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mouse anti human caix (R&D Systems)

Name: mouse anti human caix
Brand: R&D Systems
Rating: 92 (12 reviews)

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R&D Systems mouse anti human caix

<t>CAIX</t> associates with SLC1A5 in hypoxic cancer cells. A, Expression of SLC1A5 and CAIX shown by Western blotting of MDA-MB-231 WT and CA9 KO cell lysates. Cells were cultured at 1% O 2 for 72 hours. Blots are representative of two independent experiments. B, Expression of SLC1A5 and CAIX shown by Western blotting of SUM159 WT, NTC (non target control) and CA9 KO cell lysates. Cells were cultured at 1% O 2 for 72 hours. Blots are representative of two independent experiments. C, Interaction of SLC1A5 and CAIX shown by co-IP analyses in MDA-MB-231 CAIXBirA* cell lysates. Cells were cultured at 21% O 2 for 72 hours. IgG2a was used as an isotype-specific control for the IP. Blots are representative of two independent experiments. D, Co-localization (yellow; arrows) of SLC1A5 (green) and exogenously expressed CAIX (red) in MDA-MB-231 CAIXBirA* cells shown by IF staining. Cells were cultured at 21% O 2 for 72 hours. Scale bar, 10 μm. E, Interaction of SLC1A5 and endogenous CAIX shown by co-IP analyses in SUM159PT and A549 cell lysates. Cells were cultured at 1% O 2 for 72 hours. IgG2a was used as an isotype-specific control for the IP. Blots are representative of two independent experiments. F, Co-localization (yellow; arrows) of SLC1A5 (green) and endogenous CAIX (red) in SUM159PT and A549 cells shown by IF staining. Cells were cultured at 1% O 2 for 72 hours. Scale bars, 50 μm; 10 μm (zoomed image). G, Interaction of SLC1A5 and CAIX (Red puncta) in SUM159 and A549 cells shown by PLA. Cells were cultured at 1% O 2 for 72 hours. Treatments with only SLC1A5 antibody (CAIX − SLC1A5 + ) and only CAIX antibody (CAIX + SLC1A5 − ) were used as negative controls for the assay. Scale bars, 50 μm; 10 μm (zoomed image). H, Quantification of the puncta from ( G ) for SUM159PT and A549 cells. Puncta were quantified from three images for each condition, having at least 20 cells in each image. See also Supplementary Fig. S1.

Mouse Anti Human Caix, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "A Carbonic Anhydrase IX/SLC1A5 Axis Regulates Glutamine Metabolism Dependent Ferroptosis in Hypoxic Tumor Cells"

Article Title: A Carbonic Anhydrase IX/SLC1A5 Axis Regulates Glutamine Metabolism Dependent Ferroptosis in Hypoxic Tumor Cells

Journal: Molecular Cancer Therapeutics

doi: 10.1158/1535-7163.MCT-23-0041

CAIX associates with SLC1A5 in hypoxic cancer cells. A, Expression of SLC1A5 and CAIX shown by Western blotting of MDA-MB-231 WT and CA9 KO cell lysates. Cells were cultured at 1% O 2 for 72 hours. Blots are representative of two independent experiments. B, Expression of SLC1A5 and CAIX shown by Western blotting of SUM159 WT, NTC (non target control) and CA9 KO cell lysates. Cells were cultured at 1% O 2 for 72 hours. Blots are representative of two independent experiments. C, Interaction of SLC1A5 and CAIX shown by co-IP analyses in MDA-MB-231 CAIXBirA* cell lysates. Cells were cultured at 21% O 2 for 72 hours. IgG2a was used as an isotype-specific control for the IP. Blots are representative of two independent experiments. D, Co-localization (yellow; arrows) of SLC1A5 (green) and exogenously expressed CAIX (red) in MDA-MB-231 CAIXBirA* cells shown by IF staining. Cells were cultured at 21% O 2 for 72 hours. Scale bar, 10 μm. E, Interaction of SLC1A5 and endogenous CAIX shown by co-IP analyses in SUM159PT and A549 cell lysates. Cells were cultured at 1% O 2 for 72 hours. IgG2a was used as an isotype-specific control for the IP. Blots are representative of two independent experiments. F, Co-localization (yellow; arrows) of SLC1A5 (green) and endogenous CAIX (red) in SUM159PT and A549 cells shown by IF staining. Cells were cultured at 1% O 2 for 72 hours. Scale bars, 50 μm; 10 μm (zoomed image). G, Interaction of SLC1A5 and CAIX (Red puncta) in SUM159 and A549 cells shown by PLA. Cells were cultured at 1% O 2 for 72 hours. Treatments with only SLC1A5 antibody (CAIX − SLC1A5 + ) and only CAIX antibody (CAIX + SLC1A5 − ) were used as negative controls for the assay. Scale bars, 50 μm; 10 μm (zoomed image). H, Quantification of the puncta from ( G ) for SUM159PT and A549 cells. Puncta were quantified from three images for each condition, having at least 20 cells in each image. See also Supplementary Fig. S1.

Figure Legend Snippet: CAIX associates with SLC1A5 in hypoxic cancer cells. A, Expression of SLC1A5 and CAIX shown by Western blotting of MDA-MB-231 WT and CA9 KO cell lysates. Cells were cultured at 1% O 2 for 72 hours. Blots are representative of two independent experiments. B, Expression of SLC1A5 and CAIX shown by Western blotting of SUM159 WT, NTC (non target control) and CA9 KO cell lysates. Cells were cultured at 1% O 2 for 72 hours. Blots are representative of two independent experiments. C, Interaction of SLC1A5 and CAIX shown by co-IP analyses in MDA-MB-231 CAIXBirA* cell lysates. Cells were cultured at 21% O 2 for 72 hours. IgG2a was used as an isotype-specific control for the IP. Blots are representative of two independent experiments. D, Co-localization (yellow; arrows) of SLC1A5 (green) and exogenously expressed CAIX (red) in MDA-MB-231 CAIXBirA* cells shown by IF staining. Cells were cultured at 21% O 2 for 72 hours. Scale bar, 10 μm. E, Interaction of SLC1A5 and endogenous CAIX shown by co-IP analyses in SUM159PT and A549 cell lysates. Cells were cultured at 1% O 2 for 72 hours. IgG2a was used as an isotype-specific control for the IP. Blots are representative of two independent experiments. F, Co-localization (yellow; arrows) of SLC1A5 (green) and endogenous CAIX (red) in SUM159PT and A549 cells shown by IF staining. Cells were cultured at 1% O 2 for 72 hours. Scale bars, 50 μm; 10 μm (zoomed image). G, Interaction of SLC1A5 and CAIX (Red puncta) in SUM159 and A549 cells shown by PLA. Cells were cultured at 1% O 2 for 72 hours. Treatments with only SLC1A5 antibody (CAIX − SLC1A5 + ) and only CAIX antibody (CAIX + SLC1A5 − ) were used as negative controls for the assay. Scale bars, 50 μm; 10 μm (zoomed image). H, Quantification of the puncta from ( G ) for SUM159PT and A549 cells. Puncta were quantified from three images for each condition, having at least 20 cells in each image. See also Supplementary Fig. S1.

