hmgb1  (Proteintech)

Journal: Redox Biology

Article Title: Combination of YAP inhibition and photodynamic therapy induces dual DNA damage and activates STING pathway to enhance immunotherapy in uveal melanoma

doi: 10.1016/j.redox.2025.103965

Figure Lengend Snippet: HANP/VP induces ICD via PDT in vitro. (A) Schematic illustration of ICD induction by HANP/VP. n = 3/group. (B) Apoptosis was analyzed by Annxin V/PI staining after corresponding treatments. After HANP/VP combined laser irradiation treatment, the proportions of early apoptosis and late apoptosis were much higher than those in the free VP combined laser irradiation-treated group, n = 3/group. (C) Chemiluminescence was used to detect ATP release after corresponding treatments. After HANP/VP combined laser irradiation treatment, the extracellular ATP concentration was much higher than that in the VP combined laser irradiation treatment group, n = 3/group. (D) Immunofluorescence staining was performed to assess the expression of HMGB1 (purple) after corresponding treatments. The level of nuclear HMGB1 expression was reduced after HANP/VP combined laser irradiation, n = 3/group. (E) The expression level of HMGB1 and HSP70 in cell culture supernatants was detected by Western blot after corresponding treatments. Compared with the VP with Laser group, the levels of HMGB1 and HSP70 in the cell culture supernatant were much higher after HANP/VP combined laser irradiation, n = 3/group. (F) Immunofluorescence staining was used to detect ecto-CRT expression (purple) after corresponding treatments. After HANP/VP combined laser irradiation, significant ecto-CRT signals appeared at a higher level than the VP combined laser irradiation treatment, and no ecto-CRT signals were observed in the control group, n = 3/group. (G) The proportion of ecto-CRT-positive cells was analyzed by flow cytometry after corresponding treatments. After HANP/VP combined laser irradiation treatment, the proportion of ecto-CRT-positive cells was significantly higher than that in the VP combined laser irradiation treatment group (30.23 ± 12.13 %), and there were almost no ecto-CRT-positive cells in the Control group, n = 3/group. Statistical significance in panels (C – G) was determined by one-way ANOVA (∗∗ p < 0.01, ∗∗∗ p < 0.001).

Article Snippet: Cultured cells or tumor tissue sections were incubated overnight at 4 °C with Abs against Lamin A/C (1:500; ZenBio, Beijing, China), γH2AX (1:300; Wuhan Servicebio Technology Co., Ltd., Wuhan, China), HMGB1 (1:300; Proteintech, Shanghai, China), CRT (1:300; Abcam, Cambridge, UK), Ki-67 (1:500; Servicebio), Caspase-3 (1:500; Servicebio), p-STING (1:100; Cell Signaling Technology [CST], Danvers, MA, USA), CD8 (1:500; Servicebio), CD3 (1:500; Servicebio), CD68 (1:300; Servicebio), CD163 (1:300; Servicebio), CD11b (1:300; Servicebio), CD11c (1:300; Servicebio), CD83 (1:300; ABclonal Technology, Wuhan, China), CD31 (1:500; Servicebio), and PD-L1 (1:300; Abcam); then with fluorescein isothiocyanate (FITC) or Alexa Fluor 594–conjugated secondary Abs (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at 25 °C.

Techniques: In Vitro, Staining, Irradiation, Concentration Assay, Immunofluorescence, Expressing, Cell Culture, Western Blot, Control, Flow Cytometry