Journal: Molecular medicine (Cambridge, Mass.)

Article Title: β-caryophyllene reduces inflammation to protect against ischemic stroke by suppressing HMGB1 signaling.

doi: 10.1186/s10020-025-01171-z

Figure Lengend Snippet: Fig. 2 After hypoxia reoxygenation experiments, mouse primary cortical neurons were tested for proinflammatory factors and HMGB 1 expression. A Proinflammatory factor levels in primary cortical neurons in control and hypoxic reoxygenation group. B HMGB1 mRNA levels in primary cortical neurons in the control and hypoxic reoxygenation groups. C The HMGB1 protein levels in primary cortical neurons in control and hypoxia relative to the internal parameters. D HMGB1 immunofluorescence levels in primary cortical neurons in control and hypoxia reoxygenation groups. DAPI localized the nucleus and MAP 2 as a neuronal marker. n = 4, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Then the membranes were blocked with non-fat milk (5%) and incubated overnight at 4◦C with the following primary antibodies: rabbit polyclonal antibody against HMGB1 (10,829–1-AP, Proteintech, 1:250), TLR4 (19,811–1-AP, Proteintech, 1:250), RAGE (AF5309, Affinity, 1:1000), and mouse internal ginseng antibody (beta-actin; 10,829–1-AP, Proteintech, 1:1000).

Techniques: Expressing, Control, Immunofluorescence, Marker

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: β-caryophyllene reduces inflammation to protect against ischemic stroke by suppressing HMGB1 signaling.

doi: 10.1186/s10020-025-01171-z

Figure Lengend Snippet: Fig. 6 β-caryophyllene play a protective role in cerebral ischemia and reperfusion maybe related to the effect on HMGB1 expression. A HMGB1 mRNA levels in ipsilateral brain tissues in different groups. B Protein levels of HMGB1 relative to internal reference in ipsilateral brain tissue from different groups mice. C HMGB1 mRNA levels in primary cortical neurons in different groups. D The HMGB1 protein levels in primary cortical neurons in different groups. E Detection of proinflammatory factors in ipsilateral brain tissue of different group mice. F Detection of proinflammatory factors in culture supernatant of primary neuronal cells. n = 6 in vivo experiments and n = 4 in vitro experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then the membranes were blocked with non-fat milk (5%) and incubated overnight at 4◦C with the following primary antibodies: rabbit polyclonal antibody against HMGB1 (10,829–1-AP, Proteintech, 1:250), TLR4 (19,811–1-AP, Proteintech, 1:250), RAGE (AF5309, Affinity, 1:1000), and mouse internal ginseng antibody (beta-actin; 10,829–1-AP, Proteintech, 1:1000).

Techniques: Expressing, In Vivo, In Vitro

Journal: The Journal of Cell Biology

Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation

doi: 10.1083/jcb.200304135

Figure Lengend Snippet: Effect of HMGB1 on embryonic mesoangioblast proliferation. (A) D16 cells were grown in RPMI medium containing no addition, HMGB1 at the indicated concentrations, or 20% FCS. HMGB1 induced cell proliferation at all concentrations tested, but the cell number reached a plateau after 48 h. Each point represents the mean ± SD ( n = 3). The experiment was repeated three times. (B) D16 cell division was analyzed by FACS ® . After 6 h in the presence of 30 ng/ml HMGB1 the DNA content increases, but returns to the normal diploid content after 24 h. Asterisk indicates statistical significance (P < 0.001). (C) 3T3 fibroblasts (treated as the D16 cells in A) do not divide in the presence of HMGB1.

Article Snippet: Serial muscle sections were fixed in 4% PFA, permeabilized, saturated, and processed for immunofluorescence with rabbit anti-HMGB1 antibody (1:500 dilution) followed by Alexa Fluor ® 488–conjugated goat anti–rabbit Ig (Molecular Probes, Inc.).