Techniques Used: Expressing, Western Blot, Cell Culture, Control, Co-Immunoprecipitation Assay, Staining

Loss of CAIX activity increases Gln uptake in hypoxic cancer cells. A, Proliferation of the indicated cells in the presence (+) or absence (−) of 4 mmol/L Gln-containing media. B, Expression of SLC1A5 and CAIX shown by Western blotting in cell lysates from the indicated cell lines. C and D, Gln uptake in the indicated cell lines in the presence (+) or absence (−) of 100 μmol/L SLC-0111. E, Cellular expression of CAIX shown by Western blotting in cell lysates from WT and CA9 KO of MDA-MB-231 and SUM159PT cell lines. F, Gln uptake in the WT and CA9 KO of MDA-MB-231 and SUM159PT cell lines. G, Cellular expression of SLC1A5 shown by Western blotting in cell lysates from SUM159PT cells expressing shNS and shSLC1A5, cultured in the presence (+) or absence (−) of 0.5 μg/mL Dox. All samples are derived from the same blot and dashed lines indicate juxtaposition of samples from noncontiguous lanes. H, Gln uptake in the shNS and shSLC1A5–1 SUM159PT cell lines with the indicated treatments; Dox, 0.5 μg/mL and SLC-0111, 100 μmol/L. All treatments were carried out at 1% O 2 for 72 hours prior to the assay. For all graphs, bars represent means ± SD. For ( A ) statistical significance was assessed using Kruskal–Wallis test on the data from two independent experiments with n = 3 (technical replicates) for each experiment. Gln uptake data were assessed by performing Mann–Whitney for ( C ), ( D ), and ( F ), and one-way ANOVA test for ( H ) on data from at least two independent experiments with n = 3 for each experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant. See also Supplementary Fig. S2.

Figure Legend Snippet: Loss of CAIX activity increases Gln uptake in hypoxic cancer cells. A, Proliferation of the indicated cells in the presence (+) or absence (−) of 4 mmol/L Gln-containing media. B, Expression of SLC1A5 and CAIX shown by Western blotting in cell lysates from the indicated cell lines. C and D, Gln uptake in the indicated cell lines in the presence (+) or absence (−) of 100 μmol/L SLC-0111. E, Cellular expression of CAIX shown by Western blotting in cell lysates from WT and CA9 KO of MDA-MB-231 and SUM159PT cell lines. F, Gln uptake in the WT and CA9 KO of MDA-MB-231 and SUM159PT cell lines. G, Cellular expression of SLC1A5 shown by Western blotting in cell lysates from SUM159PT cells expressing shNS and shSLC1A5, cultured in the presence (+) or absence (−) of 0.5 μg/mL Dox. All samples are derived from the same blot and dashed lines indicate juxtaposition of samples from noncontiguous lanes. H, Gln uptake in the shNS and shSLC1A5–1 SUM159PT cell lines with the indicated treatments; Dox, 0.5 μg/mL and SLC-0111, 100 μmol/L. All treatments were carried out at 1% O 2 for 72 hours prior to the assay. For all graphs, bars represent means ± SD. For ( A ) statistical significance was assessed using Kruskal–Wallis test on the data from two independent experiments with n = 3 (technical replicates) for each experiment. Gln uptake data were assessed by performing Mann–Whitney for ( C ), ( D ), and ( F ), and one-way ANOVA test for ( H ) on data from at least two independent experiments with n = 3 for each experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant. See also Supplementary Fig. S2.

Techniques Used: Activity Assay, Expressing, Western Blot, Cell Culture, Derivative Assay, MANN-WHITNEY

The combined loss of CAIX/XII activity and Gln metabolism increases cytotoxicity of hypoxic cancer cells. A, Cytotoxicity analyses of SUM159PT WT and CA9 KO cells cultured in the presence (+) or absence (−) of the indicated treatments; SLC-0111, 100 μmol/L, and Gln, 4 mmol/L. B, Representative incucyte images for ( A ). Green, cytotoxicity; red, nuclei. C, Cytotoxicity analyses of SUM159PT WT cells cultured with indicated concentrations of Gln and SLC-0111. D, Graphical representation of the cotargeting strategy and inhibitors used. E and F, Cell cytotoxicity data for the indicated cell lines cultured with the combination of SLC-0111 and V9302 at the indicated concentrations. G–J, Cell cytotoxicity data for the indicated cell lines cultured with the combination of SLC-0111 and CB839 at indicated concentrations. K, Cellular expression of GLS1 shown by Western blotting in cell lysates from SUM159PT cells expressing shGLS1, cultured in the presence (+) or absence (−) of 0.5 μg/mL Dox. L, Cell cytotoxicity data for SUM159PT sh-GLS1 and 3 cell lines cultured at the indicated SLC-0111 concentrations. All treatments were carried out at 1% O 2 for 72 hours prior to the assay. For all graphs, bars represent means ± SD. Synergy scores and interpretation of the score are provided in boxed insets for each graph. Statistical significance was assessed using two-way ANOVA. Data shown are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. See also Supplementary Figs. S3 and S4.

Figure Legend Snippet: The combined loss of CAIX/XII activity and Gln metabolism increases cytotoxicity of hypoxic cancer cells. A, Cytotoxicity analyses of SUM159PT WT and CA9 KO cells cultured in the presence (+) or absence (−) of the indicated treatments; SLC-0111, 100 μmol/L, and Gln, 4 mmol/L. B, Representative incucyte images for ( A ). Green, cytotoxicity; red, nuclei. C, Cytotoxicity analyses of SUM159PT WT cells cultured with indicated concentrations of Gln and SLC-0111. D, Graphical representation of the cotargeting strategy and inhibitors used. E and F, Cell cytotoxicity data for the indicated cell lines cultured with the combination of SLC-0111 and V9302 at the indicated concentrations. G–J, Cell cytotoxicity data for the indicated cell lines cultured with the combination of SLC-0111 and CB839 at indicated concentrations. K, Cellular expression of GLS1 shown by Western blotting in cell lysates from SUM159PT cells expressing shGLS1, cultured in the presence (+) or absence (−) of 0.5 μg/mL Dox. L, Cell cytotoxicity data for SUM159PT sh-GLS1 and 3 cell lines cultured at the indicated SLC-0111 concentrations. All treatments were carried out at 1% O 2 for 72 hours prior to the assay. For all graphs, bars represent means ± SD. Synergy scores and interpretation of the score are provided in boxed insets for each graph. Statistical significance was assessed using two-way ANOVA. Data shown are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. See also Supplementary Figs. S3 and S4.

Techniques Used: Activity Assay, Cell Culture, Expressing, Western Blot

Loss of CAIX/XII activity increases cellular GSH and prevent cytotoxicity through a GSH/GPX4 axis. A and B, GSH level in ( A ) SUM159PT-WT and ( B ) 4T1Luc with the treatment of SLC-0111 at indicated concentrations. 0.1 mmol/L diethyl maleate (DEM) was used as a positive control for the assay. C, Graphical representation of the cotargeting strategy and inhibitors used. D – F, Cell cytotoxicity data of the indicated cell lines with the combination of SLC-0111 and BSO at indicated concentrations. Synergy scores and interpretation of the score are provided in boxed insets for each graph. G, Cellular expression of GLS1 shown by Western blotting in cell lysates from SUM159PT cells expressing shGLS1, cultured in the presence (+) or absence (−) of 0.5 μg/mL Dox. H, Cell cytotoxicity data of SUM159 sh-GCLC-3 and 5 cell lines at the indicated SLC-0111 concentrations. I – K, Cell cytotoxicity data of the indicated cell lines with the combination of SLC-0111 and RSL3 at indicated concentrations. Synergy scores and interpretation of the score are provided in boxed insets for each graph. All treatments were carried out at 1% O 2 for 72 hours prior to the assay. For all graphs, bars indicate means ± SD. Statistical significance was assessed for ( A and B ) using one-way ANOVA; Kruskal–Wallis on the data from three independent experiments with n = 3. All the cytotoxicity plots were assessed using two-way ANOVA. Data shown are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. See also Supplementary Fig. S5.

