Journal: Cell Death & Disease

Article Title: Dedifferentiation process driven by radiotherapy-induced HMGB1/TLR2/YAP/HIF-1α signaling enhances pancreatic cancer stemness

doi: 10.1038/s41419-019-1956-8

Figure Lengend Snippet: Sorted CD133 − cancer cells (reporter) were treated with the following agents: (i) Different concentration of rhHMGB1 (100, 200, 250, and 300 ng/mL); (ii) 20Gy X-ray irradiated HMGB1 wide type cancer cells (iHMGB1 + ); (iii) 20Gy X-ray irradiated HMGB1 knock down cancer cells (iHMGB1 shRNA1); (iv) 250 ng/mL rhHMGB1+iHMGB1shRNA1; (v) 250 ng/mL rhHMGB1+EP; (vi) PBS for the indicated time. a Western blot analyses the expression of TLR2 and TLR4 in treated CD133 − cancer cells for the indicated time (0, 6, 12, 24, and 48 h). b Co-IP analyses the binding between HMGB1 and TLR2 in CD133 − cancer cells. c , d Sorted CD133 − cancer cells treated with rhHMGB1 (250 ng/ml) and with or without Stevioside (TLR2 inhibitor) or sh-TLR2 silenced CD133 − cancer cells for the indicated time (0, 6, 12, and 24 h). qRT-PCR and Western blot analyses the expression of stem cell-related markers (CD133, Oct4, Sox2, C-myc, and Nanog) at protein and gene level. e Sphere forming ability of sorted CD133 − cancer cells or sh-TLR2 silenced CD133 − cancer cells treated with rhHMGB1 (250 ng/ml) and with or without Stevioside. Experiments were repeated three times and the data were expressed as mean ± SEM. Student's t -test, oneway, and two-sided ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Stevioside (TLR2 antagonist) purchased from Topscience (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO).

Techniques: Concentration Assay, Irradiation, Knockdown, Western Blot, Expressing, Co-Immunoprecipitation Assay, Binding Assay, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Dedifferentiation process driven by radiotherapy-induced HMGB1/TLR2/YAP/HIF-1α signaling enhances pancreatic cancer stemness

doi: 10.1038/s41419-019-1956-8

Figure Lengend Snippet: a Western blot analyses the expression of YAP, p-YAP and HIF-1α in sorted CD133 − cancer cells (reporter cells) treated with the following agents: (i) Different concentration of rhHMGB1 (100, 200, 250, and 300 ng/mL); (ii) 20Gy X-ray irradiated HMGB1 wide type cancer cells (iHMGB1 + ); (iii) 20 Gy X-ray irradiated HMGB1 knock down cancer cells (iHMGB1 shRNA1); (iv) 250 ng/mL rhHMGB1+iHMGB1shRNA1; (v) 250 ng/mL rhHMGB1+ EP; (vi) PBS for the indicated time. b qRT-PCR analyses the expression of YAP and HIF-1α in cocultured CD133 − reporter cell population. c Western blot analyses the expression of Hippo-YAP pathway related marker (p-MOB1, MOB1, p-YAP, and YAP), HIF-1α, and CD133 in CD133 − cancer cells treated with rhHMGB1 (250 ng/mL) for the indicated time (0, 6, 12, and 24 h). d Relative YAP luciferase activity in sorted CD133 − cancer cells in the presence and absence of rhHMGB1 or Flag-YAP. e qRT-PCR analyses the expression of YAP downstream target genes (cyclin E, SPP1, and CTGF) in CD133 − cell population in the presence or absence of rhHMGB1. f Western blot analyses the expression of p-YAP, YAP, and HIF-1α in CD133 − cancer cells or sh-TLR2 silenced CD133 − cancer cells treated with rhHMGB1(250 ng/ml) and with or without Stevioside. g Western blot analyses the expression of TLR2, CD133, Sox2, and Nanog in CD133 − cancer cells and YAP silenced CD133 − cancer cells treated with rhHMGB1 (250 ng/ml). h Sphere forming ability of sorted CD133 − cancer cells or YAP silenced CD133 − cancer cells treated with or without rhHMGB1(250 ng/ml). Experiments were repeated three times and the data were expressed as mean ± SEM. Student's t -test, oneway, and two-sided ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Stevioside (TLR2 antagonist) purchased from Topscience (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO).

Techniques: Western Blot, Expressing, Concentration Assay, Irradiation, Knockdown, Quantitative RT-PCR, Marker, Luciferase, Activity Assay

Journal: Cell Death & Disease

Article Title: Dedifferentiation process driven by radiotherapy-induced HMGB1/TLR2/YAP/HIF-1α signaling enhances pancreatic cancer stemness

doi: 10.1038/s41419-019-1956-8

Figure Lengend Snippet: a CD133 − cancer cells were cocultured with irradiated parental cancer cells HMGB1 + cells (HMGB1 positive, HP), irradiated HMGB1 knock down cancer cells (iHMGB1shRNA1, HN), rhHMGB1 (150 ng/mL), and the same empty medium (PBS). The tumor initiating capacity and volume were measured. b , c CD133 − PaTu8988 cancer cells, TLR2, YAP, or HIF-1α knockdown PaTu8988 CD133 − cancer cells were implanted subcutaneously into the right dorsal flanks of nude mice, respectively and treated with rhHMGB1 for 2 weeks. Flow cytometry analysis the percentage of CD133 + cancer cells ( b ). Western blot analyses the protein expression level of Nanog and Oct4 in the fresh tumor tissues ( c ). d The subcutaneous tumor mice models were received 20Gy X-ray irradiation and injected of PBS, EP (HMGB1 inhibitor), Stevioside (TLR2 inhibitor), Verteporfin (YAP inhibitor), or LW6 (HIF-1α inhibitor). The tumor volume and growth speed were measured. Experiments were repeated three times and the data were expressed as mean ± SEM. Student's t -test, oneway, two-sided ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Stevioside (TLR2 antagonist) purchased from Topscience (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO).

Techniques: Irradiation, Knockdown, Flow Cytometry, Western Blot, Expressing, Injection