs 24h post-treatment with 40 μM erastin ± 2μM ferrostatin. Circles indicate replicates, asterisks indicate significance (*p≤0.05, n=4; One-way ANOVA). E) Live cell counts (trypan blue exclusion) of CAFs stably expressing scramble-shRNA or SLC7A11-shRNA seq 1, 72h post-seeding and 24h post-treatment with tBHP. Circles indicate replicate experiments. Asterisks represent significance (**p≤0.01, ***p≤0.001, ****p≤0.0001; n=3; One-way ANOVA). F) As per C, except CAFs stably expressed scramble-shRNA or SLC7A11-shRNA sequence 1 and were treated with 40uM tBHP for 9h, instead of erastin (**p≤0.01; n=3; One-way ANOVA). G-H) β-galactosidase positive cells (senescent cells) as a fraction of total cells (mean+s.e.m.): (G) 72h after transfection with control siRNA (ns-siRNA) or SLC7A11-siRNA or (H) 48h post-treatment with SSZ. Circles indicate replicates, asterisks indicate significance (**p≤0.01; G: n=4, student t-test; H: n=3, One-way ANOVA). Bars and lines in all graphs are mean±s.e.m. Replicate numbers in all panels refer to experiments performed using independent CAF cells isolated from different PDAC patients.

    Techniques Used: Transfection, Sequencing, Concentration Assay, Activity Assay, Stable Transfection, Expressing, shRNA, Isolation

    A-B) Schematic diagram of 3D co-culture spheroid outgrowth assay and quantification of 3D co-culture spheroid outgrowth post-transfection with control-siRNA (ns-siRNA) or SLC7A11-siRNA pool. Labels: PDAC ns CAF ns = non-silencing controls; PDAC ns CAF slc = SLC7A11 knockdown in CAFs only; PDAC slc CAF ns = SLC7A11 knockdown in PDAC cells only; PDAC slc CAF slc = SLC7A11 knockdown in both PDAC cells and CAFs. Representative photos are shown above each bar with the core circled in white dashed lines and outgrowth in red dashed lines (bars in photos = 300μm). Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (ns=not significant, *p≤0.05, **p≤0.01, ***p≤0.001; One-way ANOVA). C) Schematic diagram of 3D co-culture growth assay using stable shRNA cell lines and D) representative photos (bars in photos = 200μm) and quantification of 3D co-culture spheroid growth. Labels: PDAC scr CAF scr = scramble-shRNA controls; PDAC scr CAF slc = SLC7A11-shRNA seq 1 in CAFs only; PDAC slc CAF scr = SLC7A11-shRNA seq 1 in PDAC cells only; PDAC slc CAF slc = SLC7A11-shRNA seq 1 in both PDAC cells and CAFs. Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (ns=not significant, *p≤0.05, **p≤0.01, ****p≤0.0001, n=4-6; One-way ANOVA). Replicate numbers in panel B refer to independent experiments performed using MiaPaCa-2 combined with CAF cells isolated from different PDAC patients. Replicate numbers in panels D refer to replicate spheroids performed using MiaPaCa-2 PDAC cells combined with an immortalised CAF line.
    Figure Legend Snippet: A-B) Schematic diagram of 3D co-culture spheroid outgrowth assay and quantification of 3D co-culture spheroid outgrowth post-transfection with control-siRNA (ns-siRNA) or SLC7A11-siRNA pool. Labels: PDAC ns CAF ns = non-silencing controls; PDAC ns CAF slc = SLC7A11 knockdown in CAFs only; PDAC slc CAF ns = SLC7A11 knockdown in PDAC cells only; PDAC slc CAF slc = SLC7A11 knockdown in both PDAC cells and CAFs. Representative photos are shown above each bar with the core circled in white dashed lines and outgrowth in red dashed lines (bars in photos = 300μm). Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (ns=not significant, *p≤0.05, **p≤0.01, ***p≤0.001; One-way ANOVA). C) Schematic diagram of 3D co-culture growth assay using stable shRNA cell lines and D) representative photos (bars in photos = 200μm) and quantification of 3D co-culture spheroid growth. Labels: PDAC scr CAF scr = scramble-shRNA controls; PDAC scr CAF slc = SLC7A11-shRNA seq 1 in CAFs only; PDAC slc CAF scr = SLC7A11-shRNA seq 1 in PDAC cells only; PDAC slc CAF slc = SLC7A11-shRNA seq 1 in both PDAC cells and CAFs. Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (ns=not significant, *p≤0.05, **p≤0.01, ****p≤0.0001, n=4-6; One-way ANOVA). Replicate numbers in panel B refer to independent experiments performed using MiaPaCa-2 combined with CAF cells isolated from different PDAC patients. Replicate numbers in panels D refer to replicate spheroids performed using MiaPaCa-2 PDAC cells combined with an immortalised CAF line.