Figure Legend Snippet: Loss of CAIX/XII activity increases cellular GSH and prevent cytotoxicity through a GSH/GPX4 axis. A and B, GSH level in ( A ) SUM159PT-WT and ( B ) 4T1Luc with the treatment of SLC-0111 at indicated concentrations. 0.1 mmol/L diethyl maleate (DEM) was used as a positive control for the assay. C, Graphical representation of the cotargeting strategy and inhibitors used. D – F, Cell cytotoxicity data of the indicated cell lines with the combination of SLC-0111 and BSO at indicated concentrations. Synergy scores and interpretation of the score are provided in boxed insets for each graph. G, Cellular expression of GLS1 shown by Western blotting in cell lysates from SUM159PT cells expressing shGLS1, cultured in the presence (+) or absence (−) of 0.5 μg/mL Dox. H, Cell cytotoxicity data of SUM159 sh-GCLC-3 and 5 cell lines at the indicated SLC-0111 concentrations. I – K, Cell cytotoxicity data of the indicated cell lines with the combination of SLC-0111 and RSL3 at indicated concentrations. Synergy scores and interpretation of the score are provided in boxed insets for each graph. All treatments were carried out at 1% O 2 for 72 hours prior to the assay. For all graphs, bars indicate means ± SD. Statistical significance was assessed for ( A and B ) using one-way ANOVA; Kruskal–Wallis on the data from three independent experiments with n = 3. All the cytotoxicity plots were assessed using two-way ANOVA. Data shown are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. See also Supplementary Fig. S5.

Techniques Used: Activity Assay, Positive Control, Expressing, Western Blot, Cell Culture

Combined inhibition of CAIX/XII and Gln metabolism increases lipid peroxidation and induce ferroptosis. A and B, Lipid peroxidation in ( A ) 4T1Luc and ( B ) SUM159PT cells using BODIPY C11 staining with the indicated treatments; SLC-0111, 100 μmol/L, CB839, 5 μmol/L, BSO, 1.5 μmol/L, RSL3, 10 nmol/L. C – G, Cell viability of the indicated cell lines with the combination of CB839 and SLC-0111 ( C and D ) or the combination of BSO and SLC-0111 ( E – G ), in the presence of Fer1 (2 μmol/L), Nec1s (50 μmol/L) Z-VAD-fmk (25 μmol/L), DFO (10 μmol/L), Trolox (100 μmol/L). Bars indicate means ± SD. For the lipid peroxidation experiment, statistical significance was assessed using one-way ANOVA on the data from two experiments with n = 3. For the cytotoxicity experiments, statistical significance was assessed using two-way ANOVA. Data shown are representative of three independent experiments. All treatments were carried out in hypoxia for 72 hours. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure Legend Snippet: Combined inhibition of CAIX/XII and Gln metabolism increases lipid peroxidation and induce ferroptosis. A and B, Lipid peroxidation in ( A ) 4T1Luc and ( B ) SUM159PT cells using BODIPY C11 staining with the indicated treatments; SLC-0111, 100 μmol/L, CB839, 5 μmol/L, BSO, 1.5 μmol/L, RSL3, 10 nmol/L. C – G, Cell viability of the indicated cell lines with the combination of CB839 and SLC-0111 ( C and D ) or the combination of BSO and SLC-0111 ( E – G ), in the presence of Fer1 (2 μmol/L), Nec1s (50 μmol/L) Z-VAD-fmk (25 μmol/L), DFO (10 μmol/L), Trolox (100 μmol/L). Bars indicate means ± SD. For the lipid peroxidation experiment, statistical significance was assessed using one-way ANOVA on the data from two experiments with n = 3. For the cytotoxicity experiments, statistical significance was assessed using two-way ANOVA. Data shown are representative of three independent experiments. All treatments were carried out in hypoxia for 72 hours. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Techniques Used: Inhibition, Staining

Cotargeting CAIX/XII activity and GSH synthesis decreases the tumor growth. A, Cytotoxicity (green) in tumor spheroids treated with CB839 (1 μmol/L), SLC0111 (100 μmol/L) or the combination (CB839+SLC-0111). Tumor spheroids were cultured at 21% O 2 for 10 days and monitored real time. B, Quantification of cytotoxicity in tumor speroids from ( A ). C, Schematic of the in vivo study design and treatment. D, IHC staining showing the expression of GCLC and CAIX in representative tumor sections from the indicated treatment groups. Scale bar, 50 μm. E, Tumor growth curve of SUM159PT sh-GCLC tumors with indicated treatments, n = 8 to 9 per group. F, Tumor growth at endpoint of SUM159PT sh-GCLC tumors with indicated treatments, n = 8 to 9 per group. G, Survival plot for SUM159-shGCLC tumors in the indicated treatment groups. H, IHC staining showing the expression of CD71 (Transferrin receptor) in representative tumor sections from the indicated treatment groups. Scale bar 50 μm for 20× magnification and 25 μm for 40× magnification images. I, Quantification of the mean intensity of CD71 signal from IHC images of tumor sections in the indicated treatment groups. Quantification was performed on at least 4 images per tumor section with n = 4 mice per group. Bars in panels B , E , and F indicate means ± SEM. Bars in panel I indicate means ± SD. The statistical significance for tumor growth curve was assessed using two-way ANOVA. Tumor growth at endpoint and the mean intensity for CD71 were assessed using one-way ANOVA. *, P < 0.05; ****, P < 0.0001. See also Supplementary Fig. S6.

Figure Legend Snippet: Cotargeting CAIX/XII activity and GSH synthesis decreases the tumor growth. A, Cytotoxicity (green) in tumor spheroids treated with CB839 (1 μmol/L), SLC0111 (100 μmol/L) or the combination (CB839+SLC-0111). Tumor spheroids were cultured at 21% O 2 for 10 days and monitored real time. B, Quantification of cytotoxicity in tumor speroids from ( A ). C, Schematic of the in vivo study design and treatment. D, IHC staining showing the expression of GCLC and CAIX in representative tumor sections from the indicated treatment groups. Scale bar, 50 μm. E, Tumor growth curve of SUM159PT sh-GCLC tumors with indicated treatments, n = 8 to 9 per group. F, Tumor growth at endpoint of SUM159PT sh-GCLC tumors with indicated treatments, n = 8 to 9 per group. G, Survival plot for SUM159-shGCLC tumors in the indicated treatment groups. H, IHC staining showing the expression of CD71 (Transferrin receptor) in representative tumor sections from the indicated treatment groups. Scale bar 50 μm for 20× magnification and 25 μm for 40× magnification images. I, Quantification of the mean intensity of CD71 signal from IHC images of tumor sections in the indicated treatment groups. Quantification was performed on at least 4 images per tumor section with n = 4 mice per group. Bars in panels B , E , and F indicate means ± SEM. Bars in panel I indicate means ± SD. The statistical significance for tumor growth curve was assessed using two-way ANOVA. Tumor growth at endpoint and the mean intensity for CD71 were assessed using one-way ANOVA. *, P < 0.05; ****, P < 0.0001. See also Supplementary Fig. S6.