Techniques:

Journal: The Journal of Cell Biology

Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation

doi: 10.1083/jcb.200304135

Figure Lengend Snippet: Continued HMGB1 stimulation sustains mesoangioblast proliferation. D16 cells were placed at time 0 in RPMI medium containing 30 ng/ml HMGB1; a similar amount of HMGB1 was also added at the times indicated with a triangle. Multiply stimulated D16 cells kept growing. Each point represents the average ± SD of two experiments performed in duplicate. Inset: Western blot of HMGB1 in the medium bathing D16 cells 48 h after the beginning of the experiment. HMGB1 was still present in the medium of restimulated cells, but not in the medium of cells stimulated once at time 0. This experiment was repeated two times with similar results.

Article Snippet: Serial muscle sections were fixed in 4% PFA, permeabilized, saturated, and processed for immunofluorescence with rabbit anti-HMGB1 antibody (1:500 dilution) followed by Alexa Fluor ® 488–conjugated goat anti–rabbit Ig (Molecular Probes, Inc.).

Techniques: Western Blot

Journal: The Journal of Cell Biology

Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation

doi: 10.1083/jcb.200304135

Figure Lengend Snippet: HMGB1 has chemotactic activity on embryonic mesoangioblasts. (A) D16 cells were subjected to chemotaxis assays with 10, 50, or 100 ng/ml HMGB1. Data represent the average ± SD of four experiments performed in duplicate; the effect of increasing HMGB1 concentrations is highly significant (P < 0.001 in ANOVA analysis). Addition of anti-HMGB1 antibodies recognizing the peptide 166–181 significantly reduced the chemotactic response (P < 0.05 in comparison to the sample without antibody), whereas the addition of monoclonal anti-box A antibodies had no effect. (B) Chemotactic activity on D16 cells of various HMGB1 fragments (all at 10 ng/ml). ABbt has a chemotactic effect comparable with full-length HMGB1 (P < 0.05 of proteins vs. medium alone). In contrast, boxes A and B and the AB didomain have no significant chemotactic activity. Bars represent the average ± SD of three experiments performed in duplicate. Asterisks indicate statistical significance (P< 0.01). (C) Schematic representation of full-length HMGB1, boxes A and B, the didomain AB, and tailless HMGB1 (ABbt). (D) Western blot with anti-RAGE antibodies on total D16 cell extract. (E) D16 cells transfected with dnRAGE-expressing plasmid or pCDNA3 empty vector were assayed for chemotaxis in response to medium with or without HMGB1. Cells transfected with dnRAGE exhibited a significant decrease in migration in comparison to cells transfected with control plasmid. Asterisks indicate statistical significance (P < 0.05).

Article Snippet: Serial muscle sections were fixed in 4% PFA, permeabilized, saturated, and processed for immunofluorescence with rabbit anti-HMGB1 antibody (1:500 dilution) followed by Alexa Fluor ® 488–conjugated goat anti–rabbit Ig (Molecular Probes, Inc.).

Techniques: Activity Assay, Chemotaxis Assay, Western Blot, Transfection, Expressing, Plasmid Preparation, Migration

Journal: The Journal of Cell Biology

Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation

doi: 10.1083/jcb.200304135

Figure Lengend Snippet: HMGB1 induces the transit of mesoangioblasts through an endothelial monolayer. D16 cells were placed in the upper compartment of Boyden apparatuses. The lower chambers contained RPMI alone (medium), RPMI plus 100 ng/ml HMGB1, or RPMI plus 10 ng/ml VEGF; chambers were separated by a confluent endothelial cell monolayer grown on polycarbonate filters. HMGB1 significantly stimulated D16 transmigration (P < 0.01). Bars represent the aver- age ± SD of three experiments performed in duplicate. Panels beside the bar graph show D16 cells stained with Giemsa after migration, toward medium alone or containing HMGB1. Asterisk indicates statistical significance (P < 0.05).

Article Snippet: Serial muscle sections were fixed in 4% PFA, permeabilized, saturated, and processed for immunofluorescence with rabbit anti-HMGB1 antibody (1:500 dilution) followed by Alexa Fluor ® 488–conjugated goat anti–rabbit Ig (Molecular Probes, Inc.).