    Techniques Used: Co-Culture Assay, Transfection, Growth Assay, shRNA, Isolation

    A) Schematic diagram of assay and representative photos of collagen plugs contracted by CAFs transfected with control-siRNA (ns-siRNA) or SLC7A11-siRNA pool over 6 days are shown. The line graph shows the average area of contracted plugs at the indicated time points (mean±s.e.m.; *p≤0.05, n=4; One-way ANOVA). B-E) Analysis of collagen content in collagen plugs contracted by CAFs transfected with ns-siRNA or SLC7A11-siRNA at assay endpoint. B) Average picrosirius red signal. Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (**p≤0.01, n=4; student t-test). Representative images of picrosirius red staining are shown. C) Average total birefringence. Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (*p≤0.05, n=4; student t-test). Representative birefringence images are shown. D) Average % of total birefringence that was high (red-orange), medium (yellow) and low (green). Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (*p≤0.05, n=4; student t-test). E) Left graph shows the average maximum second harmonics generation (SHG) signal detected by two-photon confocal microscopy of collagen plugs. Representative SHG images are shown. Right graph shows the average correlation based on GLCM analysis of SHG maximum intensity projections. Circles indicate biological replicates, lines indicate mean±s.e.m., asterisks indicate significance (ns=not significant, *p≤0.05, n=4; student t-test). Replicate numbers in all panels refer to independent experiments performed using independent CAF cells isolated from different PDAC patients.
    Figure Legend Snippet: A) Schematic diagram of assay and representative photos of collagen plugs contracted by CAFs transfected with control-siRNA (ns-siRNA) or SLC7A11-siRNA pool over 6 days are shown. The line graph shows the average area of contracted plugs at the indicated time points (mean±s.e.m.; *p≤0.05, n=4; One-way ANOVA). B-E) Analysis of collagen content in collagen plugs contracted by CAFs transfected with ns-siRNA or SLC7A11-siRNA at assay endpoint. B) Average picrosirius red signal. Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (**p≤0.01, n=4; student t-test). Representative images of picrosirius red staining are shown. C) Average total birefringence. Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (*p≤0.05, n=4; student t-test). Representative birefringence images are shown. D) Average % of total birefringence that was high (red-orange), medium (yellow) and low (green). Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (*p≤0.05, n=4; student t-test). E) Left graph shows the average maximum second harmonics generation (SHG) signal detected by two-photon confocal microscopy of collagen plugs. Representative SHG images are shown. Right graph shows the average correlation based on GLCM analysis of SHG maximum intensity projections. Circles indicate biological replicates, lines indicate mean±s.e.m., asterisks indicate significance (ns=not significant, *p≤0.05, n=4; student t-test). Replicate numbers in all panels refer to independent experiments performed using independent CAF cells isolated from different PDAC patients.

    Techniques Used: Transfection, Staining, Confocal Microscopy, Isolation

    A) Quantification of Pancreatic Intraepithelial Neoplasia (PanINs) 1A-3 from KC mice (n=7) and KC mice with SLC7A11 conditional KO under Pdx1 -promoter (KC Slc7a11 fl/fl ; n=7) at 70 days of age (mean±s.e.m.). B) Kaplan-Meier analysis showing survival percentage of KPC (n=26) and KPC Slc7a11 fl/fl mice (n=24) mice. C) Representative photos of KPC and KPC Slc7a11 fl/fl tumour sections probed for αSMA (brown). The quantification of αSMA staining is shown in the graph (mean±s.e.m.), based on ImageJ analysis of representative regions from each tumour section (n=5 mice per group). Scale bars = 400μm. D) Representative photos of KPC and KPC Slc7a11 fl/fl tumour sections. The quantification of picrosirius red staining is shown in the bar graph (mean±s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Scale bars = 400μm. Asterisks indicate significance (*p≤0.05; n=5 mice per group; student t-test). E-F) Live cell counts (mean±s.e.m.) of (E) KPC PDAC cells and (F) KPC CAFs 72h post-transfection with control-siRNA (ns-siRNA) or mouse SLC7A11-siRNA pool (SLC7A11 pool). Asterisks indicate significance (*p≤0.05, **p≤0.01; n=3; one-way ANOVA).
    Figure Legend Snippet: A) Quantification of Pancreatic Intraepithelial Neoplasia (PanINs) 1A-3 from KC mice (n=7) and KC mice with SLC7A11 conditional KO under Pdx1 -promoter (KC Slc7a11 fl/fl ; n=7) at 70 days of age (mean±s.e.m.). B) Kaplan-Meier analysis showing survival percentage of KPC (n=26) and KPC Slc7a11 fl/fl mice (n=24) mice. C) Representative photos of KPC and KPC Slc7a11 fl/fl tumour sections probed for αSMA (brown). The quantification of αSMA staining is shown in the graph (mean±s.e.m.), based on ImageJ analysis of representative regions from each tumour section (n=5 mice per group). Scale bars = 400μm. D) Representative photos of KPC and KPC Slc7a11 fl/fl tumour sections. The quantification of picrosirius red staining is shown in the bar graph (mean±s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Scale bars = 400μm. Asterisks indicate significance (*p≤0.05; n=5 mice per group; student t-test). E-F) Live cell counts (mean±s.e.m.) of (E) KPC PDAC cells and (F) KPC CAFs 72h post-transfection with control-siRNA (ns-siRNA) or mouse SLC7A11-siRNA pool (SLC7A11 pool). Asterisks indicate significance (*p≤0.05, **p≤0.01; n=3; one-way ANOVA).

    Techniques Used: Staining, Transfection

    All orthotopic tumours were co-injections of PDAC cells and CAFs. A) Orthotopic pancreatic tumours were treated with STAR nanoparticles + control-siRNA or SLC7A11 siRNA single sequence (SLC7A11 single seq) in the regimen shown. Representative photos of immunohistochemistry for SLC7A11 in tumour tissue at the model endpoint are shown. Graph shows optical density (staining intensity) calculated from average pixel intensity measurements from 3 representative images per tumour, using ImageJ. Circles indicate individual mice, lines indicate mean±s.e.m., asterisks indicate significance (*p≤0.05; One-way ANOVA). B) Treatment regimen for therapeutic model analysed in panels (C-D). Circles and triangles in all dot plots in panels C-D represent individual mice. C) Tumour volume at therapeutic model endpoint, as assessed by calliper measurement ex vivo (mean±s.e.m.). Asterisks indicate significance (*p≤0.05; One-way ANOVA). D) Representative photos of metastases confirmed by H&E staining following detection at model endpoint by ex vivo luminescence imaging of organs. Graph shows metastatic sites per mouse (mean±s.e.m.) for each treatment group. Scale bars in all figures = 200μm.
    Figure Legend Snippet: All orthotopic tumours were co-injections of PDAC cells and CAFs. A) Orthotopic pancreatic tumours were treated with STAR nanoparticles + control-siRNA or SLC7A11 siRNA single sequence (SLC7A11 single seq) in the regimen shown. Representative photos of immunohistochemistry for SLC7A11 in tumour tissue at the model endpoint are shown. Graph shows optical density (staining intensity) calculated from average pixel intensity measurements from 3 representative images per tumour, using ImageJ. Circles indicate individual mice, lines indicate mean±s.e.m., asterisks indicate significance (*p≤0.05; One-way ANOVA). B) Treatment regimen for therapeutic model analysed in panels (C-D). Circles and triangles in all dot plots in panels C-D represent individual mice. C) Tumour volume at therapeutic model endpoint, as assessed by calliper measurement ex vivo (mean±s.e.m.). Asterisks indicate significance (*p≤0.05; One-way ANOVA). D) Representative photos of metastases confirmed by H&E staining following detection at model endpoint by ex vivo luminescence imaging of organs. Graph shows metastatic sites per mouse (mean±s.e.m.) for each treatment group. Scale bars in all figures = 200μm.

