cdc mrsa  (ATCC)

Journal: Aging (Albany NY)

Article Title: TNFSF9 promotes metastasis of pancreatic cancer through Wnt/Snail signaling and M2 polarization of macrophages

doi: 10.18632/aging.203497

Figure Lengend Snippet: The expression of TNFSF9 in pancreatic cancer tissues is significantly higher than that in non-tumor tissues adjacent to cancer. ( A ) Representative HE staining and immunohistochemical staining pictures of cancer tissues and non-tumor tissues adjacent to pancreatic cancer ( n = 30) and immune response score ( B ). ( C ) Immunohistochemical staining pictures of pancreatic cancer tissue with metastasis and pancreatic cancer tissue without metastasis. ( D , E ) Western blot analysis of the protein expression of TNFSF9 in pancreatic cancer cells COLO357, ASPC-1, PANC-1 and BXPC-3 and normal pancreatic epithelial cells HPDE6-C7. ( F ) QPCR analysis of the mRNA expression of TNFSF9 in COLO357, ASPC-1, PANC-1, BXPC-3 and HPDE6-C7. NT, non-tumor tissue adjacent to pancreatic cancer. PC, pancreatic cancer. IRS was used to evaluate tissue staining. The staining intensity is 0 to 3 points, 0 is no staining, 1 is low staining, 2 is medium staining, and 3 is high staining. The percentage of positive cells ranges from 0 to 4 points, 0 < 1%, 1 is 1 to 10%, 2 is 11 to 50%, 3 is 51 to 80%, and 4 is >80%. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Then, 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) was used for electrophoresis, and the protein was transferred to polyvinylidene (PVDF) and sealed with 5% skimmed milk for 1 h. It was then incubated overnight at 4°C with the following primary antibodies: GAPDH (1:10000, ProteinTech), TNFSF9 (1:2000, ProteinTech), β-catenin (1:1000, ProteinTech), Wnt (1:1000, Wanleibio), ERK (1:500, Wanleibio), phosphorylated-ERK (1:1000, Santa), JNK (1:1000, Wanleibio), phosphorylated-JNK (1:1000, Wanleibio), AKT (1:1000, Santa), phosphorylated-AKT (1:1000, Santa), NF-ΚB (1:1000, Wanleibio), phosphorylated-NF-ΚB (1:1000, Wanleibio), Snail (1:1500, Wanleibio), focal adhesion kinase (FAK) (1:2000, ProteinTech), proto-oncogene tyrosine-protein kinase Src (Src) (1:800, ProteinTech), IL-6 (1:5000, Wanleibio), IL-8 (1:1000, Wanleibio), TNF-α (1:1000, Santa), E-cadherin (ProteinTech, 1:10000), N-cadherin (ProteinTech, 1:3000), and Vimentin (ProteinTech, 1:2000).

Techniques: Expressing, Staining, Immunohistochemical staining, Western Blot

Journal: Aging (Albany NY)

Article Title: TNFSF9 promotes metastasis of pancreatic cancer through Wnt/Snail signaling and M2 polarization of macrophages

doi: 10.18632/aging.203497

Figure Lengend Snippet: Correlations between TNFSF9 expression and clinicopathological features in pancreatic cancer patients.

Article Snippet: Then, 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) was used for electrophoresis, and the protein was transferred to polyvinylidene (PVDF) and sealed with 5% skimmed milk for 1 h. It was then incubated overnight at 4°C with the following primary antibodies: GAPDH (1:10000, ProteinTech), TNFSF9 (1:2000, ProteinTech), β-catenin (1:1000, ProteinTech), Wnt (1:1000, Wanleibio), ERK (1:500, Wanleibio), phosphorylated-ERK (1:1000, Santa), JNK (1:1000, Wanleibio), phosphorylated-JNK (1:1000, Wanleibio), AKT (1:1000, Santa), phosphorylated-AKT (1:1000, Santa), NF-ΚB (1:1000, Wanleibio), phosphorylated-NF-ΚB (1:1000, Wanleibio), Snail (1:1500, Wanleibio), focal adhesion kinase (FAK) (1:2000, ProteinTech), proto-oncogene tyrosine-protein kinase Src (Src) (1:800, ProteinTech), IL-6 (1:5000, Wanleibio), IL-8 (1:1000, Wanleibio), TNF-α (1:1000, Santa), E-cadherin (ProteinTech, 1:10000), N-cadherin (ProteinTech, 1:3000), and Vimentin (ProteinTech, 1:2000).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: TNFSF9 promotes metastasis of pancreatic cancer through Wnt/Snail signaling and M2 polarization of macrophages

