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spink1 protein levels  (R&D Systems)


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    R&D Systems spink1 protein levels
    Figure 3. <t>SPINK1</t> secreted from cells induces cancer radioresistance in a paracrine manner. (A) HeLa tumor xenografts were stained with antibodies against a hypoxia marker, pimonidazole (green), or SPINK1 (red). Blue, counter staining with Hoechst 33342. The dotted line represents the outside edge of the pimonidazole-positive regions. Scale bar: 50 μm. (B–E) After 24 hours serum starvation, DU145 cells were precultured in the presence or absence of 100 ng/mL rSPINK1 for 24 hours, treated with the indicated dose of γ-ray irradiation, and subjected to the clonogenic survival assay (B), col- orimetric cell viability assay (C), and FACS analysis for the cell cycle status (D) and sub-G1 fraction (E). The cells were precultured and irradiated under the indicated oxygen conditions in C. (F and G) After transfection with either pcDNA4/SPINK1 (SPINK1) or its EV, the indicated cells were precultured under mild hypoxic conditions (O2 = 3%) for 48 hours, treated with the indicated dose of γ-ray irradiation under the same oxygen conditions as the
    Spink1 Protein Levels, supplied by R&D Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization."

    Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization.

    Journal: JCI insight

    doi: 10.1172/jci.insight.148135

    Figure 3. SPINK1 secreted from cells induces cancer radioresistance in a paracrine manner. (A) HeLa tumor xenografts were stained with antibodies against a hypoxia marker, pimonidazole (green), or SPINK1 (red). Blue, counter staining with Hoechst 33342. The dotted line represents the outside edge of the pimonidazole-positive regions. Scale bar: 50 μm. (B–E) After 24 hours serum starvation, DU145 cells were precultured in the presence or absence of 100 ng/mL rSPINK1 for 24 hours, treated with the indicated dose of γ-ray irradiation, and subjected to the clonogenic survival assay (B), col- orimetric cell viability assay (C), and FACS analysis for the cell cycle status (D) and sub-G1 fraction (E). The cells were precultured and irradiated under the indicated oxygen conditions in C. (F and G) After transfection with either pcDNA4/SPINK1 (SPINK1) or its EV, the indicated cells were precultured under mild hypoxic conditions (O2 = 3%) for 48 hours, treated with the indicated dose of γ-ray irradiation under the same oxygen conditions as the
    Figure Legend Snippet: Figure 3. SPINK1 secreted from cells induces cancer radioresistance in a paracrine manner. (A) HeLa tumor xenografts were stained with antibodies against a hypoxia marker, pimonidazole (green), or SPINK1 (red). Blue, counter staining with Hoechst 33342. The dotted line represents the outside edge of the pimonidazole-positive regions. Scale bar: 50 μm. (B–E) After 24 hours serum starvation, DU145 cells were precultured in the presence or absence of 100 ng/mL rSPINK1 for 24 hours, treated with the indicated dose of γ-ray irradiation, and subjected to the clonogenic survival assay (B), col- orimetric cell viability assay (C), and FACS analysis for the cell cycle status (D) and sub-G1 fraction (E). The cells were precultured and irradiated under the indicated oxygen conditions in C. (F and G) After transfection with either pcDNA4/SPINK1 (SPINK1) or its EV, the indicated cells were precultured under mild hypoxic conditions (O2 = 3%) for 48 hours, treated with the indicated dose of γ-ray irradiation under the same oxygen conditions as the

    Techniques Used: Staining, Marker, Irradiation, Clonogenic Cell Survival Assay, Viability Assay, Transfection

