Journal: International journal of oncology

Article Title: Chitinase 3-like 1 secreted from cancer-associated fibroblasts promotes tumor angiogenesis via interleukin-8 secretion in colorectal cancer.

doi: 10.3892/ijo.2021.5293

Figure Lengend Snippet: Figure 4. Changes in IL-8 and VEGFA following the addition of 100 ng/ml CHI3L1. (A) RT-qPCR indicated that IL-8 mRNA expression was higher in CAFs than in NFs and cancer cells and that 24-h treatment with 100 ng/ml CHI3L1 increased IL-8 expression in CAFs. (B) ELISA of the cell culture supernatants revealed that CAFs secreted more CHI3L1 than NFs and cancer cells, and that 48-h treatment with 100 ng/ml CHI3L1 further increased IL-8 secretion from CAFs. (C) RT-qPCR demonstrated that VEGFA mRNA expression was slightly stronger in CAFs than in NFs, although the difference was not significant. Treatment of CAFs with CHI3L1 did not affect VEGFA expression. (D) In contrast to RT-qPCR, ELISA of the cell culture supernatants demonstrated significant differences in VEGFA secretion between untreated CAFs and CAFs treated with 100 ng/ml CHI3L1; however, VEGFA secretion from CAFs was not particularly high compared with that from cancer cell lines. (E) RT-qPCR revealed no significant changes in IL-8 mRNA expression in the three cancer cell lines, between groups treated with and without 100 ng/ml CHI3L1. (F) RT-qPCR revealed no significant changes in VEGFA mRNA expression in the three cancer cells, between groups treated with and without 100 ng/ml CHI3L1. (G) RT-qPCR demonstrated that IL-13Rα2 expression in CAFs was suppressed after transfection with IL-13Rα2 siRNA. (H) RT-qPCR indicated that IL-13Rα2 knockdown suppressed the enhanced IL-8 mRNA expression induced by CHI3L1 treatment in CAFs. (I) RT-qPCR revealed that IL-13Rα2 knockdown and the addition of CHI3L1 did not change VEGFA mRNA expression in CAFs. Data are presented as the mean ± SEM. *P<0.05, **P<0.01. The relative mRNA expression levels of IL-8, VEGFA and IL-13Rα2 were normalized to GAPDH expression in each sample. CAFs, cancer-associated fibroblasts; CHI3L1, chitinase 3-like 1; IL-13Rα2, interleukin-13 receptor α2; NC, negative control; NFs, normal fibroblasts; NS, not significant; RT-qPCR, reverse transcription-quantitative PCR; si/siRNA, small interfering RNA; VEGFA, vascular endothelial growth factor-A.

Article Snippet: CHI3L1 small interfering RNA (siRNA) (s3000), interleukin-13 receptor α2 (IL-13Rα2) siRNA (s7376), VEGFA siRNA (s462) and non-targeting nega‐ tive control siRNA (Silencer Select Negative Control No. 1; cat. no. 4390843) were pre-designed siRNAs purchased from Invitrogen; Thermo Fisher Scientific, Inc. IL-8 siRNA (cat. no. sc-39631) was also pre-designed siRNA purchased from Santa Cruz Biotechnology, Inc. CAFs were seeded at 1x105 cells/well in 6-well plates for RT-qPCR, 2.5x104 cells/well in 24-well plates for ELISA and the angio‐ genesis assay, and ~80% confluent in 10-cm dishes for western blotting the day before siRNA transfection.

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Transfection, Knockdown, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: International journal of oncology

Article Title: Chitinase 3-like 1 secreted from cancer-associated fibroblasts promotes tumor angiogenesis via interleukin-8 secretion in colorectal cancer.

doi: 10.3892/ijo.2021.5293

Figure Lengend Snippet: Figure 5. Changes in IL-8 and VEGFA after CHI3L1 knockdown in CAFs. (A) RT-qPCR indicated that CHI3L1 mRNA expression in CAFs was suppressed by transfection with CHI3L1 siRNA. (B) ELISA of the cell culture supernatants demonstrated that CHI3L1 secretion in CAFs was suppressed by transfec‑ tion with CHI3L1 siRNA. (C) Western blotting revealed that CHI3L1 protein expression in CAFs was suppressed after transfection with CHI3L1 siRNA. (D) RT-qPCR indicated that IL-8 mRNA expression in CAFs was suppressed by transfection with CHI3L1 siRNA. (E) ELISA of the cell culture supernatants demonstrated that IL-8 secretion in CAFs was suppressed by transfection with CHI3L1 siRNA. (F) RT-qPCR revealed that VEGFA mRNA expression in CAFs was suppressed by transfection with CHI3L1 siRNA. (G) ELISA of the cell culture supernatants indicated that VEGFA secretion in CAFs was suppressed by transfection with CHI3L1 siRNA. Data are presented as the mean ± SEM. *P<0.05, **P<0.01. The relative mRNA expression levels of CHI3L1, IL-8 and VEGFA were normalized to GAPDH expression in each sample. CAFs, cancer-associated fibroblasts; CHI3L1, chitinase 3-like 1; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; si/siRNA, small interfering RNA; VEGFA, vascular endothelial growth factor-A.

Article Snippet: CHI3L1 small interfering RNA (siRNA) (s3000), interleukin-13 receptor α2 (IL-13Rα2) siRNA (s7376), VEGFA siRNA (s462) and non-targeting nega‐ tive control siRNA (Silencer Select Negative Control No. 1; cat. no. 4390843) were pre-designed siRNAs purchased from Invitrogen; Thermo Fisher Scientific, Inc. IL-8 siRNA (cat. no. sc-39631) was also pre-designed siRNA purchased from Santa Cruz Biotechnology, Inc. CAFs were seeded at 1x105 cells/well in 6-well plates for RT-qPCR, 2.5x104 cells/well in 24-well plates for ELISA and the angio‐ genesis assay, and ~80% confluent in 10-cm dishes for western blotting the day before siRNA transfection.

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: International journal of oncology

Article Title: Chitinase 3-like 1 secreted from cancer-associated fibroblasts promotes tumor angiogenesis via interleukin-8 secretion in colorectal cancer.

doi: 10.3892/ijo.2021.5293

Figure Lengend Snippet: Figure 6. Effects of CHI3L1 on tube formation in vascular endothelial cells. (A) EA.hy926 cells were cultured in serum‑free DMEM, serum‑free DMEM with 100 ng/ml CHI3L1, serum‑free DMEM with 100 ng/ml IL-8 and serum‑free DMEM with 50 ng/ml VEGFA. The cells incubated with serum‑free DMEM alone were used as a control (magnification, x40). (B) IL-8 and VEGFA increased tube formation by EA.hy926 cells, and CHI3L1 tended to increase tube formation by EA.hy926 cells, although no significant difference was observed. (C) EA.hy926 cells were cultured in DMEM containing 5% FBS, conditioned medium from cancer cells (HT-29), NF or CAF cultures, conditioned medium from CAFs treated with control IgG or mAY at 10 µg/ml, and conditioned medium from CAFs transfected with negative control, CHI3L1, IL-8 or VEGFA siRNA. The cells incubated only with DMEM containing 5% FBS were used as controls (magnification, x40). (D) CM from CAFs increased tube formation by EA.hy926 cells compared with control CM and CM from NFs. The addition of mAY or transfection of CAFs with CHI3L1, IL-8 or VEGFA siRNA suppressed the effects of CAF CM on tube formation. (E) RT-qPCR demonstrated that IL-8 mRNA expression in CAFs was suppressed by transfection with IL-8 siRNA. (F) RT-qPCR demonstrated that VEGFA mRNA expression in CAFs was suppressed by transfection with VEGFA siRNA. Data are presented as the mean ± SEM. *P<0.05, **P<0.01. The relative mRNA expression levels of IL-8 and VEGFA were normalized to GAPDH expression in each sample. CAFs, cancer-associated fibroblasts; CHI3L1, chitinase 3-like 1; CM, conditioned medium; mAY, human CHI3L1 neutralizing antibody; NC, negative control; NFs, normal fibroblasts; NS, not significant; RT-qPCR, reverse transcription-quantitative PCR; si/siRNA, small interfering RNA; VEGFA, vascular endothelial growth factor-A.