doi: 10.18632/aging.203497

Figure Lengend Snippet: TNFSF9 knockdown inhibits the proliferation of pancreatic cancer cells and promotes the apoptosis of pancreatic cancer cells. ( A , B ) Cell counting kit-8 was used to evaluate the proliferation of BXPC-3 and PANC-1 cells after TNFSF9 knockdown. ( C – F ) Colony formation was used to analyze the proliferation of BXPC-3 and PANC-1 cells after TNFSF9 knockdown. ( G – J ) Flow cytometry was used to analyze the apoptosis of BXPC-3 and PANC-1 cells after TNFSF9 knockdown. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Then, 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) was used for electrophoresis, and the protein was transferred to polyvinylidene (PVDF) and sealed with 5% skimmed milk for 1 h. It was then incubated overnight at 4°C with the following primary antibodies: GAPDH (1:10000, ProteinTech), TNFSF9 (1:2000, ProteinTech), β-catenin (1:1000, ProteinTech), Wnt (1:1000, Wanleibio), ERK (1:500, Wanleibio), phosphorylated-ERK (1:1000, Santa), JNK (1:1000, Wanleibio), phosphorylated-JNK (1:1000, Wanleibio), AKT (1:1000, Santa), phosphorylated-AKT (1:1000, Santa), NF-ΚB (1:1000, Wanleibio), phosphorylated-NF-ΚB (1:1000, Wanleibio), Snail (1:1500, Wanleibio), focal adhesion kinase (FAK) (1:2000, ProteinTech), proto-oncogene tyrosine-protein kinase Src (Src) (1:800, ProteinTech), IL-6 (1:5000, Wanleibio), IL-8 (1:1000, Wanleibio), TNF-α (1:1000, Santa), E-cadherin (ProteinTech, 1:10000), N-cadherin (ProteinTech, 1:3000), and Vimentin (ProteinTech, 1:2000).

Techniques: Cell Counting, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: TNFSF9 promotes metastasis of pancreatic cancer through Wnt/Snail signaling and M2 polarization of macrophages

doi: 10.18632/aging.203497

Figure Lengend Snippet: Knockdown of TNFSF9 inhibits the migration and invasion of pancreatic cancer cells. ( A–D ) Transwell experiment was used to analyze the migration and invasion of BXPC-3 and PANC-1 cells after TNFSF9 knockdown. ( E , F ) The cell scratch test was used to analyze the migration of BXPC-3 and PANC-1 cells after TNFSF9 knockdown. ( G–J ) Western blots were used to analyze the expression levels of EMT-associated proteins; Vimentin, N-cadherin and E-cadherin in BXPC-3 and PANC-1 cells after knockdown of TNFSF9. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Then, 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) was used for electrophoresis, and the protein was transferred to polyvinylidene (PVDF) and sealed with 5% skimmed milk for 1 h. It was then incubated overnight at 4°C with the following primary antibodies: GAPDH (1:10000, ProteinTech), TNFSF9 (1:2000, ProteinTech), β-catenin (1:1000, ProteinTech), Wnt (1:1000, Wanleibio), ERK (1:500, Wanleibio), phosphorylated-ERK (1:1000, Santa), JNK (1:1000, Wanleibio), phosphorylated-JNK (1:1000, Wanleibio), AKT (1:1000, Santa), phosphorylated-AKT (1:1000, Santa), NF-ΚB (1:1000, Wanleibio), phosphorylated-NF-ΚB (1:1000, Wanleibio), Snail (1:1500, Wanleibio), focal adhesion kinase (FAK) (1:2000, ProteinTech), proto-oncogene tyrosine-protein kinase Src (Src) (1:800, ProteinTech), IL-6 (1:5000, Wanleibio), IL-8 (1:1000, Wanleibio), TNF-α (1:1000, Santa), E-cadherin (ProteinTech, 1:10000), N-cadherin (ProteinTech, 1:3000), and Vimentin (ProteinTech, 1:2000).

Techniques: Migration, Western Blot, Expressing

Journal: Aging (Albany NY)