    Figure 4. SPINK1 accelerates tumor growth after radiotherapy. (A–C) DU145/EV and DU145/SPINK1 cells were cultured under the indicated oxygen conditions for 48 hours and subjected to qPCR (A) and the ELISA assay (B) or treated with the indicated dose of γ-ray irradiation for the clonogenic survival assay (C). (D and E) DU145/EV or SPINK1 xenografts were locally irradiated at a dose of 0 (solid lines) or 10 (dotted lines) Gy. When the volumes of the xenografts reached the same sizes as those on day 0, plasma SPINK1 levels were quantified by ELISA assays (D). Tumor growth was ana- lyzed after the treatment (E). Data are represented as mean ± SD (n = 3 in A and B, n = 6 in C, n = 5 in D, and n = 9–10 in E). Two-tailed Student’s t test. ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector.
    Figure Legend Snippet: Figure 4. SPINK1 accelerates tumor growth after radiotherapy. (A–C) DU145/EV and DU145/SPINK1 cells were cultured under the indicated oxygen conditions for 48 hours and subjected to qPCR (A) and the ELISA assay (B) or treated with the indicated dose of γ-ray irradiation for the clonogenic survival assay (C). (D and E) DU145/EV or SPINK1 xenografts were locally irradiated at a dose of 0 (solid lines) or 10 (dotted lines) Gy. When the volumes of the xenografts reached the same sizes as those on day 0, plasma SPINK1 levels were quantified by ELISA assays (D). Tumor growth was ana- lyzed after the treatment (E). Data are represented as mean ± SD (n = 3 in A and B, n = 6 in C, n = 5 in D, and n = 9–10 in E). Two-tailed Student’s t test. ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector.

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Irradiation, Clonogenic Cell Survival Assay, Clinical Proteomics, Two Tailed Test, Plasmid Preparation

    Figure 5. SPINK1 decreases radiation-induced DNA damage and enhances radioresistance of cancer cells in a EGFR-dependent manner. (A–D) Four days after being transfected with either pcDNA4/SPINK1 or its EV, DU145/EGFP-53BP1-M (A and B) and DU145 (C and D) cells were irradiated with 0 or 4 Gy of γ-rays in the presence or absence of EGFR-I III (D), and the DNA DSBs detected as EGFP-53BP1 foci (A and B) or as γH2AX foci (C and D) were analyzed 2 hours (A and B) or 15 minutes (C and D) after the radiation. (A) Immunocytochemical analysis. Green, EGFP-53BP1 foci; blue, counter staining using Hoechst 33342. Scale bar: 10 μm. (B–D) The number of foci increased by 4 Gy γ-IR was calculated by subtracting the number of foci at 0 Gy from that at 4 Gy under each condition and represented as dot plots with mean ± SD. (E and F) DU145 cells were irradiated with 0 or 4 Gy of γ-ray in the presence or absence of 100 ng/mL rSPINK1 in combination with DMSO or 0.5 μM EGFR-I III (E), or with control IgG or 10 μg/mL cetuximab (F), and subjected to colori- metric cell viability assays. (G) The same experiment as in Figure 3B was conducted in the presence or absence of EGFR-I III. Data are represented as mean (n > 1000 in B–D) and mean ± SD (n = 3 in E and F, and n = 6 in G). Two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector. EV, empty vector; EGFR-I III, EGFR Inhibitor III; DSBs, double-strand breaks.
    Figure Legend Snippet: Figure 5. SPINK1 decreases radiation-induced DNA damage and enhances radioresistance of cancer cells in a EGFR-dependent manner. (A–D) Four days after being transfected with either pcDNA4/SPINK1 or its EV, DU145/EGFP-53BP1-M (A and B) and DU145 (C and D) cells were irradiated with 0 or 4 Gy of γ-rays in the presence or absence of EGFR-I III (D), and the DNA DSBs detected as EGFP-53BP1 foci (A and B) or as γH2AX foci (C and D) were analyzed 2 hours (A and B) or 15 minutes (C and D) after the radiation. (A) Immunocytochemical analysis. Green, EGFP-53BP1 foci; blue, counter staining using Hoechst 33342. Scale bar: 10 μm. (B–D) The number of foci increased by 4 Gy γ-IR was calculated by subtracting the number of foci at 0 Gy from that at 4 Gy under each condition and represented as dot plots with mean ± SD. (E and F) DU145 cells were irradiated with 0 or 4 Gy of γ-ray in the presence or absence of 100 ng/mL rSPINK1 in combination with DMSO or 0.5 μM EGFR-I III (E), or with control IgG or 10 μg/mL cetuximab (F), and subjected to colori- metric cell viability assays. (G) The same experiment as in Figure 3B was conducted in the presence or absence of EGFR-I III. Data are represented as mean (n > 1000 in B–D) and mean ± SD (n = 3 in E and F, and n = 6 in G). Two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector. EV, empty vector; EGFR-I III, EGFR Inhibitor III; DSBs, double-strand breaks.

