cxcl1 proteintech  (Proteintech)

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: REAGENT or RESOURCE SOURCE IDENTIFIER Fetal Bovine Serum Cell Technologies Cat# 30070 DMEM Corning Cat# 10-013-CV Advanced DMEM/F12 Gibco Cat# 12634028 Puromycin Gibco Cat# A1113803 Lipofectamine 2000 Invitrogen Cat# 11668027 Collagenase I Gibco Cat# 17100017 Collagenase IV Gibco Cat# 17104019 Hyaluronidase Sigma-Aldrich Cat# H1115000 EDHB Sigma-Aldrich Cat# E24859 MK-2206 MCE Cat# HY-108232 SB-265610 MCE Cat# HY-50688 Human recombinant CXCL1 MCE Cat# HY-P70508 Rat tail collagen type 1 Solarbio Cat# C8062 Critical commercial assays RNA-Quick Purification Kit ES Science Cat# RN001 TB Green Premix Ex TaqTMII (Tli RNaseH Plus) TaKaRa Cat# RR820A Masson’s Trichrome Stain Kit Solarbio Cat# G1340 Meilunbio FGSuper Sensitive ECL Luminescence Reagent Meilunbio Cat# MA0186-1 PierceTM Rapid Gold BCA Thermo Fisher Scientific Cat# A53226 Human Cytokine Array GS440 Raybiotech Cat# GSH-CAA-440-1 CXCL1 ELISA Kit Proteintech Cat# KE00133 Opal 5-Color Manual IHC Kit PANOVUE Cat# 10144100100 NF-kB Activation Nuclear Translocation Assay Kit Beyotime Cat# SN368 Deposited data ESCC WES and RNA-seq data of 19 PDX donors This paper GSA-Human: HRA004329 Single-cell RNA-seq raw data of 6 PDXmice This paper GSA: https://ngdc.cncb.ac.cn/gsa/browse/ CRA010501 Experimental models: Cell lines KYSE450 Dr. Y. Shimada JCRB1430 KYSE510 Dr. Y. Shimada JCRB1436 NIH/3T3 ATCC CRL-1658 Human derived esophagus normal fibroblast (NF) This paper N/A PDX-derived primary tumor cells (PDC) This paper N/A Experimental models: Organisms/strains NSG mouse IDMO N/A Oligonucleotides shRNA targeting sequences, see Table S3 JTSBIO Co., Ltd N/A siRNA, see Table S3 JTSBIO Co., Ltd N/A qRT-PCR primers, see Table S4 Tsingke Biotechnology N/A Software and algorithms R 4.2.2 R Core Team https://www.R-project.org/ Cell Ranger 6.0.2 10x Genomics https://10xgenomics.com/ Seurat (version 4.3.0) Hao et al.46 https://github.com/satijalab/seurat (Continued on next page) 18 Cell Reports 42, 113270, October 31, 2023

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

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 5. CXCL1 affects radiosensitivity in vivo (A) Tumor growth curves and excised tumor images of control and CXCL1-knockdown tumors with or without IR treatment (n = 4 or 5 per group). TGI, percentage of tumor growth inhibition. (B) Representative mIF images of CXCL1, KRT6A, and aSMA in control and CXCL1-knockdown tumors without IR. Scale bar, 100 mm. (C and D) Quantification of CXCL1- and aSMA-positive area (n = 4 or 5 per group). (E) Tumor growth curves and excised tumor images of vector and CXCL1 overexpression tumors with or without IR treatment. Data are presented as mean ± SD (n = 5 per group). (F) Representative mIF images of CXCL1, KRT6A, and aSMA in control (vector) and CXCL1 overexpression (OE) tumors without IR. Scale bar, 100 mm. (G and H) Quantification of CXCL1- and aSMA-positive area (n = 5 per group). For all panels, data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, as determined using two-tailed Student’s t test. See also Figure S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Fetal Bovine Serum Cell Technologies Cat# 30070 DMEM Corning Cat# 10-013-CV Advanced DMEM/F12 Gibco Cat# 12634028 Puromycin Gibco Cat# A1113803 Lipofectamine 2000 Invitrogen Cat# 11668027 Collagenase I Gibco Cat# 17100017 Collagenase IV Gibco Cat# 17104019 Hyaluronidase Sigma-Aldrich Cat# H1115000 EDHB Sigma-Aldrich Cat# E24859 MK-2206 MCE Cat# HY-108232 SB-265610 MCE Cat# HY-50688 Human recombinant CXCL1 MCE Cat# HY-P70508 Rat tail collagen type 1 Solarbio Cat# C8062 Critical commercial assays RNA-Quick Purification Kit ES Science Cat# RN001 TB Green Premix Ex TaqTMII (Tli RNaseH Plus) TaKaRa Cat# RR820A Masson’s Trichrome Stain Kit Solarbio Cat# G1340 Meilunbio FGSuper Sensitive ECL Luminescence Reagent Meilunbio Cat# MA0186-1 PierceTM Rapid Gold BCA Thermo Fisher Scientific Cat# A53226 Human Cytokine Array GS440 Raybiotech Cat# GSH-CAA-440-1 CXCL1 ELISA Kit Proteintech Cat# KE00133 Opal 5-Color Manual IHC Kit PANOVUE Cat# 10144100100 NF-kB Activation Nuclear Translocation Assay Kit Beyotime Cat# SN368 Deposited data ESCC WES and RNA-seq data of 19 PDX donors This paper GSA-Human: HRA004329 Single-cell RNA-seq raw data of 6 PDXmice This paper GSA: https://ngdc.cncb.ac.cn/gsa/browse/ CRA010501 Experimental models: Cell lines KYSE450 Dr. Y. Shimada JCRB1430 KYSE510 Dr. Y. Shimada JCRB1436 NIH/3T3 ATCC CRL-1658 Human derived esophagus normal fibroblast (NF) This paper N/A PDX-derived primary tumor cells (PDC) This paper N/A Experimental models: Organisms/strains NSG mouse IDMO N/A Oligonucleotides shRNA targeting sequences, see Table S3 JTSBIO Co., Ltd N/A siRNA, see Table S3 JTSBIO Co., Ltd N/A qRT-PCR primers, see Table S4 Tsingke Biotechnology N/A Software and algorithms R 4.2.2 R Core Team https://www.R-project.org/ Cell Ranger 6.0.2 10x Genomics https://10xgenomics.com/ Seurat (version 4.3.0) Hao et al.46 https://github.com/satijalab/seurat (Continued on next page) 18 Cell Reports 42, 113270, October 31, 2023