Article Title: TNFSF9 promotes metastasis of pancreatic cancer through Wnt/Snail signaling and M2 polarization of macrophages

doi: 10.18632/aging.203497

Figure Lengend Snippet: TNFSF9 promotes the migration of pancreatic cancer cells by activating the Wnt/Snail signaling pathway. ( A–C ) Western blot analysis the expression of Wnt, β-catenin, AKT, p-AKT, JNK, p-JNK, NF-ΚB, p-NF-ΚB, ERK p-ERK, Snail, Src and FAK in BXPC-3 and PANC-1 cells after knockdown of TNFSF9. Compared with the sh-NC group, the expression of Wnt, β-catenin, AKT, p-AKT, JNK, p-JNK, NF-ΚB, p-NF-ΚB and Snail in the sh-TNFSF9-1 and sh-TNFSF9-2 groups is significantly reduced. The expression of ERK, p-ERK, Src and FAK did not change. ( D , E ) The expression of Wnt, β-catenin, and Snail increased after the addition of Wnt agonist in TNFSF9 knockdown pancreatic cancer, but the expression of TNFSF9 did not change. ( F , G ) Increased migration of TNFSF9 knockdown pancreatic cancer cells after the addition of Wnt agonist. sh-1 is sh-TNFSF9-1. sh-2 is sh-TNFSF9-2. * P < 0.05, ** P < 0.01, *** P < 0.001 and # P < 0.001.

Article Snippet: Then, 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) was used for electrophoresis, and the protein was transferred to polyvinylidene (PVDF) and sealed with 5% skimmed milk for 1 h. It was then incubated overnight at 4°C with the following primary antibodies: GAPDH (1:10000, ProteinTech), TNFSF9 (1:2000, ProteinTech), β-catenin (1:1000, ProteinTech), Wnt (1:1000, Wanleibio), ERK (1:500, Wanleibio), phosphorylated-ERK (1:1000, Santa), JNK (1:1000, Wanleibio), phosphorylated-JNK (1:1000, Wanleibio), AKT (1:1000, Santa), phosphorylated-AKT (1:1000, Santa), NF-ΚB (1:1000, Wanleibio), phosphorylated-NF-ΚB (1:1000, Wanleibio), Snail (1:1500, Wanleibio), focal adhesion kinase (FAK) (1:2000, ProteinTech), proto-oncogene tyrosine-protein kinase Src (Src) (1:800, ProteinTech), IL-6 (1:5000, Wanleibio), IL-8 (1:1000, Wanleibio), TNF-α (1:1000, Santa), E-cadherin (ProteinTech, 1:10000), N-cadherin (ProteinTech, 1:3000), and Vimentin (ProteinTech, 1:2000).

Techniques: Migration, Western Blot, Expressing

Journal: Aging (Albany NY)

Article Title: TNFSF9 promotes metastasis of pancreatic cancer through Wnt/Snail signaling and M2 polarization of macrophages

doi: 10.18632/aging.203497

Figure Lengend Snippet: TNFSF9 induces M2 polarization of macrophages and promotes the migration of pancreatic cancer cells. ( A ) U937 cells were successfully induced into macrophages. ( B , C ) The effect of wild-type pancreatic cancer cells on the polarization of U937-derived macrophages. ( D , E ) TNFSF9 knockdown pancreatic cancer cells inhibit the expression of M2 markers (IL-10 and TGF-β) mRNA levels in U937 macrophages. At the same time, it promotes the expression of M1 marker (IL-8 and TNF-α) mRNA level and inhibits the expression of IL-1β mRNA level. ( F , G ) Co-culture of U937-derived macrophages with TNFSF9 knockdown pancreatic cancer cells inhibited the migration of BXPC-3 and PANC-1 cells. ( H , I ) The migration of BXPC-3 and PANC-1 cells increased after adding recombinant human protein TGF-β. ( J – L ) Western blots were used to analyze the expression of inflammatory cytokines IL-8, IL-6, TNF-α and IL-1β on U937-derived macrophages after co-culture with pancreatic cancer cells knocked down by TNFSF9. Compared with the sh-NC group, the expression of IL-8, IL-6 and TNF-α in the sh-TNFSF9-1 and sh-TNFSF9-2 groups were significantly increased, and the expression of IL-1β was significantly reduced. sh-1 is sh-TNFSF9-1. sh-2 is sh-TNFSF9-2. * P < 0.05, ** P < 0.01, *** P < 0.001 and # P < 0.001.

Article Snippet: Then, 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) was used for electrophoresis, and the protein was transferred to polyvinylidene (PVDF) and sealed with 5% skimmed milk for 1 h. It was then incubated overnight at 4°C with the following primary antibodies: GAPDH (1:10000, ProteinTech), TNFSF9 (1:2000, ProteinTech), β-catenin (1:1000, ProteinTech), Wnt (1:1000, Wanleibio), ERK (1:500, Wanleibio), phosphorylated-ERK (1:1000, Santa), JNK (1:1000, Wanleibio), phosphorylated-JNK (1:1000, Wanleibio), AKT (1:1000, Santa), phosphorylated-AKT (1:1000, Santa), NF-ΚB (1:1000, Wanleibio), phosphorylated-NF-ΚB (1:1000, Wanleibio), Snail (1:1500, Wanleibio), focal adhesion kinase (FAK) (1:2000, ProteinTech), proto-oncogene tyrosine-protein kinase Src (Src) (1:800, ProteinTech), IL-6 (1:5000, Wanleibio), IL-8 (1:1000, Wanleibio), TNF-α (1:1000, Santa), E-cadherin (ProteinTech, 1:10000), N-cadherin (ProteinTech, 1:3000), and Vimentin (ProteinTech, 1:2000).

Techniques: Migration, Derivative Assay, Expressing, Marker, Co-Culture Assay, Recombinant, Western Blot

Journal: Aging (Albany NY)

Article Title: TNFSF9 promotes metastasis of pancreatic cancer through Wnt/Snail signaling and M2 polarization of macrophages

doi: 10.18632/aging.203497

Figure Lengend Snippet: TNFSF9 activates Wnt signal to promote M2 polarization of macrophages. ( A ) The mRNA expression levels of IL-10 and TGF-β in TNFSF9 knockdown pancreatic cancer cells were decreased, and the mRNA expression levels of IL-4 were increased. ( B ) After adding Wnt agonist, the mRNA expression level of IL-10 and TGF-β increased. ( C ) After adding Wnt agonist, pancreatic cancer cells that knock down TNFSF9 reduce the mRNA levels of M1 markers (IL-1β, IL-8 and TNF-α) in macrophages. ( D ) After adding Wnt agonist, pancreatic cancer cells that knock down TNFSF9 increase the mRNA levels of M2 markers (IL-4, IL-10 and TGF-β) in macrophages. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: Then, 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) was used for electrophoresis, and the protein was transferred to polyvinylidene (PVDF) and sealed with 5% skimmed milk for 1 h. It was then incubated overnight at 4°C with the following primary antibodies: GAPDH (1:10000, ProteinTech), TNFSF9 (1:2000, ProteinTech), β-catenin (1:1000, ProteinTech), Wnt (1:1000, Wanleibio), ERK (1:500, Wanleibio), phosphorylated-ERK (1:1000, Santa), JNK (1:1000, Wanleibio), phosphorylated-JNK (1:1000, Wanleibio), AKT (1:1000, Santa), phosphorylated-AKT (1:1000, Santa), NF-ΚB (1:1000, Wanleibio), phosphorylated-NF-ΚB (1:1000, Wanleibio), Snail (1:1500, Wanleibio), focal adhesion kinase (FAK) (1:2000, ProteinTech), proto-oncogene tyrosine-protein kinase Src (Src) (1:800, ProteinTech), IL-6 (1:5000, Wanleibio), IL-8 (1:1000, Wanleibio), TNF-α (1:1000, Santa), E-cadherin (ProteinTech, 1:10000), N-cadherin (ProteinTech, 1:3000), and Vimentin (ProteinTech, 1:2000).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: TNFSF9 promotes metastasis of pancreatic cancer through Wnt/Snail signaling and M2 polarization of macrophages

doi: 10.18632/aging.203497

Figure Lengend Snippet: TNFSF9 promotes metastasis of pancreatic cancer in vivo . ( A ) Compared with the sh-TNFSF9 group, there were obvious metastases in the liver tissue of nude mice in the sh-NC group, and no normal spleen tissue was seen. ( B ) HE staining confirmed liver metastases of pancreatic cancer and splenic implants. The red arrow indicates metastasis of liver lesions. The blue arrow indicates the spleen implanted tumor.

Article Snippet: Then, 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) was used for electrophoresis, and the protein was transferred to polyvinylidene (PVDF) and sealed with 5% skimmed milk for 1 h. It was then incubated overnight at 4°C with the following primary antibodies: GAPDH (1:10000, ProteinTech), TNFSF9 (1:2000, ProteinTech), β-catenin (1:1000, ProteinTech), Wnt (1:1000, Wanleibio), ERK (1:500, Wanleibio), phosphorylated-ERK (1:1000, Santa), JNK (1:1000, Wanleibio), phosphorylated-JNK (1:1000, Wanleibio), AKT (1:1000, Santa), phosphorylated-AKT (1:1000, Santa), NF-ΚB (1:1000, Wanleibio), phosphorylated-NF-ΚB (1:1000, Wanleibio), Snail (1:1500, Wanleibio), focal adhesion kinase (FAK) (1:2000, ProteinTech), proto-oncogene tyrosine-protein kinase Src (Src) (1:800, ProteinTech), IL-6 (1:5000, Wanleibio), IL-8 (1:1000, Wanleibio), TNF-α (1:1000, Santa), E-cadherin (ProteinTech, 1:10000), N-cadherin (ProteinTech, 1:3000), and Vimentin (ProteinTech, 1:2000).

Techniques: In Vivo, Staining