    Techniques Used: Transfection, Irradiation, Staining, Control, Two Tailed Test, Plasmid Preparation

    Figure 6. SPINK1 decreases radiation-induced DNA damage and enhances radioresistance of cancer cells in a NRF2-dependent manner. (A and B) Four days after being transfected with either pcDNA4/SPINK1 (SPINK1) or its EV, DU145 cells were subjected to qPCR. (C–I) HeLa/scramble cells and HeLa/shSPINK1-1, HeLa/shSPINK1-2, and HeLa/shSPINK1-3 cells were cultured under severe hypoxic conditions (O2 < 0.1%) for 48 hours, irradiated with 0 (C, D, and I) or 4 (E–I) Gy of γ-rays and subjected to qPCR (C–H) or the DCFDA cellular ROS assay (I). Cells were irradiated in the presence or absence of the EGFR-I III (G and H). (J) DU145 cells were irradiated with γ-rays in the presence or absence of 100 ng/mL rSPINK1 in combination with DMSO or 2 μM ML385 and subjected to the colorimetric cell viability assay. Data are represented as mean ± SD (n = 3 in A–J). Two-tailed Student’s t test (A, B, and J). One-way ANOVA with Dunnett’s test (C–I). *P < 0.05, **P < 0.01, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; DCFDA, dichlorodihydrofluorescein diacetate; EGFR-I III, EGFR Inhibitor III.
    Figure Legend Snippet: Figure 6. SPINK1 decreases radiation-induced DNA damage and enhances radioresistance of cancer cells in a NRF2-dependent manner. (A and B) Four days after being transfected with either pcDNA4/SPINK1 (SPINK1) or its EV, DU145 cells were subjected to qPCR. (C–I) HeLa/scramble cells and HeLa/shSPINK1-1, HeLa/shSPINK1-2, and HeLa/shSPINK1-3 cells were cultured under severe hypoxic conditions (O2 < 0.1%) for 48 hours, irradiated with 0 (C, D, and I) or 4 (E–I) Gy of γ-rays and subjected to qPCR (C–H) or the DCFDA cellular ROS assay (I). Cells were irradiated in the presence or absence of the EGFR-I III (G and H). (J) DU145 cells were irradiated with γ-rays in the presence or absence of 100 ng/mL rSPINK1 in combination with DMSO or 2 μM ML385 and subjected to the colorimetric cell viability assay. Data are represented as mean ± SD (n = 3 in A–J). Two-tailed Student’s t test (A, B, and J). One-way ANOVA with Dunnett’s test (C–I). *P < 0.05, **P < 0.01, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; DCFDA, dichlorodihydrofluorescein diacetate; EGFR-I III, EGFR Inhibitor III.

    Techniques Used: Transfection, Cell Culture, Irradiation, ROS Assay, Viability Assay, Two Tailed Test, Plasmid Preparation

    Figure 7. SPINK1 protein is expressed in severely hypoxic regions and secreted to the proximal regions of blood vessels. (A and B) Two pairs of serial sections of clinical human ccRCC tissues from 2 independent patients were stained with the indicated antibodies in A and B, respectively. Corresponding serial sections were stained with H&E. Scale bar: 50 μm. N, necrosis. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; ccRCC, clear cell renal cell carcinoma.
    Figure Legend Snippet: Figure 7. SPINK1 protein is expressed in severely hypoxic regions and secreted to the proximal regions of blood vessels. (A and B) Two pairs of serial sections of clinical human ccRCC tissues from 2 independent patients were stained with the indicated antibodies in A and B, respectively. Corresponding serial sections were stained with H&E. Scale bar: 50 μm. N, necrosis. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; ccRCC, clear cell renal cell carcinoma.