    Techniques Used: Sequencing, Immunohistochemistry, Staining, Ex Vivo, Imaging


    Figure Legend Snippet:

    Techniques Used:

    A) Representative photos of tumour sections probed for αSMA (brown). The quantification of αSMA staining is shown in the graph (mean+s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Asterisks indicate significance (*p≤0.05; Control-siRNA, n=7; SLC7A11-siRNA single seq, n=5; student t-test). B) Representative photos of picrosirius red and methyl green stained tumour sections. The quantification of picrosirius red staining is shown in the bar graph (mean+s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Asterisks indicate significance (*p≤0.05; Control-siRNA, n=8; SLC7A11-siRNA single seq, n=5; student t-test). C) Polarised light analysis of representative regions from picrosirius red stained specimens. Representative photos are shown. Left bar graph shows total birefringence (mean+s.e.m.; Control-siRNA, n=8; SLC7A11-siRNA single seq, n=5). Right bar graph shows the average frequency (mean+s.e.m.; Control-siRNA, n=8; SLC7A11-siRNA single seq, n=5) of low, medium and high birefringence collagen fibrils (higher birefringence = denser fibril). ns = not significant (student t-test). D) Representative photos of CD31-stained tumour sections. Red arrows indicate open blood vessels. The bar graph shows the fraction of CD31-positive blood vessels that were open (mean+s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Asterisks indicate significance (*p≤0.05; Control-siRNA, n=8; SLC7A11-siRNA single seq, n=5; student t-test). Fields of view used for analyses in all panels, provided an average area coverage of 13% of the total tumour section (excluding necrotic regions). All circles in graphs represent individual mice. All scale bars in photos = 100μm.
    Figure Legend Snippet: A) Representative photos of tumour sections probed for αSMA (brown). The quantification of αSMA staining is shown in the graph (mean+s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Asterisks indicate significance (*p≤0.05; Control-siRNA, n=7; SLC7A11-siRNA single seq, n=5; student t-test). B) Representative photos of picrosirius red and methyl green stained tumour sections. The quantification of picrosirius red staining is shown in the bar graph (mean+s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Asterisks indicate significance (*p≤0.05; Control-siRNA, n=8; SLC7A11-siRNA single seq, n=5; student t-test). C) Polarised light analysis of representative regions from picrosirius red stained specimens. Representative photos are shown. Left bar graph shows total birefringence (mean+s.e.m.; Control-siRNA, n=8; SLC7A11-siRNA single seq, n=5). Right bar graph shows the average frequency (mean+s.e.m.; Control-siRNA, n=8; SLC7A11-siRNA single seq, n=5) of low, medium and high birefringence collagen fibrils (higher birefringence = denser fibril). ns = not significant (student t-test). D) Representative photos of CD31-stained tumour sections. Red arrows indicate open blood vessels. The bar graph shows the fraction of CD31-positive blood vessels that were open (mean+s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Asterisks indicate significance (*p≤0.05; Control-siRNA, n=8; SLC7A11-siRNA single seq, n=5; student t-test). Fields of view used for analyses in all panels, provided an average area coverage of 13% of the total tumour section (excluding necrotic regions). All circles in graphs represent individual mice. All scale bars in photos = 100μm.