Article Snippet: CHI3L1 small interfering RNA (siRNA) (s3000), interleukin-13 receptor α2 (IL-13Rα2) siRNA (s7376), VEGFA siRNA (s462) and non-targeting nega‐ tive control siRNA (Silencer Select Negative Control No. 1; cat. no. 4390843) were pre-designed siRNAs purchased from Invitrogen; Thermo Fisher Scientific, Inc. IL-8 siRNA (cat. no. sc-39631) was also pre-designed siRNA purchased from Santa Cruz Biotechnology, Inc. CAFs were seeded at 1x105 cells/well in 6-well plates for RT-qPCR, 2.5x104 cells/well in 24-well plates for ELISA and the angio‐ genesis assay, and ~80% confluent in 10-cm dishes for western blotting the day before siRNA transfection.

Techniques: Cell Culture, Incubation, Control, Transfection, Negative Control, Quantitative RT-PCR, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: International Journal of Oncology

Article Title: Chitinase 3-like 1 secreted from cancer-associated fibroblasts promotes tumor angiogenesis via interleukin-8 secretion in colorectal cancer

doi: 10.3892/ijo.2021.5293

Figure Lengend Snippet: Changes in IL-8 and VEGFA following the addition of 100 ng/ml CHI3L1. (A) RT-qPCR indicated that IL-8 mRNA expression was higher in CAFs than in NFs and cancer cells and that 24-h treatment with 100 ng/ml CHI3L1 increased IL-8 expression in CAFs. (B) ELISA of the cell culture supernatants revealed that CAFs secreted more CHI3L1 than NFs and cancer cells, and that 48-h treatment with 100 ng/ml CHI3L1 further increased IL-8 secretion from CAFs. (C) RT-qPCR demonstrated that VEGFA mRNA expression was slightly stronger in CAFs than in NFs, although the difference was not significant. Treatment of CAFs with CHI3L1 did not affect VEGFA expression. (D) In contrast to RT-qPCR, ELISA of the cell culture supernatants demonstrated significant differences in VEGFA secretion between untreated CAFs and CAFs treated with 100 ng/ml CHI3L1; however, VEGFA secretion from CAFs was not particularly high compared with that from cancer cell lines. (E) RT-qPCR revealed no significant changes in IL-8 mRNA expression in the three cancer cell lines, between groups treated with and without 100 ng/ml CHI3L1. (F) RT-qPCR revealed no significant changes in VEGFA mRNA expression in the three cancer cells, between groups treated with and without 100 ng/ml CHI3L1. (G) RT-qPCR demonstrated that IL-13Rα2 expression in CAFs was suppressed after transfection with IL-13Rα2 siRNA. (H) RT-qPCR indicated that IL-13Rα2 knockdown suppressed the enhanced IL-8 mRNA expression induced by CHI3L1 treatment in CAFs. (I) RT-qPCR revealed that IL-13Rα2 knockdown and the addition of CHI3L1 did not change VEGFA mRNA expression in CAFs. Data are presented as the mean ± SEM. * P<0.05, ** P<0.01. The relative mRNA expression levels of IL-8 , VEGFA and IL-13Rα2 were normalized to GAPDH expression in each sample. CAFs, cancer-associated fibroblasts; CHI3L1, chitinase 3-like 1; IL-13Rα2, interleukin-13 receptor α2; NC, negative control; NFs, normal fibroblasts; NS, not significant; RT-qPCR, reverse transcription-quantitative PCR; si/siRNA, small interfering RNA; VEGFA, vascular endothelial growth factor-A.

Article Snippet: CHI3L1 small interfering RNA (siRNA) (s3000), interleukin-13 receptor α2 (IL-13Rα2) siRNA (s7376), VEGFA siRNA (s462) and non-targeting negative control siRNA (Silencer Select Negative Control No. 1; cat. no. 4390843) were pre-designed siRNAs purchased from Invitrogen; Thermo Fisher Scientific, Inc. IL-8 siRNA (cat. no. sc-39631) was also pre-designed siRNA purchased from Santa Cruz Biotechnology, Inc. CAFs were seeded at 1×10 5 cells/well in 6-well plates for RT-qPCR, 2.5×10 4 cells/well in 24-well plates for ELISA and the angiogenesis assay, and ~80% confluent in 10-cm dishes for western blotting the day before siRNA transfection.

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Transfection, Knockdown, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: International Journal of Oncology

Article Title: Chitinase 3-like 1 secreted from cancer-associated fibroblasts promotes tumor angiogenesis via interleukin-8 secretion in colorectal cancer

doi: 10.3892/ijo.2021.5293

Figure Lengend Snippet: Changes in IL-8 and VEGFA after CHI3L1 knockdown in CAFs. (A) RT-qPCR indicated that CHI3L1 mRNA expression in CAFs was suppressed by transfection with CHI3L1 siRNA. (B) ELISA of the cell culture supernatants demonstrated that CHI3L1 secretion in CAFs was suppressed by transfection with CHI3L1 siRNA. (C) Western blotting revealed that CHI3L1 protein expression in CAFs was suppressed after transfection with CHI3L1 siRNA. (D) RT-qPCR indicated that IL-8 mRNA expression in CAFs was suppressed by transfection with CHI3L1 siRNA. (E) ELISA of the cell culture supernatants demonstrated that IL-8 secretion in CAFs was suppressed by transfection with CHI3L1 siRNA. (F) RT-qPCR revealed that VEGFA mRNA expression in CAFs was suppressed by transfection with CHI3L1 siRNA. (G) ELISA of the cell culture supernatants indicated that VEGFA secretion in CAFs was suppressed by transfection with CHI3L1 siRNA. Data are presented as the mean ± SEM. * P<0.05, ** P<0.01. The relative mRNA expression levels of CHI3L1 , IL-8 and VEGFA were normalized to GAPDH expression in each sample. CAFs, cancer-associated fibroblasts; CHI3L1, chitinase 3-like 1; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; si/siRNA, small interfering RNA; VEGFA, vascular endothelial growth factor-A.

Article Snippet: CHI3L1 small interfering RNA (siRNA) (s3000), interleukin-13 receptor α2 (IL-13Rα2) siRNA (s7376), VEGFA siRNA (s462) and non-targeting negative control siRNA (Silencer Select Negative Control No. 1; cat. no. 4390843) were pre-designed siRNAs purchased from Invitrogen; Thermo Fisher Scientific, Inc. IL-8 siRNA (cat. no. sc-39631) was also pre-designed siRNA purchased from Santa Cruz Biotechnology, Inc. CAFs were seeded at 1×10 5 cells/well in 6-well plates for RT-qPCR, 2.5×10 4 cells/well in 24-well plates for ELISA and the angiogenesis assay, and ~80% confluent in 10-cm dishes for western blotting the day before siRNA transfection.

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: International Journal of Oncology

Article Title: Chitinase 3-like 1 secreted from cancer-associated fibroblasts promotes tumor angiogenesis via interleukin-8 secretion in colorectal cancer

doi: 10.3892/ijo.2021.5293

Figure Lengend Snippet: Effects of CHI3L1 on tube formation in vascular endothelial cells. (A) EA.hy926 cells were cultured in serum-free DMEM, serum-free DMEM with 100 ng/ml CHI3L1, serum-free DMEM with 100 ng/ml IL-8 and serum-free DMEM with 50 ng/ml VEGFA. The cells incubated with serum-free DMEM alone were used as a control (magnification, ×40). (B) IL-8 and VEGFA increased tube formation by EA.hy926 cells, and CHI3L1 tended to increase tube formation by EA.hy926 cells, although no significant difference was observed. (C) EA.hy926 cells were cultured in DMEM containing 5% FBS, conditioned medium from cancer cells (HT-29), NF or CAF cultures, conditioned medium from CAFs treated with control IgG or mAY at 10 µ g/ml, and conditioned medium from CAFs transfected with negative control, CHI3L1, IL-8 or VEGFA siRNA. The cells incubated only with DMEM containing 5% FBS were used as controls (magnification, ×40). (D) CM from CAFs increased tube formation by EA.hy926 cells compared with control CM and CM from NFs. The addition of mAY or transfection of CAFs with CHI3L1, IL-8 or VEGFA siRNA suppressed the effects of CAF CM on tube formation. (E) RT-qPCR demonstrated that IL-8 mRNA expression in CAFs was suppressed by transfection with IL-8 siRNA. (F) RT-qPCR demonstrated that VEGFA mRNA expression in CAFs was suppressed by transfection with VEGFA siRNA. Data are presented as the mean ± SEM. * P<0.05, ** P<0.01. The relative mRNA expression levels of IL-8 and VEGFA were normalized to GAPDH expression in each sample. CAFs, cancer-associated fibroblasts; CHI3L1, chitinase 3-like 1; CM, conditioned medium; mAY, human CHI3L1 neutralizing antibody; NC, negative control; NFs, normal fibroblasts; NS, not significant; RT-qPCR, reverse transcription-quantitative PCR; si/siRNA, small interfering RNA; VEGFA, vascular endothelial growth factor-A.