Techniques Used: Activity Assay, Cell Culture, In Vivo, Immunohistochemistry, Expressing

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Journal: Molecular Cancer Therapeutics

Article Title: A Carbonic Anhydrase IX/SLC1A5 Axis Regulates Glutamine Metabolism Dependent Ferroptosis in Hypoxic Tumor Cells

doi: 10.1158/1535-7163.MCT-23-0041

Figure Lengend Snippet: CAIX associates with SLC1A5 in hypoxic cancer cells. A, Expression of SLC1A5 and CAIX shown by Western blotting of MDA-MB-231 WT and CA9 KO cell lysates. Cells were cultured at 1% O 2 for 72 hours. Blots are representative of two independent experiments. B, Expression of SLC1A5 and CAIX shown by Western blotting of SUM159 WT, NTC (non target control) and CA9 KO cell lysates. Cells were cultured at 1% O 2 for 72 hours. Blots are representative of two independent experiments. C, Interaction of SLC1A5 and CAIX shown by co-IP analyses in MDA-MB-231 CAIXBirA* cell lysates. Cells were cultured at 21% O 2 for 72 hours. IgG2a was used as an isotype-specific control for the IP. Blots are representative of two independent experiments. D, Co-localization (yellow; arrows) of SLC1A5 (green) and exogenously expressed CAIX (red) in MDA-MB-231 CAIXBirA* cells shown by IF staining. Cells were cultured at 21% O 2 for 72 hours. Scale bar, 10 μm. E, Interaction of SLC1A5 and endogenous CAIX shown by co-IP analyses in SUM159PT and A549 cell lysates. Cells were cultured at 1% O 2 for 72 hours. IgG2a was used as an isotype-specific control for the IP. Blots are representative of two independent experiments. F, Co-localization (yellow; arrows) of SLC1A5 (green) and endogenous CAIX (red) in SUM159PT and A549 cells shown by IF staining. Cells were cultured at 1% O 2 for 72 hours. Scale bars, 50 μm; 10 μm (zoomed image). G, Interaction of SLC1A5 and CAIX (Red puncta) in SUM159 and A549 cells shown by PLA. Cells were cultured at 1% O 2 for 72 hours. Treatments with only SLC1A5 antibody (CAIX − SLC1A5 + ) and only CAIX antibody (CAIX + SLC1A5 − ) were used as negative controls for the assay. Scale bars, 50 μm; 10 μm (zoomed image). H, Quantification of the puncta from ( G ) for SUM159PT and A549 cells. Puncta were quantified from three images for each condition, having at least 20 cells in each image. See also Supplementary Fig. S1.

Article Snippet: Mouse anti-human CAIX (R and D Systems, MAB2188, RRID:AB_2066530); mouse IgG2A (R and D Systems, MAB003, RRID:AB_357345); goat anti-human CAIX (R and D Systems, AF2188, RRID:AB_416562) – 1:1,000; rabbit anti-human ASCT2 (Cell Signaling Technology, 8057, RRID:AB_10891440) – 1:500; mouse anti-vinculin (Millipore, MAB3574, RRID:AB_2304338) – 1:1,000; rabbit anti-human glutaminase-1 (GLS1; Abcam, ab156876, RRID:AB_2721038) – 1:500; mouse anti-human GCLC (Santa Cruz Biotechnology, sc-390811, RRID:AB_2736837) – 1:500; mouse anti ß-actin (Sigma-Aldrich, A5441, RRID:AB_476744).

Techniques: Expressing, Western Blot, Cell Culture, Control, Co-Immunoprecipitation Assay, Staining

Journal: Molecular Cancer Therapeutics

Article Title: A Carbonic Anhydrase IX/SLC1A5 Axis Regulates Glutamine Metabolism Dependent Ferroptosis in Hypoxic Tumor Cells

doi: 10.1158/1535-7163.MCT-23-0041

Figure Lengend Snippet: Loss of CAIX activity increases Gln uptake in hypoxic cancer cells. A, Proliferation of the indicated cells in the presence (+) or absence (−) of 4 mmol/L Gln-containing media. B, Expression of SLC1A5 and CAIX shown by Western blotting in cell lysates from the indicated cell lines. C and D, Gln uptake in the indicated cell lines in the presence (+) or absence (−) of 100 μmol/L SLC-0111. E, Cellular expression of CAIX shown by Western blotting in cell lysates from WT and CA9 KO of MDA-MB-231 and SUM159PT cell lines. F, Gln uptake in the WT and CA9 KO of MDA-MB-231 and SUM159PT cell lines. G, Cellular expression of SLC1A5 shown by Western blotting in cell lysates from SUM159PT cells expressing shNS and shSLC1A5, cultured in the presence (+) or absence (−) of 0.5 μg/mL Dox. All samples are derived from the same blot and dashed lines indicate juxtaposition of samples from noncontiguous lanes. H, Gln uptake in the shNS and shSLC1A5–1 SUM159PT cell lines with the indicated treatments; Dox, 0.5 μg/mL and SLC-0111, 100 μmol/L. All treatments were carried out at 1% O 2 for 72 hours prior to the assay. For all graphs, bars represent means ± SD. For ( A ) statistical significance was assessed using Kruskal–Wallis test on the data from two independent experiments with n = 3 (technical replicates) for each experiment. Gln uptake data were assessed by performing Mann–Whitney for ( C ), ( D ), and ( F ), and one-way ANOVA test for ( H ) on data from at least two independent experiments with n = 3 for each experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant. See also Supplementary Fig. S2.

Techniques: Activity Assay, Expressing, Western Blot, Cell Culture, Derivative Assay, MANN-WHITNEY

Journal: Molecular Cancer Therapeutics

Article Title: A Carbonic Anhydrase IX/SLC1A5 Axis Regulates Glutamine Metabolism Dependent Ferroptosis in Hypoxic Tumor Cells

doi: 10.1158/1535-7163.MCT-23-0041

Figure Lengend Snippet: The combined loss of CAIX/XII activity and Gln metabolism increases cytotoxicity of hypoxic cancer cells. A, Cytotoxicity analyses of SUM159PT WT and CA9 KO cells cultured in the presence (+) or absence (−) of the indicated treatments; SLC-0111, 100 μmol/L, and Gln, 4 mmol/L. B, Representative incucyte images for ( A ). Green, cytotoxicity; red, nuclei. C, Cytotoxicity analyses of SUM159PT WT cells cultured with indicated concentrations of Gln and SLC-0111. D, Graphical representation of the cotargeting strategy and inhibitors used. E and F, Cell cytotoxicity data for the indicated cell lines cultured with the combination of SLC-0111 and V9302 at the indicated concentrations. G–J, Cell cytotoxicity data for the indicated cell lines cultured with the combination of SLC-0111 and CB839 at indicated concentrations. K, Cellular expression of GLS1 shown by Western blotting in cell lysates from SUM159PT cells expressing shGLS1, cultured in the presence (+) or absence (−) of 0.5 μg/mL Dox. L, Cell cytotoxicity data for SUM159PT sh-GLS1 and 3 cell lines cultured at the indicated SLC-0111 concentrations. All treatments were carried out at 1% O 2 for 72 hours prior to the assay. For all graphs, bars represent means ± SD. Synergy scores and interpretation of the score are provided in boxed insets for each graph. Statistical significance was assessed using two-way ANOVA. Data shown are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. See also Supplementary Figs. S3 and S4.

Techniques: Activity Assay, Cell Culture, Expressing, Western Blot

Journal: Molecular Cancer Therapeutics

Article Title: A Carbonic Anhydrase IX/SLC1A5 Axis Regulates Glutamine Metabolism Dependent Ferroptosis in Hypoxic Tumor Cells

doi: 10.1158/1535-7163.MCT-23-0041

Figure Lengend Snippet: Loss of CAIX/XII activity increases cellular GSH and prevent cytotoxicity through a GSH/GPX4 axis. A and B, GSH level in ( A ) SUM159PT-WT and ( B ) 4T1Luc with the treatment of SLC-0111 at indicated concentrations. 0.1 mmol/L diethyl maleate (DEM) was used as a positive control for the assay. C, Graphical representation of the cotargeting strategy and inhibitors used. D – F, Cell cytotoxicity data of the indicated cell lines with the combination of SLC-0111 and BSO at indicated concentrations. Synergy scores and interpretation of the score are provided in boxed insets for each graph. G, Cellular expression of GLS1 shown by Western blotting in cell lysates from SUM159PT cells expressing shGLS1, cultured in the presence (+) or absence (−) of 0.5 μg/mL Dox. H, Cell cytotoxicity data of SUM159 sh-GCLC-3 and 5 cell lines at the indicated SLC-0111 concentrations. I – K, Cell cytotoxicity data of the indicated cell lines with the combination of SLC-0111 and RSL3 at indicated concentrations. Synergy scores and interpretation of the score are provided in boxed insets for each graph. All treatments were carried out at 1% O 2 for 72 hours prior to the assay. For all graphs, bars indicate means ± SD. Statistical significance was assessed for ( A and B ) using one-way ANOVA; Kruskal–Wallis on the data from three independent experiments with n = 3. All the cytotoxicity plots were assessed using two-way ANOVA. Data shown are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. See also Supplementary Fig. S5.