    Techniques Used: Staining



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    OriGene slc7a11 shrna sequence 1
    A) Quantitative real-time PCR analysis of <t>SLC7A11</t> and SLC3A2 expression in total RNA extracts from normal pancreatic fibroblasts (isolated from n=8 patients with benign pancreatic conditions) and CAFs (isolated from n=10 PDAC patients). Bars show mean+s.e.m., circles indicate independent replicates (*p≤0.05, student t-test). B) Western blot of SLC7A11 in total protein extracts from PDAC cells and human CAFs (Cell lines 1-6). α-tubulin was used as a loading control. C) Immunoflourescence for DAPI, αSMA (CAF marker) and SLC7A11 in a human PDAC tissue specimen obtained through the Australian Pancreatic Cancer Genome Initiative (APGI). (D-G) Human PDA tissue microarrays obtained through the APGI (International Cancer Genome Consortium cohort) were stained for SLC7A11 by immunohistochemistry. D) Samples selected as references for scoring (0,1,2,3) for tumour and stromal compartments are shown (insets show magnified view of cells). Scores of 0-1 were classified as low SLC7A11 expression (“Tumour low ” and “Stroma low ”), scores of 2-3 were classified as high SLC7A11 expression (“Tumour high ” and “Stroma high ”). E-G) Kaplan-Meier survival curves showing the correlation between SLC7A11 expression in tumour cells (E), stroma (F), or a combination of both (G) with overall patient survival (days survived post-diagnosis). Patients that were deceased due to other causes or that were still alive were censored (shown as black ticks on each line graph). Total patient numbers for each group are indicated in the graph keys. Asterisks indicate significance based on (F) multivariate analysis (G) univariate Log-Rank test (*p≤0.05). H) Representative photos of Tumour low Stroma low , Tumour high Stroma low , Tumour low Stroma high , and Tumour high Stroma high groups. Scale bars in all photos = 100μm.
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    A) Quantitative real-time PCR analysis of SLC7A11 and SLC3A2 expression in total RNA extracts from normal pancreatic fibroblasts (isolated from n=8 patients with benign pancreatic conditions) and CAFs (isolated from n=10 PDAC patients). Bars show mean+s.e.m., circles indicate independent replicates (*p≤0.05, student t-test). B) Western blot of SLC7A11 in total protein extracts from PDAC cells and human CAFs (Cell lines 1-6). α-tubulin was used as a loading control. C) Immunoflourescence for DAPI, αSMA (CAF marker) and SLC7A11 in a human PDAC tissue specimen obtained through the Australian Pancreatic Cancer Genome Initiative (APGI). (D-G) Human PDA tissue microarrays obtained through the APGI (International Cancer Genome Consortium cohort) were stained for SLC7A11 by immunohistochemistry. D) Samples selected as references for scoring (0,1,2,3) for tumour and stromal compartments are shown (insets show magnified view of cells). Scores of 0-1 were classified as low SLC7A11 expression (“Tumour low ” and “Stroma low ”), scores of 2-3 were classified as high SLC7A11 expression (“Tumour high ” and “Stroma high ”). E-G) Kaplan-Meier survival curves showing the correlation between SLC7A11 expression in tumour cells (E), stroma (F), or a combination of both (G) with overall patient survival (days survived post-diagnosis). Patients that were deceased due to other causes or that were still alive were censored (shown as black ticks on each line graph). Total patient numbers for each group are indicated in the graph keys. Asterisks indicate significance based on (F) multivariate analysis (G) univariate Log-Rank test (*p≤0.05). H) Representative photos of Tumour low Stroma low , Tumour high Stroma low , Tumour low Stroma high , and Tumour high Stroma high groups. Scale bars in all photos = 100μm.

    Journal: bioRxiv

    Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

    doi: 10.1101/2020.07.12.199638

    Figure Lengend Snippet: A) Quantitative real-time PCR analysis of SLC7A11 and SLC3A2 expression in total RNA extracts from normal pancreatic fibroblasts (isolated from n=8 patients with benign pancreatic conditions) and CAFs (isolated from n=10 PDAC patients). Bars show mean+s.e.m., circles indicate independent replicates (*p≤0.05, student t-test). B) Western blot of SLC7A11 in total protein extracts from PDAC cells and human CAFs (Cell lines 1-6). α-tubulin was used as a loading control. C) Immunoflourescence for DAPI, αSMA (CAF marker) and SLC7A11 in a human PDAC tissue specimen obtained through the Australian Pancreatic Cancer Genome Initiative (APGI). (D-G) Human PDA tissue microarrays obtained through the APGI (International Cancer Genome Consortium cohort) were stained for SLC7A11 by immunohistochemistry. D) Samples selected as references for scoring (0,1,2,3) for tumour and stromal compartments are shown (insets show magnified view of cells). Scores of 0-1 were classified as low SLC7A11 expression (“Tumour low ” and “Stroma low ”), scores of 2-3 were classified as high SLC7A11 expression (“Tumour high ” and “Stroma high ”). E-G) Kaplan-Meier survival curves showing the correlation between SLC7A11 expression in tumour cells (E), stroma (F), or a combination of both (G) with overall patient survival (days survived post-diagnosis). Patients that were deceased due to other causes or that were still alive were censored (shown as black ticks on each line graph). Total patient numbers for each group are indicated in the graph keys. Asterisks indicate significance based on (F) multivariate analysis (G) univariate Log-Rank test (*p≤0.05). H) Representative photos of Tumour low Stroma low , Tumour high Stroma low , Tumour low Stroma high , and Tumour high Stroma high groups. Scale bars in all photos = 100μm.

    Article Snippet: MiaPaCa-2 cells and hTERT-immortalised CAFs were then transduced with lentiviral constructs expressing scramble shRNA, SLC7A11-shRNA sequence 1 (Origene, Cat. TL309282).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Isolation, Western Blot, Marker, Staining, Immunohistochemistry

    A) Cell proliferation (based on cell counting kit 8 absorbance) of CAFs 72h post-transfection with non-silencing siRNA (ns-siRNA) or SLC7A11-siRNA pool (pool of 4 siRNA sequences). Asterisks indicate significance (****p≤0.0001; n=6, student t-test). B) Cell proliferation (cell counting kit 8 absorbance) of CAFs treated with sulfasalazine (SSZ) ± 66μM 2-mercaptoethanol (2-ME), as a % of controls. Circles indicate replicates, asterisks indicate significance (***p≤0.001, ****p≤0.0001; One-way ANOVA). C) Live cell counts of CAFs (as a fraction of controls) treated with erastin for 48h. Asterisks indicate significance (****p≤0.0001, n=4; student t-test). D-E) Radiolabelled cystine uptake as a fraction of ns-siRNA (72h post-transfection) or untreated control cells (48h post-treatment with SSZ). Asterisks indicate significance (*p≤0.05; student t-test). F-G) Intracellular glutathione levels as assessed by colorimetric assay, and as a fraction of ns-siRNA (72h post-transfection) or untreated control cells [16h post-treatment with SSZ±N-acetyl-cysteine (NAC)]. Asterisks indicate significance (*p≤0.05, **p≤0.01, n=6; One-way ANOVA). H-I) Intracellular oxidative stress in the presence or absence of tert-butyl hydroperoxide (tBHP; oxidative stress), as measured by CellROX staining and flow cytometry (as a fraction of ns-siRNA + 0 μM tBHP). Asterisks indicate significance (ns=not significant, **p≤0.01, n=3; One-way ANOVA). Circles in all graphs indicate replicates, lines and bars in all graphs represent mean±s.e.m.