    Techniques Used: Staining, Plasmid Preparation

    Figure 8. SPINK1 in plasma reflects the degree of hypoxia within tumor tissue in vivo. (A) A scatter plot for correlation analysis between mRNA levels of SPINK1 and CA9 in 36 HeLa tumor xenografts showed a good coefficient of determination, R2 = 0.9458. (B and C) After blood flow to the HeLa tumor xeno- grafts was decreased by ligaturing the leg for the indicated times, levels of SPINK1 mRNA (B) and SPINK1 protein (C) in the tumor tissues were quantified by qPCR and the ELISA assay, respectively. (D–I) After anemia treatment by phenylhydrazine administration, mRNA levels of EPO in the kidneys (D and E) and those of CA9 (E–H) and SPINK1 (F) in tumor tissues were quantified by qPCR. SPINK1 protein levels in tumors (G) and plasma (H and I) and the tumor volume (I) were measured by the ELISA assay and digital calipers, respectively. Scatter plots for correlation analysis between the 2 indicated factors (E–I). Data are represented as mean ± SD (B–D; n = 36 in A, n = 9–10 in B and C, n = 5 in D–I). Two-tailed Student’s t test (D). One-way ANOVA with Dunnett’s test (B and C). *P < 0.05, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; CA9, carbonic anhydrase 9; EPO, erythropoietin.
    Figure Legend Snippet: Figure 8. SPINK1 in plasma reflects the degree of hypoxia within tumor tissue in vivo. (A) A scatter plot for correlation analysis between mRNA levels of SPINK1 and CA9 in 36 HeLa tumor xenografts showed a good coefficient of determination, R2 = 0.9458. (B and C) After blood flow to the HeLa tumor xeno- grafts was decreased by ligaturing the leg for the indicated times, levels of SPINK1 mRNA (B) and SPINK1 protein (C) in the tumor tissues were quantified by qPCR and the ELISA assay, respectively. (D–I) After anemia treatment by phenylhydrazine administration, mRNA levels of EPO in the kidneys (D and E) and those of CA9 (E–H) and SPINK1 (F) in tumor tissues were quantified by qPCR. SPINK1 protein levels in tumors (G) and plasma (H and I) and the tumor volume (I) were measured by the ELISA assay and digital calipers, respectively. Scatter plots for correlation analysis between the 2 indicated factors (E–I). Data are represented as mean ± SD (B–D; n = 36 in A, n = 9–10 in B and C, n = 5 in D–I). Two-tailed Student’s t test (D). One-way ANOVA with Dunnett’s test (B and C). *P < 0.05, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; CA9, carbonic anhydrase 9; EPO, erythropoietin.

    Techniques Used: Clinical Proteomics, In Vivo, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Plasmid Preparation