Techniques: Transmigration Assay, Staining, Migration

Journal: The Journal of Cell Biology

Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation

doi: 10.1083/jcb.200304135

Figure Lengend Snippet: HMGB1 attracts mesoangioblasts in vivo. D16 cells were first transduced with a lentiviral vector encoding nuclear LacZ, and were then injected through the femoral artery of mice where heparin-Sepharose beads (either unloaded or loaded with HMGB1) had been injected in the tibialis anterior muscle. Mice were killed after 24 h. (A) Tibialis anterior muscles injected with HMGB1-loaded and control beads. (B–D) Cryosections of muscles treated with control heparin-Sepharose beads (control) or HMGB1-coated heparin-Sepharose beads (HMGB1). Arrows indicate the beads. Sections were stained with X-gal, and mesoangioblasts (arrowheads) appear blue. Mesoangioblasts were found in large clusters (C) or as isolated cells (D) only in muscles injected with HMGB1-coated beads. (E) Number of migrating D16 cells in tibialis anterior muscles of wild-type mice treated with HMGB1-loaded beads (gray bar; n = 2) or control beads coinjected with 1 μg LPS (black bar; n = 3). White bar represents the number of D16 cells found in tibialis anterior muscles of α-SG −/− dystrophic mice ( n = 2).

Article Snippet: Serial muscle sections were fixed in 4% PFA, permeabilized, saturated, and processed for immunofluorescence with rabbit anti-HMGB1 antibody (1:500 dilution) followed by Alexa Fluor ® 488–conjugated goat anti–rabbit Ig (Molecular Probes, Inc.).

Techniques: In Vivo, Transduction, Plasmid Preparation, Injection, Staining, Isolation

Journal: The Journal of Cell Biology

Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation

doi: 10.1083/jcb.200304135

Figure Lengend Snippet: Effect of HMGB1 on adult mesoangioblasts. (A) Mesoangioblasts of the G1 clone, obtained from mouse bone marrow, were grown in RPMI medium containing 1, 10, or 30 ng/ml HMGB1. For comparison, G1 cells were also grown in RPMI medium alone or in RPMI plus 20% FCS. (B) Migration of G1 cells toward the lower chamber of Boyden apparatuses containing RPMI (medium) or RPMI plus 10 ng/ml HMGB1 (HMGB1). In the migration experiment, the chambers were separated by a filter; in the transmigration experiment, the chambers were separated by a filter overgrown with a monolayer of endothelial cells. Each bar represents the average ± SD of three experiments, and the arrows indicate statistical significance (P < 0.05). (C) G1 cells were labeled with DiI and then injected through the femoral artery of mice where heparin-Sepharose beads (either loaded with HMGB1 or unloaded) had been implanted in the tibialis anterior muscle (arrows). Top, phase contrast; bottom, fluorescence. G1 cells (red fluorescence) migrate in the vicinity of HMGB1-loaded cells; no G1 cells are detected near control beads.

Article Snippet: Serial muscle sections were fixed in 4% PFA, permeabilized, saturated, and processed for immunofluorescence with rabbit anti-HMGB1 antibody (1:500 dilution) followed by Alexa Fluor ® 488–conjugated goat anti–rabbit Ig (Molecular Probes, Inc.).

Techniques: Migration, Transmigration Assay, Labeling, Injection, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation

doi: 10.1083/jcb.200304135

Figure Lengend Snippet: HMGB1 expression and mesoangioblast migration in α -SG − / − dystrophic muscles. (A) Western blot analysis of HMGB1 expression levels in tibialis anterior muscles from α-SG −/− dystrophic and wild-type mice. (B) Hematoxylin and eosin staining (H&E) of tibialis anterior muscle sections from α-SG −/− dystrophic and wild-type mice. Different sections from the same muscles were processed for immunofluorescence (right). A considerable number of foci containing inflammatory cells were evident in α-SG −/− dystrophic muscles, and these contained HMGB1 in the cytoplasm in addition to the nucleus (anti-HMGB1, green; DAPI, red pseudocolor). (C) Fraction of dnRAGE expressing D16 cells before and after injection and homing to tibialis anterior muscles of α-SG −/− dystrophic mice. About 500 cells were counted before and after injection in two mice. The difference before and after migration is not statistically different. (D) Number of migrating D16 cells in tibialis anterior muscles of α-SG −/− dystrophic mice injected with control beads ( n = 2) or HMGB1-loaded beads ( n = 2). Cells expressing dnRAGE and GFP are indicated in green; the nonexpressing cells are indicated in gray. The difference in total number of cells, and in cells not expressing dnRAGE, is significant (P < 0.05). The difference in cells expressing dnRAGE is not statistically significant.

Article Snippet: Serial muscle sections were fixed in 4% PFA, permeabilized, saturated, and processed for immunofluorescence with rabbit anti-HMGB1 antibody (1:500 dilution) followed by Alexa Fluor ® 488–conjugated goat anti–rabbit Ig (Molecular Probes, Inc.).

Techniques: Expressing, Migration, Western Blot, Staining, Immunofluorescence, Injection

Journal: European journal of pharmacology

Article Title: HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway.

doi: 10.1016/j.ejphar.2020.173189

Figure Lengend Snippet: Fig. 2. Upregulation of HMGB1, TLR4, pIKBα and GFAP in astrocytes and brain microvessel endothelial cells in MCAO rats. (A, B and D) Compared with the sham group, the expression of HMGB1, TLR4, pIKBα and GFAP was potentiated in the cerebral cortex tissues of MCAO rats. The scale bars represent 60 and 30 μm in (A) and (B) respectively. (C and E) Consistently, the HMGB1 (red) in astrocytes in MCAO group translocated from nucleus (Blue color represents DAPI) to the cytoplasm (Green color represents GFAP). The expression of TLR4 (yellow) and pIKBα (yellow) in brain microvessel endothelial cells (Green color represents VIII factor) in MCAO group was potentiated respectively. The expression of GFAP (green) in astrocytes (Blue color represents DAPI) in MCAO group was also increased. The scale bars represent 10 μm. (D and E) Each value represents the mean ± S.D. (n = 10) and was measured as described in “Materials and Methods”. Note: *P < 0.05 vs. sham group.

Article Snippet: Brain slices were treated with antibodies against HMGB1 (rabbit polyclonal IgG antibody; 1:100), TLR4 (rabbit polyclonal IgG antibody; 1:100), pIKBα (rabbit polyclonal IgG antibody; 1:100) and glial fibrillary acidic protein (GFAP) (rabbit polyclonal IgG antibody; 1:100) at 4 °C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques: Expressing

Journal: European journal of pharmacology

Article Title: HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway.

doi: 10.1016/j.ejphar.2020.173189

Figure Lengend Snippet: Fig. 6. OGD activated astrocytes and induced HMGB1 release into the extracellular space. (A and C) OGD potentiated GFAP expression in astrocytes. GFAP was labelled with HRP (yellow) and nucleus was stained with DAPI (blue). (B (a and b), C, D, E and F) OGD increased HMGB1 expression (red) in astrocytes and induced HMGB1 release from nucleus to the cytoplasm. GFAP was labelled with FITC (green) and nucleus was stained with DAPI (blue). (B (c and d)) Expression of HMGB1 was not observed in rBMECs in both sham and OGD groups. VIII factor was labelled with FITC (green) and nucleus was stained with DAPI (blue). (G) OGD increased HMGB1 content in the medium. (C, D (b), E (b), F (b) and G (b)) Each value represents the mean ± S.D. (n = 10) and was measured as described in “Materials and Methods”. Note: *P < 0.05 vs. sham group. Scale bars represent 20 μm.

Article Snippet: Brain slices were treated with antibodies against HMGB1 (rabbit polyclonal IgG antibody; 1:100), TLR4 (rabbit polyclonal IgG antibody; 1:100), pIKBα (rabbit polyclonal IgG antibody; 1:100) and glial fibrillary acidic protein (GFAP) (rabbit polyclonal IgG antibody; 1:100) at 4 °C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques: Expressing, Staining

Journal: European journal of pharmacology

Article Title: HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway.

doi: 10.1016/j.ejphar.2020.173189

Figure Lengend Snippet: Fig. 8. HMGB1 contributed to upregulation of P-gp via TLR4/NF-kB pathway. (A) The relationship among HMGB1 and changes of TLR4, TIRAP, NF-kB and P-gp in rBMECs after OGD was investigated using related positive agents. (B) Changes of TLR4 (green) and TIRAP (red) after OGD and treatment with positive agents in rBMECs. (C) Each value represents the mean ± S.D. (n = 10) and was measured as described in “Materials and Methods”. Note: *P < 0.05 vs. sham group; #P < 0.05 vs. OGD group. The scale bars represent 10 μm.