Techniques: In Vivo, Control, Knockdown, Inhibition, Plasmid Preparation, Over Expression, Two Tailed Test

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 7. Col1 treatment promotes the secretion of CXCL1 in ESCC cells via NF-kB pathway (A) TRRUST enrichment analysis of elevated cytokines in Col1-treated KYSE450 cells. (B) qRT-PCR analysis of CXCL1 in ESCC cells treated with Col1 and RELA knockdown. The expression level was normalized to GAPDH (n = 3 biological rep- licates). (C) Western blot analysis of NF-kB phosphorylation in ESCC cells added to Col1-coated and uncoated plates for 24 h. (D) Representative immunofluorescence images of the activation and translocation of NF-kB P65 by Col1 treatment. KYSE450 cells were added to Col1-coated and uncoated plates for 24 h and then immunostained with NF-kB P65 antibody, followed by Cy3-conjugated secondary antibody (red) and DAPI (blue). White arrows indicate NF-kB P65 translocation in cells. Scale bar, 100 mm. (E) Quantification of the percentage of NF-kB P65 activated and translocated KYSE450 cells in (D) (n = 6 biological replicates). (F) Western blot analysis shows that the Akt inhibitor MK-2206 abrogated the phosphorylation of P65 and the increase of CXCL1 promoted by Col1 treatment. 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 S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Fetal Bovine Serum Cell Technologies Cat# 30070 DMEM Corning Cat# 10-013-CV Advanced DMEM/F12 Gibco Cat# 12634028 Puromycin Gibco Cat# A1113803 Lipofectamine 2000 Invitrogen Cat# 11668027 Collagenase I Gibco Cat# 17100017 Collagenase IV Gibco Cat# 17104019 Hyaluronidase Sigma-Aldrich Cat# H1115000 EDHB Sigma-Aldrich Cat# E24859 MK-2206 MCE Cat# HY-108232 SB-265610 MCE Cat# HY-50688 Human recombinant CXCL1 MCE Cat# HY-P70508 Rat tail collagen type 1 Solarbio Cat# C8062 Critical commercial assays RNA-Quick Purification Kit ES Science Cat# RN001 TB Green Premix Ex TaqTMII (Tli RNaseH Plus) TaKaRa Cat# RR820A Masson’s Trichrome Stain Kit Solarbio Cat# G1340 Meilunbio FGSuper Sensitive ECL Luminescence Reagent Meilunbio Cat# MA0186-1 PierceTM Rapid Gold BCA Thermo Fisher Scientific Cat# A53226 Human Cytokine Array GS440 Raybiotech Cat# GSH-CAA-440-1 CXCL1 ELISA Kit Proteintech Cat# KE00133 Opal 5-Color Manual IHC Kit PANOVUE Cat# 10144100100 NF-kB Activation Nuclear Translocation Assay Kit Beyotime Cat# SN368 Deposited data ESCC WES and RNA-seq data of 19 PDX donors This paper GSA-Human: HRA004329 Single-cell RNA-seq raw data of 6 PDXmice This paper GSA: https://ngdc.cncb.ac.cn/gsa/browse/ CRA010501 Experimental models: Cell lines KYSE450 Dr. Y. Shimada JCRB1430 KYSE510 Dr. Y. Shimada JCRB1436 NIH/3T3 ATCC CRL-1658 Human derived esophagus normal fibroblast (NF) This paper N/A PDX-derived primary tumor cells (PDC) This paper N/A Experimental models: Organisms/strains NSG mouse IDMO N/A Oligonucleotides shRNA targeting sequences, see Table S3 JTSBIO Co., Ltd N/A siRNA, see Table S3 JTSBIO Co., Ltd N/A qRT-PCR primers, see Table S4 Tsingke Biotechnology N/A Software and algorithms R 4.2.2 R Core Team https://www.R-project.org/ Cell Ranger 6.0.2 10x Genomics https://10xgenomics.com/ Seurat (version 4.3.0) Hao et al.46 https://github.com/satijalab/seurat (Continued on next page) 18 Cell Reports 42, 113270, October 31, 2023

Techniques: Quantitative RT-PCR, Knockdown, Expressing, Western Blot, Phospho-proteomics, Activation Assay, Translocation Assay, Two Tailed Test