Article Snippet: CHI3L1 small interfering RNA (siRNA) (s3000), interleukin-13 receptor α2 (IL-13Rα2) siRNA (s7376), VEGFA siRNA (s462) and non-targeting negative control siRNA (Silencer Select Negative Control No. 1; cat. no. 4390843) were pre-designed siRNAs purchased from Invitrogen; Thermo Fisher Scientific, Inc. IL-8 siRNA (cat. no. sc-39631) was also pre-designed siRNA purchased from Santa Cruz Biotechnology, Inc. CAFs were seeded at 1×10 5 cells/well in 6-well plates for RT-qPCR, 2.5×10 4 cells/well in 24-well plates for ELISA and the angiogenesis assay, and ~80% confluent in 10-cm dishes for western blotting the day before siRNA transfection.

Techniques: Cell Culture, Incubation, Control, Transfection, Negative Control, Quantitative RT-PCR, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Interleukin-8 promotes integrin β3 upregulation and cell invasion through PI3K/Akt pathway in hepatocellular carcinoma

doi: 10.1186/s13046-019-1455-x

Figure Lengend Snippet: IL-8 knockdown decreased integrin β3 expression and HCC cell invasion and migration. a Real-time PCR analysis of IL-8 mRNA level after 48 h of transfection with IL-8 siRNA or control siRNA (C siRNA). b ELISA analysis of IL-8 protein level after 48 h of transfection with IL-8 siRNA or C siRNA. c Real-time PCR analysis of integrin β3 mRNA level after 48 h of transfection with IL-8 siRNA or C siRNA. d Real-time PCR analysis of integrin αv mRNA level after 48 h of transfection with IL-8 siRNA or C siRNA. e Western blot analysis of integrin β3 expression after 48 h of transfection with IL-8 siRNA or C siRNA. f Western blot analysis of integrin αv expression after 48 h of transfection with IL-8 siRNA or C siRNA. g Invasion assay of HCCLM3 and MHCC97H cells after 48 h of transfection with IL-8 siRNA or C siRNA. h Migration assay of HCCLM3 and MHCC97H cells after 48 h of transfection with IL-8 siRNA or C siRNA. * P < 0.05 compared to cells transfected with C siRNA

Article Snippet: Human IL-8 siRNA (#sc-156,051), integrin β3 siRNA (#sc-29,375), and control siRNA (#sc-37,007) were purchased from Santa Cruz.

Techniques: Knockdown, Expressing, Migration, Real-time Polymerase Chain Reaction, Transfection, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Invasion Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Interleukin-8 promotes integrin β3 upregulation and cell invasion through PI3K/Akt pathway in hepatocellular carcinoma

doi: 10.1186/s13046-019-1455-x

Figure Lengend Snippet: IL-8 increased HCC cell invasion through CXCR1/2 receptors. a Huh-7 and HepG2 cells were incubated with various concentrations of IL-8 for 24 h, and in vitro invasion was measured with Transwell assay. b After 48 h of transfection with CXCR1 siRNA, CXCR2 siRNA or control siRNA (C siRNA), cells were incubated with IL-8 (100 ng/ml) for 24 h, and in vitro invasion was measured with Transwell assay. Results are expressed as the mean ± SEM. * P < 0.05 compared with the control group. # P < 0.05 compared with the IL-8-treated group

Article Snippet: Human IL-8 siRNA (#sc-156,051), integrin β3 siRNA (#sc-29,375), and control siRNA (#sc-37,007) were purchased from Santa Cruz.

