Journal: Cell reports

Article Title: Collagen 1-mediated CXCL1 secretion in tumor cells activates fibroblasts to promote radioresistance of esophageal cancer.

doi: 10.1016/j.celrep.2023.113270

Figure Lengend Snippet: Figure 4. CXCL1 promotes migration and activation of fibroblasts via the CXCR2-STAT3 pathway (A) Western blot analysis of myCAF- and iCAF-related markers in fibroblasts incubated with CM from CXCL1-knockdown ESCC cells. (B) Quantification of migration in (Figure S5B) (n = 3 biological replicates). (C) Western blot analysis of myCAF- and iCAF-related markers in fibroblasts treated with indicated concentration of rCXCL1 proteins for 24 h. (D) Representative images of fibroblasts migration caused by indicated concentration of rCXCL1 proteins. Scale bar, 100 mm. (E) Quantification of migration in (D) (n = 3 biological replicates). (F) Western blot analysis of CAF markers in fibroblasts pre-treated with SB-265610 for 6 h and then treated by CM of KYSE510 for 18 h. KYSE510 cells were treated with Col1 or not. (G) Western blot analysis of CAF markers in fibroblasts pre-treated with SB-265610 for 6 h and then treated by rCXCL1 for 18 h. For all panels, data are presented as mean ± SD. **p < 0.01, ***p < 0.001, and ****p < 0.0001, as determined using two-tailed Student’s t test. Each assay for western blot had three biological repeats, and the quantification results are presented below each band. See also Figure S5.

Article Snippet: For CXCR2 receptor inhibitory assay, CXCR2 inhibitor SB-265610 (MCE, HY-50688) was added to serum-free medium for 6 h after fibroblasts grew until confluency, then CXCL1 recombinant protein was added to medium for another 18 h.

Techniques: Migration, Activation Assay, Western Blot, Incubation, Knockdown, Concentration Assay, Two Tailed Test

Journal: Hepatology Communications

Article Title: Multiple cell-type interactions drive invariant NKT cell hepatitis

doi: 10.1097/HC9.0000000000000592

Figure Lengend Snippet: CXCR2 signaling inhibitor reduces α-Galcer–induced neutrophil accumulation, thus ameliorating liver injury. The mice were injected with CXCR2 signaling inhibitor AZD5069 or vehicle 3 hours before α-Galcer treatment. Serum and liver tissue samples were collected after α-Galcer treatment for 16 hours. (A) The levels of ALT and AST in serum were measured. (B) The degree of inflammatory cell infiltration in the liver was analyzed by H&E staining. (C) Liver tissues were subjected to MPO immunostaining, and the quantitation of MPO + cells per field was determined. Representative images are shown. (D) The levels of MDA and 4-HNE were determined by immunohistochemical staining. Representative images are shown. The quantitation of MDA + and 4-HNE + area per field was determined. (E) The mRNA expression of several genes in the liver, including IFN-γ, TNF-α, IL-1β, and IL-6, were analyzed by RT-qPCR. Values represent means±SD. * p <0.05, ** p <0.01, *** p <0.001. Abbreviations: α-Galcer, α-Galactosylceramide; 4-HNE, 4-hydroxynonenal; H&E, hematoxylin-eosin; IFN-γ, interferon γ; MDA, malondialdehyde; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: To inhibit different receptor-mediated signaling, the mice were treated with CXCR2 (C-X-C Motif receptor 2) inhibitor AZD5069 (10 mg/kg, MCE, Cat No. HY-19855) at 3 hours before α-Galcer treatment.

Techniques: Injection, Staining, Immunostaining, Quantitation Assay, Immunohistochemical staining, Expressing, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: CAF-secreted CXCL1 conferred radioresistance by regulating DNA damage response in a ROS-dependent manner in esophageal squamous cell carcinoma

doi: 10.1038/cddis.2017.180

Figure Lengend Snippet: Human chemokine CXCL1 was expressed in an autocrine/paracrine signaling loop. ( a ) CXCL1 was highly expressed in CAF medium compared with in matched NF medium as determined with human cytokine/chemokine array. ( b ) The fold change of CXCL1 mRNA level in different cells by qRT-PCR analysis. ( c ) The concentration of CXCL1 in culture medium of KYSE-30, KYSE-150, CAF-1, NF-1, CAF-2 and NF-2 by ELISA. ( d and e ) The mRNA level changes of CXCL1 and its receptor CXCR2 after 8 Gy of radiation in KYSE-30 and in KYSE-150 by qRT-PCR analysis. * P <0.05, ** P <0.01, compared with CXCL1 mRNA level in KYSE-30 or KYSE-150 before radiation. # P <0.05, compared with CXCR2 mRNA level in KYSE-30 or KYSE-150 before radiation. ( f ) The fold change of CXCL1 mRNA level in KYSE-30 and KYSE-150 that were cultured in CAF medium for 24 h compared with cultured in normal medium, and in CAF-1 and in CAF-2 that were cultured in the CM of KYSE-30 or KYSE-150 for 24 h compared with cultured in normal medium by qRT-PCR analysis. Negative control (NC): the cells that were transfected with scrambled siRNA. * P <0.05, compared with CXCL1 mRNA level in NC that was transfected with scrambled siRNA before cultured in normal medium for 24 h

Article Snippet: The inhibitor of CXCL1 receptor CXCR2 SB225002 (#S7651), ATM kinase inhibitor Ku55933 (#S1092) and Mek1/2 kinase inhibitor U0126 (#S1102) were purchased from Selleck (Houston, TX, USA).

Techniques: Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Negative Control, Transfection

Journal: Cell Death & Disease

Article Title: CAF-secreted CXCL1 conferred radioresistance by regulating DNA damage response in a ROS-dependent manner in esophageal squamous cell carcinoma

doi: 10.1038/cddis.2017.180

Figure Lengend Snippet: CXCL1 conferred radioresistance by enhancement of DNA damage repair. ( a and b ) The determination of the radiosensitivity of KYSE-150 and KYSE-30 that were cultured in normal medium or in CAF medium with or without 500 ng/ml CXCL1 antibody for 24 h by clonogenic survival assay. * P <0.05, compared with cultured in CAF medium with 500 ng/ml CXCL1 antibody. # P <0.05, compared with cultured in normal medium. Ab, 500 ng/ml CXCL1 antibody. ( c ) The expressions of cellular DNA damage repair proteins including γ -H2AX, p-ATM, Rad50, p-Chk2 and DNA-PKcs 30 min after radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium for 24 h, by western blotting analysis. GAPDH was used as a loading control. ( d ) The expressions of γ -H2AX, p-ATM, Rad50, p-Rad50, p-Chk2, Ku80, DNA-PKcs and p-DNA-PKcs 30 min after radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium with or without 500 ng/ml CXCL1 antibody for 24 h, by western blotting analysis. GAPDH was used as a loading control. ( e ) The expressions of p-ATM and γ -H2AX 30 min after radiation in KYSE-150 and KYSE-30 that were cultured in normal medium or in CAF medium with or without 10 μ M ATM kinase inhibitor Ku55933 for 24 h, by western blotting analysis. GAPDH was used as a loading control

Article Snippet: The inhibitor of CXCL1 receptor CXCR2 SB225002 (#S7651), ATM kinase inhibitor Ku55933 (#S1092) and Mek1/2 kinase inhibitor U0126 (#S1102) were purchased from Selleck (Houston, TX, USA).

Techniques: Cell Culture, Clonogenic Cell Survival Assay, Western Blot, Control

Journal: Cell Death & Disease

Article Title: CAF-secreted CXCL1 conferred radioresistance by regulating DNA damage response in a ROS-dependent manner in esophageal squamous cell carcinoma

doi: 10.1038/cddis.2017.180

Figure Lengend Snippet: CXCL1 enhanced DNA damage repair in a SOD1–ROS-axis-dependent manner. ( a and b ) The ROS level following radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or CAF medium with or without 500 ng/ml CXCL1 antibody or 400 nM CXCR2 inhibitor SB225002 for 24 h, by immunofluorescence analysis. Magnification: × 10. ( c and d ) The fold change of SOD1 mRNA level before or after radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium with or without 500 ng/ml CXCL1 antibody or 400 nM CXCR2 inhibitor SB225002 for 24 h, by qRT-PCR analysis. * P <0.05, ** P <0.01. ( e ) The expression of SOD1 protein following radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium with or without 500 ng/ml CXCL1 antibody for 24 h, by western blotting analysis. GAPDH was used as a loading control. ( f ) The expressions of γ -H2AX, p-ATM, Rad50, p-Rad50, p-Chk2, Ku80, DNA-PKcs, p-DNA-PKcs and SOD1 following radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium with or without 100 ng/ml human SOD1 protein for 24 h, by western blotting analysis. GAPDH was used as a loading control

Article Snippet: The inhibitor of CXCL1 receptor CXCR2 SB225002 (#S7651), ATM kinase inhibitor Ku55933 (#S1092) and Mek1/2 kinase inhibitor U0126 (#S1102) were purchased from Selleck (Houston, TX, USA).