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    Figure 3. <t>SPINK1</t> secreted from cells induces cancer radioresistance in a paracrine manner. (A) HeLa tumor xenografts were stained with antibodies against a hypoxia marker, pimonidazole (green), or SPINK1 (red). Blue, counter staining with Hoechst 33342. The dotted line represents the outside edge of the pimonidazole-positive regions. Scale bar: 50 μm. (B–E) After 24 hours serum starvation, DU145 cells were precultured in the presence or absence of 100 ng/mL rSPINK1 for 24 hours, treated with the indicated dose of γ-ray irradiation, and subjected to the clonogenic survival assay (B), col- orimetric cell viability assay (C), and FACS analysis for the cell cycle status (D) and sub-G1 fraction (E). The cells were precultured and irradiated under the indicated oxygen conditions in C. (F and G) After transfection with either pcDNA4/SPINK1 (SPINK1) or its EV, the indicated cells were precultured under mild hypoxic conditions (O2 = 3%) for 48 hours, treated with the indicated dose of γ-ray irradiation under the same oxygen conditions as the
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    Figure 3. <t>SPINK1</t> secreted from cells induces cancer radioresistance in a paracrine manner. (A) HeLa tumor xenografts were stained with antibodies against a hypoxia marker, pimonidazole (green), or SPINK1 (red). Blue, counter staining with Hoechst 33342. The dotted line represents the outside edge of the pimonidazole-positive regions. Scale bar: 50 μm. (B–E) After 24 hours serum starvation, DU145 cells were precultured in the presence or absence of 100 ng/mL rSPINK1 for 24 hours, treated with the indicated dose of γ-ray irradiation, and subjected to the clonogenic survival assay (B), col- orimetric cell viability assay (C), and FACS analysis for the cell cycle status (D) and sub-G1 fraction (E). The cells were precultured and irradiated under the indicated oxygen conditions in C. (F and G) After transfection with either pcDNA4/SPINK1 (SPINK1) or its EV, the indicated cells were precultured under mild hypoxic conditions (O2 = 3%) for 48 hours, treated with the indicated dose of γ-ray irradiation under the same oxygen conditions as the
    Spink1 Protein Levels, supplied by Boster Bio, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Figure 3. SPINK1 secreted from cells induces cancer radioresistance in a paracrine manner. (A) HeLa tumor xenografts were stained with antibodies against a hypoxia marker, pimonidazole (green), or SPINK1 (red). Blue, counter staining with Hoechst 33342. The dotted line represents the outside edge of the pimonidazole-positive regions. Scale bar: 50 μm. (B–E) After 24 hours serum starvation, DU145 cells were precultured in the presence or absence of 100 ng/mL rSPINK1 for 24 hours, treated with the indicated dose of γ-ray irradiation, and subjected to the clonogenic survival assay (B), col- orimetric cell viability assay (C), and FACS analysis for the cell cycle status (D) and sub-G1 fraction (E). The cells were precultured and irradiated under the indicated oxygen conditions in C. (F and G) After transfection with either pcDNA4/SPINK1 (SPINK1) or its EV, the indicated cells were precultured under mild hypoxic conditions (O2 = 3%) for 48 hours, treated with the indicated dose of γ-ray irradiation under the same oxygen conditions as the

    Journal: JCI insight

    Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization.

    doi: 10.1172/jci.insight.148135

    Figure Lengend Snippet: Figure 3. SPINK1 secreted from cells induces cancer radioresistance in a paracrine manner. (A) HeLa tumor xenografts were stained with antibodies against a hypoxia marker, pimonidazole (green), or SPINK1 (red). Blue, counter staining with Hoechst 33342. The dotted line represents the outside edge of the pimonidazole-positive regions. Scale bar: 50 μm. (B–E) After 24 hours serum starvation, DU145 cells were precultured in the presence or absence of 100 ng/mL rSPINK1 for 24 hours, treated with the indicated dose of γ-ray irradiation, and subjected to the clonogenic survival assay (B), col- orimetric cell viability assay (C), and FACS analysis for the cell cycle status (D) and sub-G1 fraction (E). The cells were precultured and irradiated under the indicated oxygen conditions in C. (F and G) After transfection with either pcDNA4/SPINK1 (SPINK1) or its EV, the indicated cells were precultured under mild hypoxic conditions (O2 = 3%) for 48 hours, treated with the indicated dose of γ-ray irradiation under the same oxygen conditions as the

    Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

    Techniques: Staining, Marker, Irradiation, Clonogenic Cell Survival Assay, Viability Assay, Transfection

