Figure 7.
Figure 7. CXCR3 inhibition results in an attenuated transformed phenotype. (A) CXCL11 protein amounts in conditioned medium from A431 SCR or KRT17 shRNA cells were measured by ELISA. Data from three experimental repeats (each performed in 10 replicates) are represented as mean ± SEM (error bars). *, P < 0.016. (B) CXCL9 protein amounts in conditioned medium from primary keratinocytes isolated from ear lesions of P81 Krt17+/+ or Krt17−/− Gli2tg/+ mice were measured by ELISA. Data from four experimental repeats (each performed in triplicate) are represented as mean ± SEM (error bars). *, P < 0.04. (C) CXCL11 protein amounts in conditioned medium from A431 SCR shRNA cells transfected with either NS or hnRNP K siRNA (RPK1) were measured by ELISA. Data from three experimental repeats (each performed in 10 replicates) are represented as mean ± SEM (error bars). *, P < 0.04. (D) Immunoblotting performed on lysates from A431 cells stably expressing SCR or KRT17 shRNA with antibodies against the indicated proteins. (E) Soft agar colony assay using A431 SCR shRNA cells. Cells were grown in conditioned medium from A431 SCR or KRT17 shRNA cells, supplemented with anti-CXCR3 neutralizing antibody (0.5 ng/ml) or IgG control. Bar, 1 cm. (F) Colony numbers from E were quantitated using ImageJ and normalized to those grown in IgG-supplemented conditioned medium from A431 SCR shRNA cells. Data from four experimental repeats (each performed in triplicate) are represented as mean ± SEM (error bars). *, P < 0.03. (G) Soft agar colony assay using A431 SCR shRNA cells. Cells were grown in conditioned medium from A431 SCR shRNA cells, supplemented with DMSO or CXCR3 antagonist (10 µM). Colony numbers were quantitated using ImageJ and normalized to those grown in DMSO control. Data from three experimental repeats (each performed in triplicate) are represented as mean ± SEM (error bars). *, P < 0.05. (H) Soft agar colony assay using A431 SCR shRNA cells. Cells were grown in conditioned medium from A431 KRT17 shRNA cells, supplemented with or without 10 ng/ml recombinant CXCL11. Bar, 1 cm. (I) Colony numbers from H were quantitated using ImageJ and normalized to those grown without CXCL11. Data from five experimental repeats (each performed in triplicate) are represented as mean ± SEM (error bars). *, P < 0.02. (J) Soft agar colony assay using A431 SCR shRNA cells. Cells were grown in conditioned medium from A431 cells transfected with mCherry-C1 vector or hnRNP K, supplemented with anti-CXCR3 neutralizing antibody (0.5 ng/ml) or IgG control. Colony numbers were quantitated using ImageJ and normalized to those grown in conditioned medium from vector-expressing cells. Data from four experimental repeats (each performed in triplicate) are represented as mean ± SEM (error bars). *, P < 0.02; **, P < 0.05.

CXCR3 inhibition results in an attenuated transformed phenotype. (A) CXCL11 protein amounts in conditioned medium from A431 SCR or KRT17 shRNA cells were measured by ELISA. Data from three experimental repeats (each performed in 10 replicates) are represented as mean ± SEM (error bars). *, P < 0.016. (B) CXCL9 protein amounts in conditioned medium from primary keratinocytes isolated from ear lesions of P81 Krt17+/+ or Krt17−/− Gli2tg/+ mice were measured by ELISA. Data from four experimental repeats (each performed in triplicate) are represented as mean ± SEM (error bars). *, P < 0.04. (C) CXCL11 protein amounts in conditioned medium from A431 SCR shRNA cells transfected with either NS or hnRNP K siRNA (RPK1) were measured by ELISA. Data from three experimental repeats (each performed in 10 replicates) are represented as mean ± SEM (error bars). *, P < 0.04. (D) Immunoblotting performed on lysates from A431 cells stably expressing SCR or KRT17 shRNA with antibodies against the indicated proteins. (E) Soft agar colony assay using A431 SCR shRNA cells. Cells were grown in conditioned medium from A431 SCR or KRT17 shRNA cells, supplemented with anti-CXCR3 neutralizing antibody (0.5 ng/ml) or IgG control. Bar, 1 cm. (F) Colony numbers from E were quantitated using ImageJ and normalized to those grown in IgG-supplemented conditioned medium from A431 SCR shRNA cells. Data from four experimental repeats (each performed in triplicate) are represented as mean ± SEM (error bars). *, P < 0.03. (G) Soft agar colony assay using A431 SCR shRNA cells. Cells were grown in conditioned medium from A431 SCR shRNA cells, supplemented with DMSO or CXCR3 antagonist (10 µM). Colony numbers were quantitated using ImageJ and normalized to those grown in DMSO control. Data from three experimental repeats (each performed in triplicate) are represented as mean ± SEM (error bars). *, P < 0.05. (H) Soft agar colony assay using A431 SCR shRNA cells. Cells were grown in conditioned medium from A431 KRT17 shRNA cells, supplemented with or without 10 ng/ml recombinant CXCL11. Bar, 1 cm. (I) Colony numbers from H were quantitated using ImageJ and normalized to those grown without CXCL11. Data from five experimental repeats (each performed in triplicate) are represented as mean ± SEM (error bars). *, P < 0.02. (J) Soft agar colony assay using A431 SCR shRNA cells. Cells were grown in conditioned medium from A431 cells transfected with mCherry-C1 vector or hnRNP K, supplemented with anti-CXCR3 neutralizing antibody (0.5 ng/ml) or IgG control. Colony numbers were quantitated using ImageJ and normalized to those grown in conditioned medium from vector-expressing cells. Data from four experimental repeats (each performed in triplicate) are represented as mean ± SEM (error bars). *, P < 0.02; **, P < 0.05.

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