Techniques: Incubation, In Vitro, Transwell Assay, Transfection, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Interleukin-8 promotes integrin β3 upregulation and cell invasion through PI3K/Akt pathway in hepatocellular carcinoma

doi: 10.1186/s13046-019-1455-x

Figure Lengend Snippet: Integrin β3 was involved in IL-8/CXCR1/2-induced invasion of HCC cells. a Real-time PCR and Western blot analysis of integrin β3 expression in Huh-7 and HepG2 cells after incubation with different concentrations of IL-8 for 24 h. b Huh-7 and HepG2 cells were transfected with integrin β3 siRNA (si-β3) or control siRNA (C siRNA) for 48 h, and in vitro invasion was measured with Transwell assay in the presence or absence of IL-8 (100 ng/ml) for 24 h. c HCCLM3 cells were transfected using IL-8 siRNA with or without integrin β3 cDNA, and in vitro invasion was measured with Transwell assay. d Real-time PCR and Western blot analysis of integrin β3 expression in Huh-7 and HepG2 cells after transfection of CXCR1 siRNA, CXCR2 siRNA or C siRNA followed by stimulation with IL-8 (100 ng/ml) for 24 h. Results are expressed as the mean ± SEM. * P < 0.05 compared with the control group. # P < 0.05 compared with the IL-8-treated group. ※ P < 0.05 compared with the IL-8 siRNA-treated group

Article Snippet: Human IL-8 siRNA (#sc-156,051), integrin β3 siRNA (#sc-29,375), and control siRNA (#sc-37,007) were purchased from Santa Cruz.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Incubation, Transfection, Control, In Vitro, Transwell Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Interleukin-8 promotes integrin β3 upregulation and cell invasion through PI3K/Akt pathway in hepatocellular carcinoma

doi: 10.1186/s13046-019-1455-x

Figure Lengend Snippet: PI3K/Akt signaling pathway was involved in IL-8 mediated HCC cell invasion and integrin β3 upregulation. a Western blot analysis of total and phosphorylated PI3K and Akt in C siRNA and IL-8 siRNA transfected cells. b After pretreatment with PI3K-specific inhibitor LY294002 (25 μM) for 30 min, cells were stimulated with IL-8 (100 ng/ml) for 24 h, and in vitro invasion was measured with Transwell assay. c After pretreatment with LY294002 (25 μM) for 30 min, cells were stimulated with IL-8 (100 ng/ml) for 24 h, and expression of integrin β3, PI3K, p-PI3K, Akt and p-Akt was measured by western blot. Results are expressed as the mean ± SEM. * P < 0.05 compared with the control group. # P < 0.05 compared with the IL-8-treated group

Article Snippet: Human IL-8 siRNA (#sc-156,051), integrin β3 siRNA (#sc-29,375), and control siRNA (#sc-37,007) were purchased from Santa Cruz.

Techniques: Western Blot, Transfection, In Vitro, Transwell Assay, Expressing, Control

Journal: Cancer letters

Article Title: Sorafenib improves alkylating therapy by blocking induced inflammation, invasion and angiogenesis in breast cancer cells

doi: 10.1016/j.canlet.2018.03.037

Figure Lengend Snippet: (A) ELISA assays showing sorafenib inhibition of basal and MMS-induced IL8, MMP1, MMP3, CXCL2 and PGE2 levels as determined in the culture medium of MDA-MB231 after 12 h treatment. Inflammatory protein levels in MCF-7 and SKBR3 cells are also shown. COX2 immunocontent was determined by western blot. (B) AP-1- and NFκB-luciferase reporter gene assays showing the effect of sorafenib and UO126 upon basal and MMS-induced NFkB and AP-1 transcription factor activity in MDA-MB231 cell line. Unless otherwise specified, sorafenib was used at 1 μM; UO126 at 10 μM; and alkylating agent at IC50 concentration. Legends: Srfn (sorafenib); ND (not detected); UO126 (10 μM). Data are presented as mean±SD. * different from untreated controls; &different from control and from MMS-treated cells (1-way-ANOVA-Tukey; p<0.05, n=3 in duplicate).

Article Snippet: 2.5. siRNA-mediated knockdown Small-interference RNA (siRNA) transfections were performed using Lipofectamine RNAi MAX Reagent following manufacturer instructions (Invitrogen). siRNA duplexes targeting human IL8 (sc-39631), CXCL2 (sc-43934), MMP1 (sc-41552), VEGFA (sc-29520) PTGS2/COX-2 (sc-29279) and scrambled siRNA-A (sc-37007) were purchased from Santa Cruz Biotechnology, and incubated at 20 to 50 nM siRNA range for 24 h prior to treatments.