    Journal: bioRxiv

    Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

    doi: 10.1101/2020.07.12.199638

    Figure Lengend Snippet: A) Cell proliferation (based on cell counting kit 8 absorbance) of CAFs 72h post-transfection with non-silencing siRNA (ns-siRNA) or SLC7A11-siRNA pool (pool of 4 siRNA sequences). Asterisks indicate significance (****p≤0.0001; n=6, student t-test). B) Cell proliferation (cell counting kit 8 absorbance) of CAFs treated with sulfasalazine (SSZ) ± 66μM 2-mercaptoethanol (2-ME), as a % of controls. Circles indicate replicates, asterisks indicate significance (***p≤0.001, ****p≤0.0001; One-way ANOVA). C) Live cell counts of CAFs (as a fraction of controls) treated with erastin for 48h. Asterisks indicate significance (****p≤0.0001, n=4; student t-test). D-E) Radiolabelled cystine uptake as a fraction of ns-siRNA (72h post-transfection) or untreated control cells (48h post-treatment with SSZ). Asterisks indicate significance (*p≤0.05; student t-test). F-G) Intracellular glutathione levels as assessed by colorimetric assay, and as a fraction of ns-siRNA (72h post-transfection) or untreated control cells [16h post-treatment with SSZ±N-acetyl-cysteine (NAC)]. Asterisks indicate significance (*p≤0.05, **p≤0.01, n=6; One-way ANOVA). H-I) Intracellular oxidative stress in the presence or absence of tert-butyl hydroperoxide (tBHP; oxidative stress), as measured by CellROX staining and flow cytometry (as a fraction of ns-siRNA + 0 μM tBHP). Asterisks indicate significance (ns=not significant, **p≤0.01, n=3; One-way ANOVA). Circles in all graphs indicate replicates, lines and bars in all graphs represent mean±s.e.m.

    Article Snippet: MiaPaCa-2 cells and hTERT-immortalised CAFs were then transduced with lentiviral constructs expressing scramble shRNA, SLC7A11-shRNA sequence 1 (Origene, Cat. TL309282).

    Techniques: Cell Counting, Transfection, Colorimetric Assay, Staining, Flow Cytometry

    A) Live cell counts of CAFs 72h post-transfection with non-silencing-siRNA (ns-siRNA), SLC7A11-siRNA pool or SLC7A11-siRNA single sequence (SLC7A11-siRNA single seq) and 24h post-treatment with tert-butyl hydroperoxide (tBHP). Circles indicate replicates, asterisks and hashes indicate significance (*p≤0.05, ** p≤0.01, **** p≤0.0001; # p≤0.05, ## p≤0.01, relative to ns-siRNA of the same tBHP concentration; n=4; One-way ANOVA). B) Frequency of AnnexinV+DAPI positive (apoptotic) cells, as a fraction of ns-siRNA+0μM tBHP controls, 72h post-transfection and 24h post-tBHP treatment. Circles indicate replicates, asterisks indicate significance (ns=not significant, **p≤0.01, ***p≤0.001; One-way ANOVA). C) Glutathione peroxidase activity of CAFs treated with erastin (9h) as a % of controls. Circles indicate replicates, asterisks indicate significance (*p≤0.05; n=4; student t-test). D) Live cell counts of CAFs 24h post-treatment with 40 μM erastin ± 2μM ferrostatin. Circles indicate replicates, asterisks indicate significance (*p≤0.05, n=4; One-way ANOVA). E) Live cell counts (trypan blue exclusion) of CAFs stably expressing scramble-shRNA or SLC7A11-shRNA seq 1, 72h post-seeding and 24h post-treatment with tBHP. Circles indicate replicate experiments. Asterisks represent significance (**p≤0.01, ***p≤0.001, ****p≤0.0001; n=3; One-way ANOVA). F) As per C, except CAFs stably expressed scramble-shRNA or SLC7A11-shRNA sequence 1 and were treated with 40uM tBHP for 9h, instead of erastin (**p≤0.01; n=3; One-way ANOVA). G-H) β-galactosidase positive cells (senescent cells) as a fraction of total cells (mean+s.e.m.): (G) 72h after transfection with control siRNA (ns-siRNA) or SLC7A11-siRNA or (H) 48h post-treatment with SSZ. Circles indicate replicates, asterisks indicate significance (**p≤0.01; G: n=4, student t-test; H: n=3, One-way ANOVA). Bars and lines in all graphs are mean±s.e.m. Replicate numbers in all panels refer to experiments performed using independent CAF cells isolated from different PDAC patients.

    Journal: bioRxiv

    Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

    doi: 10.1101/2020.07.12.199638

    Figure Lengend Snippet: A) Live cell counts of CAFs 72h post-transfection with non-silencing-siRNA (ns-siRNA), SLC7A11-siRNA pool or SLC7A11-siRNA single sequence (SLC7A11-siRNA single seq) and 24h post-treatment with tert-butyl hydroperoxide (tBHP). Circles indicate replicates, asterisks and hashes indicate significance (*p≤0.05, ** p≤0.01, **** p≤0.0001; # p≤0.05, ## p≤0.01, relative to ns-siRNA of the same tBHP concentration; n=4; One-way ANOVA). B) Frequency of AnnexinV+DAPI positive (apoptotic) cells, as a fraction of ns-siRNA+0μM tBHP controls, 72h post-transfection and 24h post-tBHP treatment. Circles indicate replicates, asterisks indicate significance (ns=not significant, **p≤0.01, ***p≤0.001; One-way ANOVA). C) Glutathione peroxidase activity of CAFs treated with erastin (9h) as a % of controls. Circles indicate replicates, asterisks indicate significance (*p≤0.05; n=4; student t-test). D) Live cell counts of CAFs 24h post-treatment with 40 μM erastin ± 2μM ferrostatin. Circles indicate replicates, asterisks indicate significance (*p≤0.05, n=4; One-way ANOVA). E) Live cell counts (trypan blue exclusion) of CAFs stably expressing scramble-shRNA or SLC7A11-shRNA seq 1, 72h post-seeding and 24h post-treatment with tBHP. Circles indicate replicate experiments. Asterisks represent significance (**p≤0.01, ***p≤0.001, ****p≤0.0001; n=3; One-way ANOVA). F) As per C, except CAFs stably expressed scramble-shRNA or SLC7A11-shRNA sequence 1 and were treated with 40uM tBHP for 9h, instead of erastin (**p≤0.01; n=3; One-way ANOVA). G-H) β-galactosidase positive cells (senescent cells) as a fraction of total cells (mean+s.e.m.): (G) 72h after transfection with control siRNA (ns-siRNA) or SLC7A11-siRNA or (H) 48h post-treatment with SSZ. Circles indicate replicates, asterisks indicate significance (**p≤0.01; G: n=4, student t-test; H: n=3, One-way ANOVA). Bars and lines in all graphs are mean±s.e.m. Replicate numbers in all panels refer to experiments performed using independent CAF cells isolated from different PDAC patients.