Techniques: Cell Culture, Immunofluorescence, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: Cell Death & Disease

Article Title: CAF-secreted CXCL1 conferred radioresistance by regulating DNA damage response in a ROS-dependent manner in esophageal squamous cell carcinoma

doi: 10.1038/cddis.2017.180

Figure Lengend Snippet: CXCL1 conferred radioresistance by activation of Mek/Erk signaling pathway. ( a ) The expressions of Erk1/2, p-Erk1/2 and p-Mek1/2 following radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium for 24 h, by western blotting analysis. GAPDH was used as a loading control. ( b ) The expressions of Erk1/2, p-Erk1/2 and p-Mek1/2 following radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium with or without 500 ng/ml CXCL1 antibody for 24 h, by western blotting analysis. GAPDH was used as a loading control. ( c ) The expressions of Erk1/2, p-Erk1/2, p-Mek1/2 and γ -H2AX following radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium with or without 10 μ M Mek1/2 kinase inhibitor U0126 for 24 h, by western blotting analysis. GAPDH was used as a loading control. ( d ) The radiosensitivity of KYSE-150 and KYSE-30 that were cultured in normal medium or in CAF medium with or without 10 μ M Mek1/2 kinase inhibitor U0126 for 24 h as determined by clonogenic survival assay. * P <0.05, compared with cultured in CAF medium with 10 μ M Mek1/2 kinase inhibitor U0126. # P <0.05, compared with cultured in normal medium without 10 μ M Mek1/2 kinase inhibitor U0126

Article Snippet: The inhibitor of CXCL1 receptor CXCR2 SB225002 (#S7651), ATM kinase inhibitor Ku55933 (#S1092) and Mek1/2 kinase inhibitor U0126 (#S1102) were purchased from Selleck (Houston, TX, USA).

Techniques: Activation Assay, Cell Culture, Western Blot, Control, Clonogenic Cell Survival Assay

Journal: Cell Death & Disease

Article Title: CAF-secreted CXCL1 conferred radioresistance by regulating DNA damage response in a ROS-dependent manner in esophageal squamous cell carcinoma

doi: 10.1038/cddis.2017.180

Figure Lengend Snippet: Blockage of CAF-secreted CXCL1 reversed radioresistance of xenograft tumor models. ( a and b ) The growth curve of xenograft tumors implanted with KYSE-150 or KYSE-30 alone or combined with CAFs after treatment with tumor injection of 1 μ g/ml CXCL1 antibody, fractionated radiation at a total dose of 12 Gy alone or their combinations. Tumors treated with only PBS was chosen as a control. ( c and d ) The weight of excised xenograft tumors in ( a ) and ( b ) at the end of experiment at day 28. ( e and f ) The photos of excised xenograft tumors in ( a ) and ( b ) at the end of experiment at day 28

Article Snippet: The inhibitor of CXCL1 receptor CXCR2 SB225002 (#S7651), ATM kinase inhibitor Ku55933 (#S1092) and Mek1/2 kinase inhibitor U0126 (#S1102) were purchased from Selleck (Houston, TX, USA).

Techniques: Injection, Control

Journal: Cell Death & Disease

Article Title: CAF-secreted CXCL1 conferred radioresistance by regulating DNA damage response in a ROS-dependent manner in esophageal squamous cell carcinoma

doi: 10.1038/cddis.2017.180

Figure Lengend Snippet: CXCL1 expressed in CAFs is an independent prognostic factor of ESCC patients treated with chemoradiotherapy. ( a ) The concentration of CXCL1 in plasma of ESCC patients ( n =35) and in healthy controls ( n =29) by ELISA. P =0.04207. ( b ) Kaplan–Meier analysis showed overall survival of ESCC patients with positive CXCL1 expression in CAFs ( n =75) was significantly poorer than those CXCL1-negative patients in CAFs ( n =66) after chemoradiotherapy. The grading of CXCL1 expression was described in ‘Materials and Methods’. ( c ) One representative result of CXCL1 expression in CAFs and in tumor tissues in 141 ESCC patients by IHC analysis. CD31 and vimentin were used for staining blood vessels and fibroblasts, respectively

Article Snippet: The inhibitor of CXCL1 receptor CXCR2 SB225002 (#S7651), ATM kinase inhibitor Ku55933 (#S1092) and Mek1/2 kinase inhibitor U0126 (#S1102) were purchased from Selleck (Houston, TX, USA).

Techniques: Concentration Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Expressing, Staining

Journal: Cell Death & Disease

Article Title: CAF-secreted CXCL1 conferred radioresistance by regulating DNA damage response in a ROS-dependent manner in esophageal squamous cell carcinoma

doi: 10.1038/cddis.2017.180

Figure Lengend Snippet: The association of CXCL1 expression in CAFs and clinicopathological characteristics of ESCC patients

Article Snippet: The inhibitor of CXCL1 receptor CXCR2 SB225002 (#S7651), ATM kinase inhibitor Ku55933 (#S1092) and Mek1/2 kinase inhibitor U0126 (#S1102) were purchased from Selleck (Houston, TX, USA).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: CAF-secreted CXCL1 conferred radioresistance by regulating DNA damage response in a ROS-dependent manner in esophageal squamous cell carcinoma

doi: 10.1038/cddis.2017.180

Figure Lengend Snippet: The association of clinicopathological characteristics with overall survival of ESCC patients treated with chemoradiotherapy

Article Snippet: The inhibitor of CXCL1 receptor CXCR2 SB225002 (#S7651), ATM kinase inhibitor Ku55933 (#S1092) and Mek1/2 kinase inhibitor U0126 (#S1102) were purchased from Selleck (Houston, TX, USA).

Techniques: Expressing