Techniques: Activity Assay, Positive Control, Expressing, Western Blot, Cell Culture

Journal: Molecular Cancer Therapeutics

Article Title: A Carbonic Anhydrase IX/SLC1A5 Axis Regulates Glutamine Metabolism Dependent Ferroptosis in Hypoxic Tumor Cells

doi: 10.1158/1535-7163.MCT-23-0041

Figure Lengend Snippet: Combined inhibition of CAIX/XII and Gln metabolism increases lipid peroxidation and induce ferroptosis. A and B, Lipid peroxidation in ( A ) 4T1Luc and ( B ) SUM159PT cells using BODIPY C11 staining with the indicated treatments; SLC-0111, 100 μmol/L, CB839, 5 μmol/L, BSO, 1.5 μmol/L, RSL3, 10 nmol/L. C – G, Cell viability of the indicated cell lines with the combination of CB839 and SLC-0111 ( C and D ) or the combination of BSO and SLC-0111 ( E – G ), in the presence of Fer1 (2 μmol/L), Nec1s (50 μmol/L) Z-VAD-fmk (25 μmol/L), DFO (10 μmol/L), Trolox (100 μmol/L). Bars indicate means ± SD. For the lipid peroxidation experiment, statistical significance was assessed using one-way ANOVA on the data from two experiments with n = 3. For the cytotoxicity experiments, statistical significance was assessed using two-way ANOVA. Data shown are representative of three independent experiments. All treatments were carried out in hypoxia for 72 hours. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Techniques: Inhibition, Staining

Journal: Molecular Cancer Therapeutics

Article Title: A Carbonic Anhydrase IX/SLC1A5 Axis Regulates Glutamine Metabolism Dependent Ferroptosis in Hypoxic Tumor Cells

doi: 10.1158/1535-7163.MCT-23-0041

Figure Lengend Snippet: Cotargeting CAIX/XII activity and GSH synthesis decreases the tumor growth. A, Cytotoxicity (green) in tumor spheroids treated with CB839 (1 μmol/L), SLC0111 (100 μmol/L) or the combination (CB839+SLC-0111). Tumor spheroids were cultured at 21% O 2 for 10 days and monitored real time. B, Quantification of cytotoxicity in tumor speroids from ( A ). C, Schematic of the in vivo study design and treatment. D, IHC staining showing the expression of GCLC and CAIX in representative tumor sections from the indicated treatment groups. Scale bar, 50 μm. E, Tumor growth curve of SUM159PT sh-GCLC tumors with indicated treatments, n = 8 to 9 per group. F, Tumor growth at endpoint of SUM159PT sh-GCLC tumors with indicated treatments, n = 8 to 9 per group. G, Survival plot for SUM159-shGCLC tumors in the indicated treatment groups. H, IHC staining showing the expression of CD71 (Transferrin receptor) in representative tumor sections from the indicated treatment groups. Scale bar 50 μm for 20× magnification and 25 μm for 40× magnification images. I, Quantification of the mean intensity of CD71 signal from IHC images of tumor sections in the indicated treatment groups. Quantification was performed on at least 4 images per tumor section with n = 4 mice per group. Bars in panels B , E , and F indicate means ± SEM. Bars in panel I indicate means ± SD. The statistical significance for tumor growth curve was assessed using two-way ANOVA. Tumor growth at endpoint and the mean intensity for CD71 were assessed using one-way ANOVA. *, P < 0.05; ****, P < 0.0001. See also Supplementary Fig. S6.

Techniques: Activity Assay, Cell Culture, In Vivo, Immunohistochemistry, Expressing

Figure 3. Specific cytotoxicity exhibited by CAIX‑specific CAR‑NK92 cells against CAIX+ target cells. The cytotoxic activity of CAR‑NK92 and Ctrl‑NK92 cells against CAIX‑ or CAIX+ cancer cells was determined using LDH release assay after a 4‑h incubation with CAIX‑ or CAIX+ target cells at an E/T ratio of 1:1, 3:1, 10:1 and 30:1. *P<0.05; **P<0.01; ***P<0.001. CAR, chimeric antigen receptor; LDH, lactate dehydrogenase; E/T, effector‑to‑target; ns, not significant.

Journal: Oncology reports

Article Title: Bortezomib improves adoptive carbonic anhydrase IX‑specific chimeric antigen receptor‑modified NK92 cell therapy in mouse models of human renal cell carcinoma.

doi: 10.3892/or.2018.6731

Figure Lengend Snippet: Figure 3. Specific cytotoxicity exhibited by CAIX‑specific CAR‑NK92 cells against CAIX+ target cells. The cytotoxic activity of CAR‑NK92 and Ctrl‑NK92 cells against CAIX‑ or CAIX+ cancer cells was determined using LDH release assay after a 4‑h incubation with CAIX‑ or CAIX+ target cells at an E/T ratio of 1:1, 3:1, 10:1 and 30:1. *P<0.05; **P<0.01; ***P<0.001. CAR, chimeric antigen receptor; LDH, lactate dehydrogenase; E/T, effector‑to‑target; ns, not significant.

Article Snippet: For the membrane surface expression of CAIX in renal cancer cell lines, 0.5x106 cells were suspended in 50 μl FACS buffer containing 2 μl FITC-conjugated mouse anti-human CAIX antibody (dilution 1:10; cat. no. FAB2188F; R&D Systems, Inc., Minneapolis, MN, USA) and incubated at 4 ̊C for 30 min. A FITC‐conjugated mouse IgG2A isotype antibody (dilution 1:10; cat. no. IC003F R&D Systems, Inc.) was used as the control.

Techniques: Activity Assay, Lactate Dehydrogenase Assay, Incubation

Figure 2. Specific cytokine release of CAIX‑specific CAR‑NK92 cells against CAIX+ cells. (A) FACS was used to assess the surface expression of CAIX proteins in human renal cancer ACHN, Ketr‑3, and OSRC‑2 cell lines and in the 293 cells. (B) The levels of cytokines, released by Ctrl‑NK92 and CAR‑NK92 cells, were assessed by enzyme‑linked immunosorbent assay (ELISA) after 24 h of incubation with CAIX‑ or CAIX+ target cells at an effector‑to‑target (E/T) ratio of 1:1. **P<0.01; ***P<0.001. CAR, chimeric antigen receptor; ns, not significant.

Journal: Oncology reports

Article Title: Bortezomib improves adoptive carbonic anhydrase IX‑specific chimeric antigen receptor‑modified NK92 cell therapy in mouse models of human renal cell carcinoma.

doi: 10.3892/or.2018.6731

Figure Lengend Snippet: Figure 2. Specific cytokine release of CAIX‑specific CAR‑NK92 cells against CAIX+ cells. (A) FACS was used to assess the surface expression of CAIX proteins in human renal cancer ACHN, Ketr‑3, and OSRC‑2 cell lines and in the 293 cells. (B) The levels of cytokines, released by Ctrl‑NK92 and CAR‑NK92 cells, were assessed by enzyme‑linked immunosorbent assay (ELISA) after 24 h of incubation with CAIX‑ or CAIX+ target cells at an effector‑to‑target (E/T) ratio of 1:1. **P<0.01; ***P<0.001. CAR, chimeric antigen receptor; ns, not significant.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Incubation

Identification of the CAIX Interactome using the BioID proximal ligation strategy and validation of high confidence proximal CAIX-associating proteins by co-immunoprecipitation. ( a ) Schematic showing the domain structure of the CAIX-BirA* fusion protein used for the BioID studies. SP, signal peptide; PG, proteoglycan-like domain; CA, catalytic domain; TM, transmembrane domain; IC, intracellular domain. ( b ) FACS analysis showing levels of constitutive expression of the CAIX-BirA* fusion protein expressed by MDA-MB-231 cells (MDA-MB-231-CAIX-BirA*) cultured in normoxia compared to levels of endogenous CAIX expression by WT MDA-MB-231 cells cultured in either normoxia or hypoxia. The filled (gray) histograms indicate specific CAIX staining whereas the dotted lined histograms indicate staining by isotype-specific control IgG. ( c ) Image showing exogenous CAIX (red) localized at the cell surface of MDA-MB-231-CAIX-BirA* cells grown in normoxia. Scale bar, 10 μ M . ( d – g ) Co-IP of high confidence proximal CAIX-associating proteins ( d ) ITGB1, ( e ) ITGA2 ( f ) CD98hc and ( g ) MMP14 with exogenous CAIX expressed by MDA-MB-231-CAIX-BirA* cells cultured in normoxia. ( h – j ) Co-IP of ( h ) ITGB1, ( i ) ITGA2 and ( j ) MMP14 with endogenous CAIX expressed by WT MDA-MB-231 cells cultured in either normoxia (N) or hypoxia (H). Isotype-specific IgG was used as a control for co-IPs and data in ( d ) to ( j ) are representative of at least two independent experiments. IgG, immunoglobulin G.

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: Identification of the CAIX Interactome using the BioID proximal ligation strategy and validation of high confidence proximal CAIX-associating proteins by co-immunoprecipitation. ( a ) Schematic showing the domain structure of the CAIX-BirA* fusion protein used for the BioID studies. SP, signal peptide; PG, proteoglycan-like domain; CA, catalytic domain; TM, transmembrane domain; IC, intracellular domain. ( b ) FACS analysis showing levels of constitutive expression of the CAIX-BirA* fusion protein expressed by MDA-MB-231 cells (MDA-MB-231-CAIX-BirA*) cultured in normoxia compared to levels of endogenous CAIX expression by WT MDA-MB-231 cells cultured in either normoxia or hypoxia. The filled (gray) histograms indicate specific CAIX staining whereas the dotted lined histograms indicate staining by isotype-specific control IgG. ( c ) Image showing exogenous CAIX (red) localized at the cell surface of MDA-MB-231-CAIX-BirA* cells grown in normoxia. Scale bar, 10 μ M . ( d – g ) Co-IP of high confidence proximal CAIX-associating proteins ( d ) ITGB1, ( e ) ITGA2 ( f ) CD98hc and ( g ) MMP14 with exogenous CAIX expressed by MDA-MB-231-CAIX-BirA* cells cultured in normoxia. ( h – j ) Co-IP of ( h ) ITGB1, ( i ) ITGA2 and ( j ) MMP14 with endogenous CAIX expressed by WT MDA-MB-231 cells cultured in either normoxia (N) or hypoxia (H). Isotype-specific IgG was used as a control for co-IPs and data in ( d ) to ( j ) are representative of at least two independent experiments. IgG, immunoglobulin G.

Article Snippet: Cells were incubated with either anti-mouse CAIX (R&D Systems, MAB2188) or a control anti-mouse IgG 2a (R&D Systems, MAB003) at 1:100 for 30 min on ice.

Techniques: Ligation, Biomarker Discovery, Immunoprecipitation, Expressing, Cell Culture, Staining, Control, Co-Immunoprecipitation Assay

Selected CAIX-BirA*FLAG high-confidence proximal associating proteins identified by BioID in MDA-MB-231 cells

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: Selected CAIX-BirA*FLAG high-confidence proximal associating proteins identified by BioID in MDA-MB-231 cells

Article Snippet: Cells were incubated with either anti-mouse CAIX (R&D Systems, MAB2188) or a control anti-mouse IgG 2a (R&D Systems, MAB003) at 1:100 for 30 min on ice.

Techniques:

CAIX colocalizes with integrins and MMP14 in pseudopodia-like protrusions resembling lamellipodia. ( a ) Images of MDA-MB-231-CAIX-BirA* cells cultured on type 1 collagen in normoxia, showing colocalization (yellow, arrows in merge) of exogenously expressed CAIX (red) with ITGB1, ITGA2, MMP14 and cofilin (each marker in green), but not with FAK (green; arrowheads). Scale bar, 10 μm. ( b ) Images of WT MDA-MB-231 cells grown in either normoxia or hypoxia, showing colocalization of hypoxia-induced CAIX (red) with F-actin (green) in pseudopodia-like protrusions (arrows, merge). Scale bar, 10 μm. ( c ) Images of WT MDA-MB-231 cells cultured on type 1 collagen in hypoxia, showing colocalization of hypoxia-induced CAIX (red) with F-actin, ITGA2 and MMP14 (green) at leading edges of cells (arrows, merge), but not at focal adhesions. Scale bar, 10 μm. ( d and e ) 3D maximum projections of 2D images of MDA-MB-231-CAIX-BirA* cells transfected with GFP and cultured on type 1 collagen in normoxia, showing robust colocalization of CAIX (red) with ( d ) ITGA2 and ( e ) MMP14 (blue) in pseudopodia-like protrusions and depletion of cytoplasmic GFP (green) from these regions. Images for each channel are shown in the upper panels and merged images are shown in the lower panels. Scale bar, 10 μm.

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX colocalizes with integrins and MMP14 in pseudopodia-like protrusions resembling lamellipodia. ( a ) Images of MDA-MB-231-CAIX-BirA* cells cultured on type 1 collagen in normoxia, showing colocalization (yellow, arrows in merge) of exogenously expressed CAIX (red) with ITGB1, ITGA2, MMP14 and cofilin (each marker in green), but not with FAK (green; arrowheads). Scale bar, 10 μm. ( b ) Images of WT MDA-MB-231 cells grown in either normoxia or hypoxia, showing colocalization of hypoxia-induced CAIX (red) with F-actin (green) in pseudopodia-like protrusions (arrows, merge). Scale bar, 10 μm. ( c ) Images of WT MDA-MB-231 cells cultured on type 1 collagen in hypoxia, showing colocalization of hypoxia-induced CAIX (red) with F-actin, ITGA2 and MMP14 (green) at leading edges of cells (arrows, merge), but not at focal adhesions. Scale bar, 10 μm. ( d and e ) 3D maximum projections of 2D images of MDA-MB-231-CAIX-BirA* cells transfected with GFP and cultured on type 1 collagen in normoxia, showing robust colocalization of CAIX (red) with ( d ) ITGA2 and ( e ) MMP14 (blue) in pseudopodia-like protrusions and depletion of cytoplasmic GFP (green) from these regions. Images for each channel are shown in the upper panels and merged images are shown in the lower panels. Scale bar, 10 μm.

Article Snippet: Cells were incubated with either anti-mouse CAIX (R&D Systems, MAB2188) or a control anti-mouse IgG 2a (R&D Systems, MAB003) at 1:100 for 30 min on ice.