    Figure 4. SPINK1 accelerates tumor growth after radiotherapy. (A–C) DU145/EV and DU145/SPINK1 cells were cultured under the indicated oxygen conditions for 48 hours and subjected to qPCR (A) and the ELISA assay (B) or treated with the indicated dose of γ-ray irradiation for the clonogenic survival assay (C). (D and E) DU145/EV or SPINK1 xenografts were locally irradiated at a dose of 0 (solid lines) or 10 (dotted lines) Gy. When the volumes of the xenografts reached the same sizes as those on day 0, plasma SPINK1 levels were quantified by ELISA assays (D). Tumor growth was ana- lyzed after the treatment (E). Data are represented as mean ± SD (n = 3 in A and B, n = 6 in C, n = 5 in D, and n = 9–10 in E). Two-tailed Student’s t test. ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector.

    Journal: JCI insight

    Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization.

    doi: 10.1172/jci.insight.148135

    Figure Lengend Snippet: Figure 4. SPINK1 accelerates tumor growth after radiotherapy. (A–C) DU145/EV and DU145/SPINK1 cells were cultured under the indicated oxygen conditions for 48 hours and subjected to qPCR (A) and the ELISA assay (B) or treated with the indicated dose of γ-ray irradiation for the clonogenic survival assay (C). (D and E) DU145/EV or SPINK1 xenografts were locally irradiated at a dose of 0 (solid lines) or 10 (dotted lines) Gy. When the volumes of the xenografts reached the same sizes as those on day 0, plasma SPINK1 levels were quantified by ELISA assays (D). Tumor growth was ana- lyzed after the treatment (E). Data are represented as mean ± SD (n = 3 in A and B, n = 6 in C, n = 5 in D, and n = 9–10 in E). Two-tailed Student’s t test. ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector.

    Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Irradiation, Clonogenic Cell Survival Assay, Clinical Proteomics, Two Tailed Test, Plasmid Preparation

    Figure 5. SPINK1 decreases radiation-induced DNA damage and enhances radioresistance of cancer cells in a EGFR-dependent manner. (A–D) Four days after being transfected with either pcDNA4/SPINK1 or its EV, DU145/EGFP-53BP1-M (A and B) and DU145 (C and D) cells were irradiated with 0 or 4 Gy of γ-rays in the presence or absence of EGFR-I III (D), and the DNA DSBs detected as EGFP-53BP1 foci (A and B) or as γH2AX foci (C and D) were analyzed 2 hours (A and B) or 15 minutes (C and D) after the radiation. (A) Immunocytochemical analysis. Green, EGFP-53BP1 foci; blue, counter staining using Hoechst 33342. Scale bar: 10 μm. (B–D) The number of foci increased by 4 Gy γ-IR was calculated by subtracting the number of foci at 0 Gy from that at 4 Gy under each condition and represented as dot plots with mean ± SD. (E and F) DU145 cells were irradiated with 0 or 4 Gy of γ-ray in the presence or absence of 100 ng/mL rSPINK1 in combination with DMSO or 0.5 μM EGFR-I III (E), or with control IgG or 10 μg/mL cetuximab (F), and subjected to colori- metric cell viability assays. (G) The same experiment as in Figure 3B was conducted in the presence or absence of EGFR-I III. Data are represented as mean (n > 1000 in B–D) and mean ± SD (n = 3 in E and F, and n = 6 in G). Two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector. EV, empty vector; EGFR-I III, EGFR Inhibitor III; DSBs, double-strand breaks.