Article Snippet: Brain slices were treated with antibodies against HMGB1 (rabbit polyclonal IgG antibody; 1:100), TLR4 (rabbit polyclonal IgG antibody; 1:100), pIKBα (rabbit polyclonal IgG antibody; 1:100) and glial fibrillary acidic protein (GFAP) (rabbit polyclonal IgG antibody; 1:100) at 4 °C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques:

Journal: Neural regeneration research

Article Title: Maraviroc promotes recovery from traumatic brain injury in mice by suppression of neuroinflammation and activation of neurotoxic reactive astrocytes.

doi: 10.4103/1673-5374.344829

Figure Lengend Snippet: Figure 4 ｜ Effect of maraviroc on the HMGB1/NF-κB pathway and inflammatory cytokines in the perilesional cortex after traumatic brain injury. (A–F) Representative western blot bands and quantitative data of IL-1β (B), TNF-α (C), IL-6 (D), HMGB1 (E), and NF-κB p65 (F) protein expression in the perilesional cortex 3 days post- TBI. (G, H) Representative photographs of immunofluorescence staining of NF-κB p65 in the perilesional cortex at 3 days post-TBI. Scale bars: 50 µm. Maraviroc treatment alleviated neuroinflammation post-TBI compared with the TBI + vehicle group (P = 0.0005). The black boxes in the hematoxylin and eosin-stained illustration show the perilesional cortex. One- way analysis of variance followed by Tukey’s post hoc test was used. Data are expressed as the mean ± SD (n = 5/group). All experiments were repeated at least three times. DAPI: 4′,6-Diamidino-2-phenylindole; HMGB1: high mobility group protein B1; IL: interleukin; NF-κB: nuclear factor NF-kappa-B; TBI: traumatic brain injury; TNF: tumor necrosis factor.

Article Snippet: Table 1 | Primary antibody used in this study Antibody Host organism Cat# RRID Vendor Dilution MW (kDa) Application NLRP3 Rabbit ab214185 AB_ 2819003 Abcam 1:1000 117 WB ASC Rabbit 67824 AB _2799736 CST 1:1000 22 WB, IF Caspase-1 p20 Mouse SC-398715 AB_ 2819181 Santa Cruz 1:1000 for WB; 1:200 for IF 17, 40 WB, IF IL-1β Mouse 12242 AB_ 2715503 CST 1:1000 17 WB IL-18 Rabbit 57058 NA CST 1:1000 17, 22 WB GSDMD Rabbit ab209845 AB_ 278550 Abcam 1:1000 32, 53 WB HMGB1 Rabbit 3935 AB_ 2295241 CST 1:1000 29 WB NF-κB p65 Rabbit 8242 AB_ 10859369 CST 1:1000 65 WB, IF TNF-α Rabbit 11948 AB_ 2687962 CST 1:1000 17, 25, 28 WB IL-6 Rabbit 12912 AB_ 2798059 CST 1:1000 24 WB Iba-1 Goat ab5076 AB_ 2224402 Abcam 1:1000 for WB; 1:500 for IF 17 WB, IF CD206 Mouse SC-58986 AB_ 2144945 Santa Cruz 1:1000 for WB; 1:200 for IF 190 WB, IF iNOS Rabbit 13120 AB_ 2687529 CST 1:1000 for WB; 1:500 for IF 130 WB, IF GFAP Mouse 3670 AB_ 561049 CST 1:1000 for WB; 1:500 for IF 50 WB, IF C3 Rat NB200-540 AB_ 10003444 Novus Biological 1:1000 for WB; 1:500 for IF 187 WB, IF Caspase-3 Rabbit 9662 AB_ 331439 CST 1:1000 17, 19, 35 WB Bax Rabbit 14796 AB_ 2716251 CST 1:1000 20 WB NeuN Rabbit ab177487 AB_ 2532109 Abcam 1:500 IF Ly6G Rat sc-53515 AB_ 783639 Santa Cruz 1:200 IF F4/80 Rat ab6640 AB_ 1140040 Abcam 1:500 IF Abcam: Abcam, Cambridge, MA, USA; CST: Cell Signaling Technology, Danvers, MA, USA; IF: immunofluorescence; MW: molecular weight; NA: not applicable; Novus Biological: Novus Biological, Littleton, CO, USA; Santa Cruz: Santa Cruz Biotechnology, Santa Cruz, CA, USA; WB: western blot.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Mitochondrial‐Targeted CS@KET/P780 Nanoplatform for Site‐Specific Delivery and High‐Efficiency Cancer Immunotherapy in Hepatocellular Carcinoma