    Article Snippet: MiaPaCa-2 cells and hTERT-immortalised CAFs were then transduced with lentiviral constructs expressing scramble shRNA, SLC7A11-shRNA sequence 1 (Origene, Cat. TL309282).

    Techniques: Transfection, Sequencing, Concentration Assay, Activity Assay, Stable Transfection, Expressing, shRNA, Isolation

    A-B) Schematic diagram of 3D co-culture spheroid outgrowth assay and quantification of 3D co-culture spheroid outgrowth post-transfection with control-siRNA (ns-siRNA) or SLC7A11-siRNA pool. Labels: PDAC ns CAF ns = non-silencing controls; PDAC ns CAF slc = SLC7A11 knockdown in CAFs only; PDAC slc CAF ns = SLC7A11 knockdown in PDAC cells only; PDAC slc CAF slc = SLC7A11 knockdown in both PDAC cells and CAFs. Representative photos are shown above each bar with the core circled in white dashed lines and outgrowth in red dashed lines (bars in photos = 300μm). Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (ns=not significant, *p≤0.05, **p≤0.01, ***p≤0.001; One-way ANOVA). C) Schematic diagram of 3D co-culture growth assay using stable shRNA cell lines and D) representative photos (bars in photos = 200μm) and quantification of 3D co-culture spheroid growth. Labels: PDAC scr CAF scr = scramble-shRNA controls; PDAC scr CAF slc = SLC7A11-shRNA seq 1 in CAFs only; PDAC slc CAF scr = SLC7A11-shRNA seq 1 in PDAC cells only; PDAC slc CAF slc = SLC7A11-shRNA seq 1 in both PDAC cells and CAFs. Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (ns=not significant, *p≤0.05, **p≤0.01, ****p≤0.0001, n=4-6; One-way ANOVA). Replicate numbers in panel B refer to independent experiments performed using MiaPaCa-2 combined with CAF cells isolated from different PDAC patients. Replicate numbers in panels D refer to replicate spheroids performed using MiaPaCa-2 PDAC cells combined with an immortalised CAF line.

    Journal: bioRxiv

    Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

    doi: 10.1101/2020.07.12.199638

    Figure Lengend Snippet: A-B) Schematic diagram of 3D co-culture spheroid outgrowth assay and quantification of 3D co-culture spheroid outgrowth post-transfection with control-siRNA (ns-siRNA) or SLC7A11-siRNA pool. Labels: PDAC ns CAF ns = non-silencing controls; PDAC ns CAF slc = SLC7A11 knockdown in CAFs only; PDAC slc CAF ns = SLC7A11 knockdown in PDAC cells only; PDAC slc CAF slc = SLC7A11 knockdown in both PDAC cells and CAFs. Representative photos are shown above each bar with the core circled in white dashed lines and outgrowth in red dashed lines (bars in photos = 300μm). Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (ns=not significant, *p≤0.05, **p≤0.01, ***p≤0.001; One-way ANOVA). C) Schematic diagram of 3D co-culture growth assay using stable shRNA cell lines and D) representative photos (bars in photos = 200μm) and quantification of 3D co-culture spheroid growth. Labels: PDAC scr CAF scr = scramble-shRNA controls; PDAC scr CAF slc = SLC7A11-shRNA seq 1 in CAFs only; PDAC slc CAF scr = SLC7A11-shRNA seq 1 in PDAC cells only; PDAC slc CAF slc = SLC7A11-shRNA seq 1 in both PDAC cells and CAFs. Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (ns=not significant, *p≤0.05, **p≤0.01, ****p≤0.0001, n=4-6; One-way ANOVA). Replicate numbers in panel B refer to independent experiments performed using MiaPaCa-2 combined with CAF cells isolated from different PDAC patients. Replicate numbers in panels D refer to replicate spheroids performed using MiaPaCa-2 PDAC cells combined with an immortalised CAF line.

    Article Snippet: MiaPaCa-2 cells and hTERT-immortalised CAFs were then transduced with lentiviral constructs expressing scramble shRNA, SLC7A11-shRNA sequence 1 (Origene, Cat. TL309282).

    Techniques: Co-Culture Assay, Transfection, Growth Assay, shRNA, Isolation

    A) Schematic diagram of assay and representative photos of collagen plugs contracted by CAFs transfected with control-siRNA (ns-siRNA) or SLC7A11-siRNA pool over 6 days are shown. The line graph shows the average area of contracted plugs at the indicated time points (mean±s.e.m.; *p≤0.05, n=4; One-way ANOVA). B-E) Analysis of collagen content in collagen plugs contracted by CAFs transfected with ns-siRNA or SLC7A11-siRNA at assay endpoint. B) Average picrosirius red signal. Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (**p≤0.01, n=4; student t-test). Representative images of picrosirius red staining are shown. C) Average total birefringence. Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (*p≤0.05, n=4; student t-test). Representative birefringence images are shown. D) Average % of total birefringence that was high (red-orange), medium (yellow) and low (green). Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (*p≤0.05, n=4; student t-test). E) Left graph shows the average maximum second harmonics generation (SHG) signal detected by two-photon confocal microscopy of collagen plugs. Representative SHG images are shown. Right graph shows the average correlation based on GLCM analysis of SHG maximum intensity projections. Circles indicate biological replicates, lines indicate mean±s.e.m., asterisks indicate significance (ns=not significant, *p≤0.05, n=4; student t-test). Replicate numbers in all panels refer to independent experiments performed using independent CAF cells isolated from different PDAC patients.

    Journal: bioRxiv

    Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

    doi: 10.1101/2020.07.12.199638

    Figure Lengend Snippet: A) Schematic diagram of assay and representative photos of collagen plugs contracted by CAFs transfected with control-siRNA (ns-siRNA) or SLC7A11-siRNA pool over 6 days are shown. The line graph shows the average area of contracted plugs at the indicated time points (mean±s.e.m.; *p≤0.05, n=4; One-way ANOVA). B-E) Analysis of collagen content in collagen plugs contracted by CAFs transfected with ns-siRNA or SLC7A11-siRNA at assay endpoint. B) Average picrosirius red signal. Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (**p≤0.01, n=4; student t-test). Representative images of picrosirius red staining are shown. C) Average total birefringence. Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (*p≤0.05, n=4; student t-test). Representative birefringence images are shown. D) Average % of total birefringence that was high (red-orange), medium (yellow) and low (green). Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (*p≤0.05, n=4; student t-test). E) Left graph shows the average maximum second harmonics generation (SHG) signal detected by two-photon confocal microscopy of collagen plugs. Representative SHG images are shown. Right graph shows the average correlation based on GLCM analysis of SHG maximum intensity projections. Circles indicate biological replicates, lines indicate mean±s.e.m., asterisks indicate significance (ns=not significant, *p≤0.05, n=4; student t-test). Replicate numbers in all panels refer to independent experiments performed using independent CAF cells isolated from different PDAC patients.

    Article Snippet: MiaPaCa-2 cells and hTERT-immortalised CAFs were then transduced with lentiviral constructs expressing scramble shRNA, SLC7A11-shRNA sequence 1 (Origene, Cat. TL309282).

    Techniques: Transfection, Staining, Confocal Microscopy, Isolation

    A) Quantification of Pancreatic Intraepithelial Neoplasia (PanINs) 1A-3 from KC mice (n=7) and KC mice with SLC7A11 conditional KO under Pdx1 -promoter (KC Slc7a11 fl/fl ; n=7) at 70 days of age (mean±s.e.m.). B) Kaplan-Meier analysis showing survival percentage of KPC (n=26) and KPC Slc7a11 fl/fl mice (n=24) mice. C) Representative photos of KPC and KPC Slc7a11 fl/fl tumour sections probed for αSMA (brown). The quantification of αSMA staining is shown in the graph (mean±s.e.m.), based on ImageJ analysis of representative regions from each tumour section (n=5 mice per group). Scale bars = 400μm. D) Representative photos of KPC and KPC Slc7a11 fl/fl tumour sections. The quantification of picrosirius red staining is shown in the bar graph (mean±s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Scale bars = 400μm. Asterisks indicate significance (*p≤0.05; n=5 mice per group; student t-test). E-F) Live cell counts (mean±s.e.m.) of (E) KPC PDAC cells and (F) KPC CAFs 72h post-transfection with control-siRNA (ns-siRNA) or mouse SLC7A11-siRNA pool (SLC7A11 pool). Asterisks indicate significance (*p≤0.05, **p≤0.01; n=3; one-way ANOVA).

    Journal: bioRxiv

    Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

    doi: 10.1101/2020.07.12.199638

    Figure Lengend Snippet: A) Quantification of Pancreatic Intraepithelial Neoplasia (PanINs) 1A-3 from KC mice (n=7) and KC mice with SLC7A11 conditional KO under Pdx1 -promoter (KC Slc7a11 fl/fl ; n=7) at 70 days of age (mean±s.e.m.). B) Kaplan-Meier analysis showing survival percentage of KPC (n=26) and KPC Slc7a11 fl/fl mice (n=24) mice. C) Representative photos of KPC and KPC Slc7a11 fl/fl tumour sections probed for αSMA (brown). The quantification of αSMA staining is shown in the graph (mean±s.e.m.), based on ImageJ analysis of representative regions from each tumour section (n=5 mice per group). Scale bars = 400μm. D) Representative photos of KPC and KPC Slc7a11 fl/fl tumour sections. The quantification of picrosirius red staining is shown in the bar graph (mean±s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Scale bars = 400μm. Asterisks indicate significance (*p≤0.05; n=5 mice per group; student t-test). E-F) Live cell counts (mean±s.e.m.) of (E) KPC PDAC cells and (F) KPC CAFs 72h post-transfection with control-siRNA (ns-siRNA) or mouse SLC7A11-siRNA pool (SLC7A11 pool). Asterisks indicate significance (*p≤0.05, **p≤0.01; n=3; one-way ANOVA).

    Article Snippet: MiaPaCa-2 cells and hTERT-immortalised CAFs were then transduced with lentiviral constructs expressing scramble shRNA, SLC7A11-shRNA sequence 1 (Origene, Cat. TL309282).

    Techniques: Staining, Transfection

    All orthotopic tumours were co-injections of PDAC cells and CAFs. A) Orthotopic pancreatic tumours were treated with STAR nanoparticles + control-siRNA or SLC7A11 siRNA single sequence (SLC7A11 single seq) in the regimen shown. Representative photos of immunohistochemistry for SLC7A11 in tumour tissue at the model endpoint are shown. Graph shows optical density (staining intensity) calculated from average pixel intensity measurements from 3 representative images per tumour, using ImageJ. Circles indicate individual mice, lines indicate mean±s.e.m., asterisks indicate significance (*p≤0.05; One-way ANOVA). B) Treatment regimen for therapeutic model analysed in panels (C-D). Circles and triangles in all dot plots in panels C-D represent individual mice. C) Tumour volume at therapeutic model endpoint, as assessed by calliper measurement ex vivo (mean±s.e.m.). Asterisks indicate significance (*p≤0.05; One-way ANOVA). D) Representative photos of metastases confirmed by H&E staining following detection at model endpoint by ex vivo luminescence imaging of organs. Graph shows metastatic sites per mouse (mean±s.e.m.) for each treatment group. Scale bars in all figures = 200μm.