Techniques: Enzyme-linked Immunosorbent Assay, Inhibition, Western Blot, Luciferase, Activity Assay, Concentration Assay

Journal: Cancer letters

Article Title: Sorafenib improves alkylating therapy by blocking induced inflammation, invasion and angiogenesis in breast cancer cells

doi: 10.1016/j.canlet.2018.03.037

Figure Lengend Snippet: (A) 4-HC induces inflammatory proteins production in MDA-MB231 cells. The cells were treated for 12 h with 25 μM 4-HC for 12 h, and afterwards the culture medium was collected for ELISA analysis. (B) Representative IHC microphotographs showing protein content of MMP1, IL8, CXCL2, COX2 and IL6 in MDA-MB231 tumor xenografts treated with cyclophosphamide, sorafenib or combination of both. IHC score (0 to 3+) for each mice/tumor (n=8) is represented as a heatmap in the bottom of each microphotograph. IL8 IHC microphotographs also show peritumoral regions weakly stained.

Article Snippet: 2.5. siRNA-mediated knockdown Small-interference RNA (siRNA) transfections were performed using Lipofectamine RNAi MAX Reagent following manufacturer instructions (Invitrogen). siRNA duplexes targeting human IL8 (sc-39631), CXCL2 (sc-43934), MMP1 (sc-41552), VEGFA (sc-29520) PTGS2/COX-2 (sc-29279) and scrambled siRNA-A (sc-37007) were purchased from Santa Cruz Biotechnology, and incubated at 20 to 50 nM siRNA range for 24 h prior to treatments.

Techniques: Enzyme-linked Immunosorbent Assay, Staining

Journal: Cancer letters

Article Title: Sorafenib improves alkylating therapy by blocking induced inflammation, invasion and angiogenesis in breast cancer cells

doi: 10.1016/j.canlet.2018.03.037

Figure Lengend Snippet: (A) Celltiter-Glo cell viability assays showing the impact of inflammatory genes knockdown and celecoxib treatment upon MMS cytotoxicity in MDA-MB231 cells after 72 h treatment. (B) Effect of inflammatory gene depletion on invasiveness of MDA-MB231 cells. siRNA transfections were performed directly in the cell seeding step on top-chambers of transwell plates and cell invasion was assessed after 48 h. Celecoxib and SB225002 were incubated directly in the top chamber of transwell plates. (C) The effect of inflammatory gene knockdown (by siRNA), COX-2 inhibition (celecoxib, 10 μM) and CXCR2 antagonist (SB225002, 15 μM) on the angiogenic potential of MDA-MB231 conditioned medium in vitro. (D) Log-rank test p-value (antilog) and Hazard ratio (HR) for the univariate analysis of correlation between sorafenib-inhibited inflammation-related genes expression and breast cancer patient overall survival (OS) as assessed by BreastMark tool. The most significant HR are annotated with the graph. (E) Kaplan-Meier plots of the top-3 survival related genes (IL8, MMP1 and VEGFA; obtained from “D”) as a single or combined predictor signatures of breast cancer patients survival. Legends: si (siRNA); rec (recombinant). Data are presented as mean±SD. * different from untreated/scrambled siRNA controls or at indicated comparisons. &different from control and from alkylation/MMS-treated cells (1way-ANOVA-Tukey; p<0.05, n=3 in duplicate). See S3 for supplementary data.

Article Snippet: 2.5. siRNA-mediated knockdown Small-interference RNA (siRNA) transfections were performed using Lipofectamine RNAi MAX Reagent following manufacturer instructions (Invitrogen). siRNA duplexes targeting human IL8 (sc-39631), CXCL2 (sc-43934), MMP1 (sc-41552), VEGFA (sc-29520) PTGS2/COX-2 (sc-29279) and scrambled siRNA-A (sc-37007) were purchased from Santa Cruz Biotechnology, and incubated at 20 to 50 nM siRNA range for 24 h prior to treatments.

Techniques: Transfection, Incubation, Inhibition, In Vitro, Expressing, Recombinant