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<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.
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<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.
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<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.
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<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.
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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.

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

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.

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.

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: 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.

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.

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: 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.

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.

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: 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.

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.

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: 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.

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.

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