Techniques: Cell Culture, Marker, Transfection

CAIX regulates migration of breast cancer cells. ( a ) Images of wound-induced cell migration by the indicated MDA-MB-231 cells cultured in normoxia. Scale bar, 300 μm. Dashed lines demarcate the wound boundary at t =0 h and yellow shading indicates the cell-free area. ( b ) Quantification of migration by the cells described in a . *** P <0.001. ( c ) Images of wound-induced migration of MDA-MB-231 CAIX-BirA* cells cultured in normoxia and treated with increasing concentrations of U-104. Scale bar, 300 μm. Wound boundaries and cell-free area are demarcated as described in a . ( d ) Quantification of migration by the cells described in c . * P <0.05, *** P <0.001. Data in panels ( b ) and ( d ) show the mean±s.e.m. of technical replicates ( n =16) and are representative of two independent experiments. ( e ) Wound-induced migration of WT MDA-MB-231 cells cultured in normoxia (black bars) or hypoxia (gray bars) and treated with U-104. Data show the mean±s.e.m. of technical replicates ( n =12) and are representative of three independent experiments. ** P <0.01, *** P <0.001. Statistical analysis was performed using Student’s t -test ( b ) or ANOVA ( d , e ).

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX regulates migration of breast cancer cells. ( a ) Images of wound-induced cell migration by the indicated MDA-MB-231 cells cultured in normoxia. Scale bar, 300 μm. Dashed lines demarcate the wound boundary at t =0 h and yellow shading indicates the cell-free area. ( b ) Quantification of migration by the cells described in a . *** P <0.001. ( c ) Images of wound-induced migration of MDA-MB-231 CAIX-BirA* cells cultured in normoxia and treated with increasing concentrations of U-104. Scale bar, 300 μm. Wound boundaries and cell-free area are demarcated as described in a . ( d ) Quantification of migration by the cells described in c . * P <0.05, *** P <0.001. Data in panels ( b ) and ( d ) show the mean±s.e.m. of technical replicates ( n =16) and are representative of two independent experiments. ( e ) Wound-induced migration of WT MDA-MB-231 cells cultured in normoxia (black bars) or hypoxia (gray bars) and treated with U-104. Data show the mean±s.e.m. of technical replicates ( n =12) and are representative of three independent experiments. ** P <0.01, *** P <0.001. Statistical analysis was performed using Student’s t -test ( b ) or ANOVA ( d , e ).

Article Snippet: Cells were incubated with either anti-mouse CAIX (R&D Systems, MAB2188) or a control anti-mouse IgG 2a (R&D Systems, MAB003) at 1:100 for 30 min on ice.

Techniques: Migration, Cell Culture

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX colocalizes with MMP14 at invadopodia and regulates type I collagen degradation. ( a ) Images of a MDA-MB-231-CAIX-BirA* cell (white dashed line shows boundary) cultured on type I collagen in normoxia, identifying a ROI (yellow box) containing invadopodia (arrows) labeled for Cortactin (cyan), CAIX (red) and MMP14 (green). Profile plots showing colocalization of CAIX and MMP14 at cortactin-positive invadopodia (1 and 2) along the indicated lines (insets) are shown at the right. Scale bar, 10 μm. ( b ) Images of an MDA-MB-231-CAIX-BirA* cell (white dashed line shows cell boundary) cultured on DQ type I collagen in normoxia, identifying an ROI (yellow box) containing regions of collagen degradation (1 and 2, arrows) that are labeled for DQ collagen (cyan), CAIX (red) and MMP14 (green). Profile plots showing colocalization of the signals along the indicated lines (insets) are shown to the right. Scale bar, 10 μm. ( c ) Images showing WT MDA-MB-231 cells cultured in hypoxia on fluorescently labeled gelatin (blue, with black areas denoting regions of degradation) and stained for CAIX (green) and markers of invadopodia (red), including cortactin, Tks5 and MMP14 to demonstrate colocalization at mature invadopodia (white arrows; yellow foci). Scale bars, 10 μm (left), 2 μm (right). ( d ) Quantification of DQ collagen degradation by the indicated MDA-MB-231 cell lines grown in normoxia. *** P <0.001. ( e ) Quantitation of degradation of DQ collagen by MDA-MB-231 CAIX KO and CAIX rescue cells cultured in normoxia. * P <0.05. Data in ( d ) and ( e ) show the mean±s.e.m. of technical replicates ( n =5) and are representative of three independent experiments. ( f ) Dose-dependent inhibition of DQ collagen degradation by CAIX rescue cells treated with U-104. Data show the mean±s.e.m. of technical replicates ( n =4) and are representative of two independent experiments. * P <0.05, ** P <0.01. ( g ) Quantification of DQ collagen degradation by WT MDA-MB-231 and CAIX KO cells cultured in hypoxia. Data show the mean±s.e.m. of technical replicates ( n =8) and are representative of three independent experiments. ** P <0.01. ( h and i ) Invasion through Matrigel by highly metastatic MDA-MB-231 LM2-4 breast cancer cells cultured in hypoxia and ( h ) expressing CAIX-targeted (shCAIX) shRNA or ( i ) treated with U-104. Data show the mean±s.e.m. of three independent experiments. *** P <0.001. Western blots of hypoxia-induced CAIX expression are shown as insets.

Article Snippet: Cells were incubated with either anti-mouse CAIX (R&D Systems, MAB2188) or a control anti-mouse IgG 2a (R&D Systems, MAB003) at 1:100 for 30 min on ice.

Techniques: Cell Culture, Labeling, Staining, Quantitation Assay, Inhibition, Expressing, shRNA, Western Blot

CAIX regulates breast cancer cell invasion and metastasis. ( a ) Schematic showing the domain structure of wild type (WT) huCAIX (459 aa) and constructs lacking either the intracellular domain (ΔIC), the extracellular proteoglycan-like domain (ΔPG) or the indicated point mutations in the IC domain. SP, signal peptide; CA, catalytic domain; TM, transmembrane domain. ( b ) Western blot analysis of the levels of expression of the indicated huCAIX constructs by 4T1shCAIX cells cultured in hypoxia. β-actin served as a loading control. ( c ) Analysis of CAIX catalytic activity using the in-cell carbonic anhydrase activity assay. Assays were performed in normoxia in the presence or absence of U-104 (50 μ M ) as indicated. Levels of CAIX catalytic activity were normalized by using the time required to achieve 50% of the total decrease in pH. Data were normalized to the spontaneous rate of reaction in the presence of buffer alone and the activity of cells expressing WT huCAIX was set to 100%. Data show the mean±s.e.m. of technical replicates ( n =3/group) and are representative of three independent experiments * P <0.05, *** P <0.001. ( d ) Invasion through Matrigel by the indicated 4T1 cell lines cultured in hypoxia. Data show the mean±s.e.m. of three independent experiments. * P <0.05, *** P <0.001. ( e ) Invasion through Matrigel by the 4T1shCAIX cell lines expressing WT, ΔIC and ΔPG variants of huCAIX and cultured in hypoxia. * P <0.05, *** P <0.001. ( f ) Invasion through Matrigel by 4T1 sh/WT huCAIX cells cultured in hypoxia and treated with U-104. *** P <0.001. Western blots of hypoxia-induced CAIX expression are shown as insets. For ( d )– ( f ), data show the mean±s.e.m. of three independent experiments. ( g – i ) Analysis of invasion through type 1 collagen by 4T1 sh/WT huCAIX cells cultured in hypoxia and treated with ( g ) U-104 (50 μ M ), ( h ) anti-MMP14 antibody (20 μg/ml) or ( i ) a combination of anti-MMP14 antibody (20 μg/ml) and U-104 (50 μ M ). * P <0.05, ** P <0.01. Data in ( g )–( i ) show the mean±s.e.m. of three independent experiments. ( j ) Analysis of spontaneous lung metastases formed by the indicated 4T1 cell lines following growth of orthotopic breast tumors. Data show the mean±s.e.m. n =6/group. ( k ) Analysis of experimental lung metastases formed by the indicated 4T1 cell lines. Mean±s.e.m. is shown. n =6/group. * P <0.05, ** P <0.01. Statistical analysis was performed using ANOVA ( c , d , e , k ) or Student’s t -test ( f , g , h ).