    Journal: bioRxiv

    Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

    doi: 10.1101/2020.07.12.199638

    Figure Lengend Snippet: All orthotopic tumours were co-injections of PDAC cells and CAFs. A) Orthotopic pancreatic tumours were treated with STAR nanoparticles + control-siRNA or SLC7A11 siRNA single sequence (SLC7A11 single seq) in the regimen shown. Representative photos of immunohistochemistry for SLC7A11 in tumour tissue at the model endpoint are shown. Graph shows optical density (staining intensity) calculated from average pixel intensity measurements from 3 representative images per tumour, using ImageJ. Circles indicate individual mice, lines indicate mean±s.e.m., asterisks indicate significance (*p≤0.05; One-way ANOVA). B) Treatment regimen for therapeutic model analysed in panels (C-D). Circles and triangles in all dot plots in panels C-D represent individual mice. C) Tumour volume at therapeutic model endpoint, as assessed by calliper measurement ex vivo (mean±s.e.m.). Asterisks indicate significance (*p≤0.05; One-way ANOVA). D) Representative photos of metastases confirmed by H&E staining following detection at model endpoint by ex vivo luminescence imaging of organs. Graph shows metastatic sites per mouse (mean±s.e.m.) for each treatment group. Scale bars in all figures = 200μm.

    Article Snippet: MiaPaCa-2 cells and hTERT-immortalised CAFs were then transduced with lentiviral constructs expressing scramble shRNA, SLC7A11-shRNA sequence 1 (Origene, Cat. TL309282).

    Techniques: Sequencing, Immunohistochemistry, Staining, Ex Vivo, Imaging

    Journal: bioRxiv

    Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

    doi: 10.1101/2020.07.12.199638

    Figure Lengend Snippet:

    Article Snippet: MiaPaCa-2 cells and hTERT-immortalised CAFs were then transduced with lentiviral constructs expressing scramble shRNA, SLC7A11-shRNA sequence 1 (Origene, Cat. TL309282).

    Techniques:

    A) Representative photos of tumour sections probed for αSMA (brown). The quantification of αSMA staining is shown in the graph (mean+s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Asterisks indicate significance (*p≤0.05; Control-siRNA, n=7; SLC7A11-siRNA single seq, n=5; student t-test). B) Representative photos of picrosirius red and methyl green stained tumour sections. The quantification of picrosirius red staining is shown in the bar graph (mean+s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Asterisks indicate significance (*p≤0.05; Control-siRNA, n=8; SLC7A11-siRNA single seq, n=5; student t-test). C) Polarised light analysis of representative regions from picrosirius red stained specimens. Representative photos are shown. Left bar graph shows total birefringence (mean+s.e.m.; Control-siRNA, n=8; SLC7A11-siRNA single seq, n=5). Right bar graph shows the average frequency (mean+s.e.m.; Control-siRNA, n=8; SLC7A11-siRNA single seq, n=5) of low, medium and high birefringence collagen fibrils (higher birefringence = denser fibril). ns = not significant (student t-test). D) Representative photos of CD31-stained tumour sections. Red arrows indicate open blood vessels. The bar graph shows the fraction of CD31-positive blood vessels that were open (mean+s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Asterisks indicate significance (*p≤0.05; Control-siRNA, n=8; SLC7A11-siRNA single seq, n=5; student t-test). Fields of view used for analyses in all panels, provided an average area coverage of 13% of the total tumour section (excluding necrotic regions). All circles in graphs represent individual mice. All scale bars in photos = 100μm.

    Journal: bioRxiv

    Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

    doi: 10.1101/2020.07.12.199638

    Figure Lengend Snippet: A) Representative photos of tumour sections probed for αSMA (brown). The quantification of αSMA staining is shown in the graph (mean+s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Asterisks indicate significance (*p≤0.05; Control-siRNA, n=7; SLC7A11-siRNA single seq, n=5; student t-test). B) Representative photos of picrosirius red and methyl green stained tumour sections. The quantification of picrosirius red staining is shown in the bar graph (mean+s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Asterisks indicate significance (*p≤0.05; Control-siRNA, n=8; SLC7A11-siRNA single seq, n=5; student t-test). C) Polarised light analysis of representative regions from picrosirius red stained specimens. Representative photos are shown. Left bar graph shows total birefringence (mean+s.e.m.; Control-siRNA, n=8; SLC7A11-siRNA single seq, n=5). Right bar graph shows the average frequency (mean+s.e.m.; Control-siRNA, n=8; SLC7A11-siRNA single seq, n=5) of low, medium and high birefringence collagen fibrils (higher birefringence = denser fibril). ns = not significant (student t-test). D) Representative photos of CD31-stained tumour sections. Red arrows indicate open blood vessels. The bar graph shows the fraction of CD31-positive blood vessels that were open (mean+s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Asterisks indicate significance (*p≤0.05; Control-siRNA, n=8; SLC7A11-siRNA single seq, n=5; student t-test). Fields of view used for analyses in all panels, provided an average area coverage of 13% of the total tumour section (excluding necrotic regions). All circles in graphs represent individual mice. All scale bars in photos = 100μm.

    Article Snippet: MiaPaCa-2 cells and hTERT-immortalised CAFs were then transduced with lentiviral constructs expressing scramble shRNA, SLC7A11-shRNA sequence 1 (Origene, Cat. TL309282).

    Techniques: Staining