    Journal: JCI insight

    Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization.

    doi: 10.1172/jci.insight.148135

    Figure Lengend Snippet: Figure 5. SPINK1 decreases radiation-induced DNA damage and enhances radioresistance of cancer cells in a EGFR-dependent manner. (A–D) Four days after being transfected with either pcDNA4/SPINK1 or its EV, DU145/EGFP-53BP1-M (A and B) and DU145 (C and D) cells were irradiated with 0 or 4 Gy of γ-rays in the presence or absence of EGFR-I III (D), and the DNA DSBs detected as EGFP-53BP1 foci (A and B) or as γH2AX foci (C and D) were analyzed 2 hours (A and B) or 15 minutes (C and D) after the radiation. (A) Immunocytochemical analysis. Green, EGFP-53BP1 foci; blue, counter staining using Hoechst 33342. Scale bar: 10 μm. (B–D) The number of foci increased by 4 Gy γ-IR was calculated by subtracting the number of foci at 0 Gy from that at 4 Gy under each condition and represented as dot plots with mean ± SD. (E and F) DU145 cells were irradiated with 0 or 4 Gy of γ-ray in the presence or absence of 100 ng/mL rSPINK1 in combination with DMSO or 0.5 μM EGFR-I III (E), or with control IgG or 10 μg/mL cetuximab (F), and subjected to colori- metric cell viability assays. (G) The same experiment as in Figure 3B was conducted in the presence or absence of EGFR-I III. Data are represented as mean (n > 1000 in B–D) and mean ± SD (n = 3 in E and F, and n = 6 in G). Two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector. EV, empty vector; EGFR-I III, EGFR Inhibitor III; DSBs, double-strand breaks.

    Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

    Techniques: Transfection, Irradiation, Staining, Control, Two Tailed Test, Plasmid Preparation

    Figure 6. SPINK1 decreases radiation-induced DNA damage and enhances radioresistance of cancer cells in a NRF2-dependent manner. (A and B) Four days after being transfected with either pcDNA4/SPINK1 (SPINK1) or its EV, DU145 cells were subjected to qPCR. (C–I) HeLa/scramble cells and HeLa/shSPINK1-1, HeLa/shSPINK1-2, and HeLa/shSPINK1-3 cells were cultured under severe hypoxic conditions (O2 < 0.1%) for 48 hours, irradiated with 0 (C, D, and I) or 4 (E–I) Gy of γ-rays and subjected to qPCR (C–H) or the DCFDA cellular ROS assay (I). Cells were irradiated in the presence or absence of the EGFR-I III (G and H). (J) DU145 cells were irradiated with γ-rays in the presence or absence of 100 ng/mL rSPINK1 in combination with DMSO or 2 μM ML385 and subjected to the colorimetric cell viability assay. Data are represented as mean ± SD (n = 3 in A–J). Two-tailed Student’s t test (A, B, and J). One-way ANOVA with Dunnett’s test (C–I). *P < 0.05, **P < 0.01, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; DCFDA, dichlorodihydrofluorescein diacetate; EGFR-I III, EGFR Inhibitor III.

    Journal: JCI insight

    Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization.

    doi: 10.1172/jci.insight.148135

    Figure Lengend Snippet: Figure 6. SPINK1 decreases radiation-induced DNA damage and enhances radioresistance of cancer cells in a NRF2-dependent manner. (A and B) Four days after being transfected with either pcDNA4/SPINK1 (SPINK1) or its EV, DU145 cells were subjected to qPCR. (C–I) HeLa/scramble cells and HeLa/shSPINK1-1, HeLa/shSPINK1-2, and HeLa/shSPINK1-3 cells were cultured under severe hypoxic conditions (O2 < 0.1%) for 48 hours, irradiated with 0 (C, D, and I) or 4 (E–I) Gy of γ-rays and subjected to qPCR (C–H) or the DCFDA cellular ROS assay (I). Cells were irradiated in the presence or absence of the EGFR-I III (G and H). (J) DU145 cells were irradiated with γ-rays in the presence or absence of 100 ng/mL rSPINK1 in combination with DMSO or 2 μM ML385 and subjected to the colorimetric cell viability assay. Data are represented as mean ± SD (n = 3 in A–J). Two-tailed Student’s t test (A, B, and J). One-way ANOVA with Dunnett’s test (C–I). *P < 0.05, **P < 0.01, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; DCFDA, dichlorodihydrofluorescein diacetate; EGFR-I III, EGFR Inhibitor III.

    Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

    Techniques: Transfection, Cell Culture, Irradiation, ROS Assay, Viability Assay, Two Tailed Test, Plasmid Preparation

    Figure 7. SPINK1 protein is expressed in severely hypoxic regions and secreted to the proximal regions of blood vessels. (A and B) Two pairs of serial sections of clinical human ccRCC tissues from 2 independent patients were stained with the indicated antibodies in A and B, respectively. Corresponding serial sections were stained with H&E. Scale bar: 50 μm. N, necrosis. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; ccRCC, clear cell renal cell carcinoma.

    Journal: JCI insight

    Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization.

    doi: 10.1172/jci.insight.148135

    Figure Lengend Snippet: Figure 7. SPINK1 protein is expressed in severely hypoxic regions and secreted to the proximal regions of blood vessels. (A and B) Two pairs of serial sections of clinical human ccRCC tissues from 2 independent patients were stained with the indicated antibodies in A and B, respectively. Corresponding serial sections were stained with H&E. Scale bar: 50 μm. N, necrosis. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; ccRCC, clear cell renal cell carcinoma.

    Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

    Techniques: Staining, Plasmid Preparation

    Figure 8. SPINK1 in plasma reflects the degree of hypoxia within tumor tissue in vivo. (A) A scatter plot for correlation analysis between mRNA levels of SPINK1 and CA9 in 36 HeLa tumor xenografts showed a good coefficient of determination, R2 = 0.9458. (B and C) After blood flow to the HeLa tumor xeno- grafts was decreased by ligaturing the leg for the indicated times, levels of SPINK1 mRNA (B) and SPINK1 protein (C) in the tumor tissues were quantified by qPCR and the ELISA assay, respectively. (D–I) After anemia treatment by phenylhydrazine administration, mRNA levels of EPO in the kidneys (D and E) and those of CA9 (E–H) and SPINK1 (F) in tumor tissues were quantified by qPCR. SPINK1 protein levels in tumors (G) and plasma (H and I) and the tumor volume (I) were measured by the ELISA assay and digital calipers, respectively. Scatter plots for correlation analysis between the 2 indicated factors (E–I). Data are represented as mean ± SD (B–D; n = 36 in A, n = 9–10 in B and C, n = 5 in D–I). Two-tailed Student’s t test (D). One-way ANOVA with Dunnett’s test (B and C). *P < 0.05, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; CA9, carbonic anhydrase 9; EPO, erythropoietin.

    Journal: JCI insight

    Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization.

    doi: 10.1172/jci.insight.148135

    Figure Lengend Snippet: Figure 8. SPINK1 in plasma reflects the degree of hypoxia within tumor tissue in vivo. (A) A scatter plot for correlation analysis between mRNA levels of SPINK1 and CA9 in 36 HeLa tumor xenografts showed a good coefficient of determination, R2 = 0.9458. (B and C) After blood flow to the HeLa tumor xeno- grafts was decreased by ligaturing the leg for the indicated times, levels of SPINK1 mRNA (B) and SPINK1 protein (C) in the tumor tissues were quantified by qPCR and the ELISA assay, respectively. (D–I) After anemia treatment by phenylhydrazine administration, mRNA levels of EPO in the kidneys (D and E) and those of CA9 (E–H) and SPINK1 (F) in tumor tissues were quantified by qPCR. SPINK1 protein levels in tumors (G) and plasma (H and I) and the tumor volume (I) were measured by the ELISA assay and digital calipers, respectively. Scatter plots for correlation analysis between the 2 indicated factors (E–I). Data are represented as mean ± SD (B–D; n = 36 in A, n = 9–10 in B and C, n = 5 in D–I). Two-tailed Student’s t test (D). One-way ANOVA with Dunnett’s test (B and C). *P < 0.05, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; CA9, carbonic anhydrase 9; EPO, erythropoietin.

    Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

    Techniques: Clinical Proteomics, In Vivo, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Plasmid Preparation