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX regulates breast cancer cell invasion and metastasis. ( a ) Schematic showing the domain structure of wild type (WT) huCAIX (459 aa) and constructs lacking either the intracellular domain (ΔIC), the extracellular proteoglycan-like domain (ΔPG) or the indicated point mutations in the IC domain. SP, signal peptide; CA, catalytic domain; TM, transmembrane domain. ( b ) Western blot analysis of the levels of expression of the indicated huCAIX constructs by 4T1shCAIX cells cultured in hypoxia. β-actin served as a loading control. ( c ) Analysis of CAIX catalytic activity using the in-cell carbonic anhydrase activity assay. Assays were performed in normoxia in the presence or absence of U-104 (50 μ M ) as indicated. Levels of CAIX catalytic activity were normalized by using the time required to achieve 50% of the total decrease in pH. Data were normalized to the spontaneous rate of reaction in the presence of buffer alone and the activity of cells expressing WT huCAIX was set to 100%. Data show the mean±s.e.m. of technical replicates ( n =3/group) and are representative of three independent experiments * P <0.05, *** P <0.001. ( d ) Invasion through Matrigel by the indicated 4T1 cell lines cultured in hypoxia. Data show the mean±s.e.m. of three independent experiments. * P <0.05, *** P <0.001. ( e ) Invasion through Matrigel by the 4T1shCAIX cell lines expressing WT, ΔIC and ΔPG variants of huCAIX and cultured in hypoxia. * P <0.05, *** P <0.001. ( f ) Invasion through Matrigel by 4T1 sh/WT huCAIX cells cultured in hypoxia and treated with U-104. *** P <0.001. Western blots of hypoxia-induced CAIX expression are shown as insets. For ( d )– ( f ), data show the mean±s.e.m. of three independent experiments. ( g – i ) Analysis of invasion through type 1 collagen by 4T1 sh/WT huCAIX cells cultured in hypoxia and treated with ( g ) U-104 (50 μ M ), ( h ) anti-MMP14 antibody (20 μg/ml) or ( i ) a combination of anti-MMP14 antibody (20 μg/ml) and U-104 (50 μ M ). * P <0.05, ** P <0.01. Data in ( g )–( i ) show the mean±s.e.m. of three independent experiments. ( j ) Analysis of spontaneous lung metastases formed by the indicated 4T1 cell lines following growth of orthotopic breast tumors. Data show the mean±s.e.m. n =6/group. ( k ) Analysis of experimental lung metastases formed by the indicated 4T1 cell lines. Mean±s.e.m. is shown. n =6/group. * P <0.05, ** P <0.01. Statistical analysis was performed using ANOVA ( c , d , e , k ) or Student’s t -test ( f , g , h ).

Article Snippet: Cells were incubated with either anti-mouse CAIX (R&D Systems, MAB2188) or a control anti-mouse IgG 2a (R&D Systems, MAB003) at 1:100 for 30 min on ice.

Techniques: Construct, Western Blot, Expressing, Cell Culture, Control, Activity Assay

CAIX associates with MMP14 through its intracellular domain and potentiates MMP14 activity. ( a ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells cultured in hypoxia and expressing WT, ΔIC or ΔPG variants of huCAIX. ( b ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells cultured in hypoxia and expressing WT huCAIX or the T443A point mutant. ( c ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells cultured in hypoxia and expressing WT or the Y449A point mutant. ( d ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells grown in hypoxia and expressing WT huCAIX, the S448A point mutant or the ΔPG mutant. ( e ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells in hypoxia expressing WT huCAIX, the ΔIC truncation or the double point mutant S448A+Y449A. Isotype-specific IgG was used as a control for co-IPs. ( f ) Time-dependent stimulation of MMP14 activity in the presence (red bars) or absence (black bars) of equimolar concentrations of CAIX. Data show the mean±s.e.m. of technical replicates ( n =5) and are representative of two independent experiments. * P <0.05, ** P <0.01. ( g ) Dose-dependent inhibition of CAIX-mediated stimulation of MMP14 activity in the presence of increasing concentrations of U-104. Mean±s.e.m. of technical replicates ( n =5). Data are representative of two independent experiments. *** P <0.001. ( h ) Inhibition of CAIX-mediated stimulation of MMP14 activity in the presence of increasing amounts of D 2 O (heavy water). Mean±s.e.m. of technical replicates ( n =5). Data are representative of three independent experiments. *** P <0.001. Statistical analysis was performed using Student’s t -test ( f ) or ANOVA ( g , h ).

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX associates with MMP14 through its intracellular domain and potentiates MMP14 activity. ( a ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells cultured in hypoxia and expressing WT, ΔIC or ΔPG variants of huCAIX. ( b ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells cultured in hypoxia and expressing WT huCAIX or the T443A point mutant. ( c ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells cultured in hypoxia and expressing WT or the Y449A point mutant. ( d ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells grown in hypoxia and expressing WT huCAIX, the S448A point mutant or the ΔPG mutant. ( e ) Co-IP of huCAIX and MMP14 from 4T1shCAIX cells in hypoxia expressing WT huCAIX, the ΔIC truncation or the double point mutant S448A+Y449A. Isotype-specific IgG was used as a control for co-IPs. ( f ) Time-dependent stimulation of MMP14 activity in the presence (red bars) or absence (black bars) of equimolar concentrations of CAIX. Data show the mean±s.e.m. of technical replicates ( n =5) and are representative of two independent experiments. * P <0.05, ** P <0.01. ( g ) Dose-dependent inhibition of CAIX-mediated stimulation of MMP14 activity in the presence of increasing concentrations of U-104. Mean±s.e.m. of technical replicates ( n =5). Data are representative of two independent experiments. *** P <0.001. ( h ) Inhibition of CAIX-mediated stimulation of MMP14 activity in the presence of increasing amounts of D 2 O (heavy water). Mean±s.e.m. of technical replicates ( n =5). Data are representative of three independent experiments. *** P <0.001. Statistical analysis was performed using Student’s t -test ( f ) or ANOVA ( g , h ).

Article Snippet: Cells were incubated with either anti-mouse CAIX (R&D Systems, MAB2188) or a control anti-mouse IgG 2a (R&D Systems, MAB003) at 1:100 for 30 min on ice.

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Cell Culture, Expressing, Mutagenesis, Control, Inhibition

CAIX stimulates the activity of MMP14. Model for CAIX-mediated regulation of MMP14 activity. CAIX, which is upregulated in hypoxia, associates through its intracellular domain with MMP14 at functional invadopodia. At these invasive structures, CAIX functions to potentiate MMP14-mediated collagen degradation by directly contributing H + ions required for MMP14 catalytic activity.

Journal: Oncogene

Article Title: The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion

doi: 10.1038/onc.2017.219

Figure Lengend Snippet: CAIX stimulates the activity of MMP14. Model for CAIX-mediated regulation of MMP14 activity. CAIX, which is upregulated in hypoxia, associates through its intracellular domain with MMP14 at functional invadopodia. At these invasive structures, CAIX functions to potentiate MMP14-mediated collagen degradation by directly contributing H + ions required for MMP14 catalytic activity.

Article Snippet: Cells were incubated with either anti-mouse CAIX (R&D Systems, MAB2188) or a control anti-mouse IgG 2a (R&D Systems, MAB003) at 1:100 for 30 min on ice.

Techniques: Activity Assay, Functional Assay

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Structured Review

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1) Product Images from "A Carbonic Anhydrase IX/SLC1A5 Axis Regulates Glutamine Metabolism Dependent Ferroptosis in Hypoxic Tumor Cells"

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