doi: 10.1002/advs.202308027

Figure Lengend Snippet: CS@KET/P780 NPs evoke apoptosis through ROS accumulation and trigger ICD in liver cancer cells (The following experimental conditions are: 808 nm for IR780, 660 nm for P780, KET/P780 NPs, and CS@KET/P780 NPs; P = 1.0 W cm −2 , irradiation time = 30 s; C KET = 4.5 × 10 −6 m , C P780 = 2.5 × 10 −6 m ). A) LSCM images to display subcellular localization of IR780 or P780 with mitochondria. Scale bar:10 µm. B) Flow cytometry analysis and C) fluorescence imaging for intracellular ROS level of Hep3B cells using DCFH‐DA as a probe. Scale bar:100 µm. D) Flow cytometry analysis for mitochondrial membrane potential of Hep3B cells following different treatments. E) ATP content in Hep3B cells following different treatments. F) Flow cytometry findings of apoptosis in Hep3B cells after various treatments. G) Western blot analysis of apoptotic markers for Hep3B cells following different treatments. H,I) Flow cytometry evaluation and immunofluorescence staining of the CRT on the surface of Hepa1‐6 cells following various treatments. Scale bar:10 µm. J) Hepa1‐6 cells were labeled with HMGB1 immunofluorescence following various treatments. Scale bar: 60 µm. K) Western blot analysis of the COX‐2 in Hepa1‐6 cells following various treatments. L) ATP content in the supernatant of Hepa1‐6 cells after various treatments. M) HMGB1, N) PGE2, O) IL‐2, and P) TNF‐α in the supernatant of Hepa1‐6 cells after various treatments. (*** P < 0.001, one‐way ANOVA).

Article Snippet: CRT Rabbit Monoclonal Antibody (WL04297), Ki67 (bsm‐33 070 M), and HMGB1 Rb pAb (WL03023) were obtained from WanLeibio.

Techniques: Irradiation, Flow Cytometry, Fluorescence, Imaging, Membrane, Western Blot, Immunofluorescence, Staining, Labeling

Journal: Advanced Science

Article Title: Mitochondrial‐Targeted CS@KET/P780 Nanoplatform for Site‐Specific Delivery and High‐Efficiency Cancer Immunotherapy in Hepatocellular Carcinoma

doi: 10.1002/advs.202308027

Figure Lengend Snippet: The CS@KET/P780 NPs‐mediated immune activation in vivo. The mice were treated with normal saline, free KET, KET/P780 NPs with laser irradiation, or CS@KET/P780 NPs with laser irradiation ( λ = 660 nm, P = 1.0 W cm −2 ; irradiation time = 3 min; C KET = 2 mg kg −1 , C P780 = 2 mg kg −1 , V = 100 µL). A) Immunohistochemical tests of CRT and HMGB1 in tumor tissues after various therapies. B) Immunohistochemical analysis of CD8a in tumor tissues after various therapies. C–P) Flow cytometry analysis and quantitative analysis of the percentage of major immune cells in tumors of mice, including C,E) CD4 + T cells, D,F) CD8 + T cells, G,I) Tregs, H,J) DCs, K,M) M1 macrophage and L,N) M2 macrophage. O,P) IL‐2 and TNF‐α content in mice serum following different treatments detected by ELISA. All data are presented as the means ± SD from at least 3 independent experiments. (** P <0.01, *** P <0.001, one‐way ANOVA).

Article Snippet: CRT Rabbit Monoclonal Antibody (WL04297), Ki67 (bsm‐33 070 M), and HMGB1 Rb pAb (WL03023) were obtained from WanLeibio.

Techniques: Activation Assay, In Vivo, Saline, Irradiation, Immunohistochemical staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay