Journal: Cancers

Article Title: Withaferin A Increases the Effectiveness of Immune Checkpoint Blocker for the Treatment of Non-Small Cell Lung Cancer

doi: 10.3390/cancers15123089

Figure Lengend Snippet: WFA induces ICD in NSCLC cell lines. Cells were treated with WFA for 48 h then collected using Accutase cell detachment solution. ( A – C ) CRT levels were determined by flow cytometry in LLC ( A ), H1650 ( B ), and A549 ( C ) cells. ( D ) The levels of secreted HMGB-1 in LLC cell treated with 0.6 µM WFA or 30 nM doxorubicin were measured by ELISA and the means of two independent experiments ± SEM are represented in the bar graph. ( E ) Bone marrow-derived DC were co-cultured with WFA-pretreated LLC cells (24 h) in a ratio 5:1 of DC:LLC cells for 24 h. DC activation markers (CD80, CD86, and MHC-II) were examined by flow cytometry. The means of at least 2 independent experiments ± SEM is represented, and one-way ANOVA was used to calculate statistical significance. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The levels of secreted HMGB-1 were measured using the HMGB-1 detection ELISA kit (Chondrex, 6010, Woodinville, WA, USA) according to the manufacturer’s protocol.

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Derivative Assay, Cell Culture, Activation Assay

Journal: Cancers

Article Title: Withaferin A Increases the Effectiveness of Immune Checkpoint Blocker for the Treatment of Non-Small Cell Lung Cancer

doi: 10.3390/cancers15123089

Figure Lengend Snippet: WFA induces ICD in NSCLC cell lines. Cells were treated with WFA for 48 h then collected using Accutase cell detachment solution. ( A – C ) CRT levels were determined by flow cytometry in LLC ( A ), H1650 ( B ), and A549 ( C ) cells. ( D ) The levels of secreted HMGB-1 in LLC cell treated with 0.6 µM WFA or 30 nM doxorubicin were measured by ELISA and the means of two independent experiments ± SEM are represented in the bar graph. ( E ) Bone marrow-derived DC were co-cultured with WFA-pretreated LLC cells (24 h) in a ratio 5:1 of DC:LLC cells for 24 h. DC activation markers (CD80, CD86, and MHC-II) were examined by flow cytometry. The means of at least 2 independent experiments ± SEM is represented, and one-way ANOVA was used to calculate statistical significance. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The levels of secreted HMGB-1 were measured using the HMGB-1 detection ELISA kit (Chondrex, 6010, Woodinville, WA, USA) according to the manufacturer’s protocol.

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Derivative Assay, Cell Culture, Activation Assay

Journal: ACS Nano

Article Title: Two-Stage SN38 Release from a Core–Shell Nanoparticle Enhances Tumor Deposition and Antitumor Efficacy for Synergistic Combination with Immune Checkpoint Blockade

doi: 10.1021/acsnano.2c09788

Figure Lengend Snippet: OxPt/SN38 induces ICD and upregulates PD-L1. CLSM images (a) and flow cytometry (b) showing DNA DSBs of MC38 cells after treatment with OxPt plus IRI or OxPt/SN38. Scale bar: 20 μm. Cellular morphology was visualized by F-actin (green) staining. (c) Western blot of γ-H2AX, IRF1, and PD-L1 of MC38 cells. (d) Schematic showing PD-L1 upregulation induced by DNA DSBs. DSBs activate STAT signaling through ATM/ATR/Chk1 kinases, which in turn induces IRF1 activation and downstream PD-L1 upregulation. Flow cytometry (e) with statistical analysis (f) of PD-L1 expression in MC38 cells, n = 3. CLSM images (g) and flow cytometry (h) with statistical analysis (i) of ROS generation in MC38 cells, n = 3. CLSM images (j) and flow cytometry with statistical analysis (k) of CRT expression in MC38 cells, n = 3. (l) HMGB-1 release from MC38 cells detected by ELISA, n = 3. In all treatments, MC38 cells were incubated with drugs at 5 μM OxPt equiv and 9 μM SN38 equiv for 24 h. Data are expressed as means ± SD, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, by one-way ANOVA with Tukey’s multiple comparisons test. MFI denotes mean fluorescent intensity.

Article Snippet: Two ×10 5 cells/well of MC38 cells were seeded in 6-well plates and then cultured with PBS, OxPt, IRI, OxPt plus IRI, ZnP/SN38, OxPt NCP, or OxPt/SN38 for 24 h. The medium was collected to detect HMGB-1 release by ELISA (Chondrex, Redmond, WA).

Techniques: Flow Cytometry, Staining, Western Blot, Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Frontiers in Immunology

Article Title: Propionic Acid, Induced in Gut by an Inulin Diet, Suppresses Inflammation and Ameliorates Liver Ischemia and Reperfusion Injury in Mice

doi: 10.3389/fimmu.2022.862503

Figure Lengend Snippet: Intraperitoneal pretreatment with propionic acid ameliorates liver IRI in mice. (A) WT mice were divided into two groups. Mice in the PA group were intraperitoneally pretreated with PA (5 mmol/kg in saline) 2 h prior to liver ischemia, whereas mice in the saline group were pretreated with saline. Then, mice in both groups were performed liver IRI model. (B) The sALT level after 6 h of reperfusion (n = 8 mice/group; ****P < .0001) (Power calculation: 1.000). (C) Macroscopic findings after 6 h of reperfusion in saline- and PA -pretreated mice. Regions (bleeding and swelling) of macroscopic changes induced by IR insult are circled by dotted lines. (D) Representative liver histology (H&E staining) after IR insult (magnification ×400). Congestion area was shown in circle by yellow dot. (E) Suzuki’s histological grading in each group (n = 8 mice/group; ****P < .0001) (Power calculation: 1.000). (F) Quantitative RT-PCR detection of inflammatory cytokines (TNF-α, IL-6, CXCL-1, CXCL-2, IL-1β, and IL-10) at 6 h of reperfusion. Data were normalized to β-actin gene expression (n = 6 mice/group; *P < .05, **P < .01) (Power calculation: TNF-α; 0.998, IL-6; 0.980, CXCL-2; 1.000, IL-1β; 0.996). The reduction of CXCL-1 and IL-10 in PA-treated group compared to saline-treated group was not significant. (G) The levels of serum HMGB-1 at 6 h after reperfusion in saline- and PA -pretreated mice (n = 8 mice/group; **P < .01) (Power calculation: 1.000).

Article Snippet: Then, bovine HMGB-1 (Chondrex, Redmond, WA, USA) (1 µg/mL) was added to the macrophages.

Techniques: Staining, Quantitative RT-PCR, Expressing

Journal: Frontiers in Immunology

Article Title: Propionic Acid, Induced in Gut by an Inulin Diet, Suppresses Inflammation and Ameliorates Liver Ischemia and Reperfusion Injury in Mice

doi: 10.3389/fimmu.2022.862503

Figure Lengend Snippet: PA suppressed HMGB-1-mediated activation of peritoneal macrophages. Peritoneal macrophages from WT mice stimulated by HMGB-1 (1 µg/mL) were cultured with or without the pretreatment with PA (1, 2, 5, and 10 mM, respectively) for 48 h. (A) TNF-α release was measured by ELISA (n = 3 samples/group; ****P < .0001) (Power calculation: 1.000). (B) Western blot assisted analyses of NF-κB signaling and MAPK signaling molecules in the macrophages stimulated by HMGB-1 (1 µg/mL) 30 min after the pretreatment with PA (5 mM). β-actin was used as the internal control.

Article Snippet: Then, bovine HMGB-1 (Chondrex, Redmond, WA, USA) (1 µg/mL) was added to the macrophages.

Techniques: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot