Journal: Canadian Journal of Gastroenterology & Hepatology

Article Title: Depletion and Reversal of Hepatocellular Carcinoma Inducing CTL through ER Stress-Dependent PERK-CHOP Signaling Pathway

doi: 10.1155/2022/6413783

Figure Lengend Snippet: Function of CTLs was induced by hepatoma cells. (a) CTLL-2 cells were cocultured with hepatoma cells. PD-1 and TIM-3 expressions were determined by western blotting analysis. (b) PRF1 and GzmB expression was analyzed by ELISA. Data are presented as the mean ± SD. ∗∗∗ P < 0.001, ns indicates no statistical significance.

Article Snippet: The supernatant was aliquoted into a centrifuged tube and used for the concentration-dependent detection of PRF1 (E-EL-M0890c, ElabScience, China) and GzmB (ELMO003, Boxbio, China) in strict accordance with the manufacturer's instructions.

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Canadian Journal of Gastroenterology & Hepatology

Article Title: Depletion and Reversal of Hepatocellular Carcinoma Inducing CTL through ER Stress-Dependent PERK-CHOP Signaling Pathway

doi: 10.1155/2022/6413783

Figure Lengend Snippet: Activation of ER stress induces CTLs depletion. (a) CTTL-2 cells were cocultured with Hepa1-6 cells for one day. Different concentrations of TM (0, 1, 2, and 4 μ M) were added to CTTL-2 cells and cultured for 1 day. Western blotting analysis was used to determine GRP78 levels. (b) Cells were treated with TM (0 and 4 μ M), and changes in ER morphology were analyzed by electron microscopy (scale bar = 2 μ m). (c) CTTL-2 cells were cocultured with Hepa1-6 cells for one day. Different concentrations of 4-PBA (0, 0.1, 0.2 and 0.4 mM) were added to CTTL-2 cells and cultured for one day. The expression of GRP78 was analyzed by western blotting. (d) Cells were treated with 4-PBA (0 and 0.4 mM), and ER morphology was examined by electron microscopy. (e) CTLL-2 cells were treated with TM, and western blotting was used to analyze PD-1 and TIM-3 expression. (f) ELISA analysis of PRF1 and GzmB expression. (g) CTLL-2 cells were treated with 4-PBA, and PD-1 and TIM-3 expression levels were measured by western blotting. (h) ELISA was used to detect the expression of PRF1 and GzmB. Data are presented as the mean ± SD. ∗∗∗ P < 0.001, ns indicates no statistical significance.

Article Snippet: The supernatant was aliquoted into a centrifuged tube and used for the concentration-dependent detection of PRF1 (E-EL-M0890c, ElabScience, China) and GzmB (ELMO003, Boxbio, China) in strict accordance with the manufacturer's instructions.

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

Journal: Canadian Journal of Gastroenterology & Hepatology

Article Title: Depletion and Reversal of Hepatocellular Carcinoma Inducing CTL through ER Stress-Dependent PERK-CHOP Signaling Pathway

doi: 10.1155/2022/6413783

Figure Lengend Snippet: CTLs depletion was induced through the ER stress PERK-CHOP pathway. After coculturing hepatoma cells with CTLL-2 cells, (a) western blotting was used to examine the expression of PERK, P-PERK, and CHOP. (b) TM-induced protein expression of PERK, P-PERK, and CHOP. (c) 4-PBA treatment induced a reduction in PERK, P-PERK, and CHOP protein levels. (d) Cells were cocultured for one day and CTLL-2 cells were treated with either GSK2656157 (0, 10, 50 and 100 nM) for one day. Western blotting analysis of ER stress-associated protein expression. (e) Electron microscopic analysis (scale bar = 2 μ m). (f) Protein expression of PD-1 and TIM-3 in CTLL-2 cells. (g) Expression of PRF1 and GzmB in CTLL-2 cells. (h) Expression of ER stress-associated proteins in CTLL-2 cells after one day of silencing CHOP plasmid (0 and 0.25 μ g/ μ L) treatment. (i) ER morphology was detected by electron microscopy (scale bar = 2 μ m). (j) Western blotting and (k) ELISA analysis. Data are presented as the means ± SD. ∗∗∗ P < 0.001, ns indicates no statistical significance.

Article Snippet: The supernatant was aliquoted into a centrifuged tube and used for the concentration-dependent detection of PRF1 (E-EL-M0890c, ElabScience, China) and GzmB (ELMO003, Boxbio, China) in strict accordance with the manufacturer's instructions.

Techniques: Western Blot, Expressing, Plasmid Preparation, Electron Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: A triple combination strategy for nasopharyngeal carcinoma: Aptamer-guided liposomal chemotherapy, engineered NK cells, and Fc-enhanced PD-L1 antibody therapy

doi: 10.1016/j.apsb.2025.10.007

Figure Lengend Snippet: Aptamer-engineered NK cells exhibit enhanced targeted cytotoxicity and effector functions against NPC cells. (A) Cytotoxicity of NK, S3-NK, P-NK, and S3-P-NK cells against 5-8F target cells, measured by LDH release assay across a range of effector-to-target (E:T) ratios after a 2 h co-culture. (B) Flow cytometry images demonstrated that NP69, 5-8F, and C666-1 cells were co-cultured with NK cells at a 10:1 E:T ratio for 2 h, washed with PBS, and co-incubated for 24 h, leading to apoptosis and necrosis. (C) Quantification of total apoptotic (early + late) and necrotic cell populations from the analysis shown in (B). (D) Schematic of the proposed mechanism for S3-P-NK cell-mediated antitumor immunity, involving targeted recognition followed by the release of cytotoxic granules (perforin, granzyme B) and immunostimulatory cytokines (IFN- γ ). (E–G) ELISA quantification of effector molecules released into the supernatant after co-culture of NK cells with 5-8F or C666-1 target cells (E:T = 10:1) for 2 h, PBS washing, and co-incubation for 24 h: (E) IFN- γ , (F) Granzyme B, (G) Perforin. Data in (A, C, E, F, G) are presented as mean ± SD ( n = 3). Statistical significance was determined by one-way ANOVA. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001; ns, not significant.

Article Snippet: ELISA kits for human IFN- γ (CSB-E08636h), granzyme B (CSB-E08718h), and perforin (CSB-E09313h) were acquired from Cusabio (Wuhan, China).

Techniques: Lactate Dehydrogenase Assay, Co-Culture Assay, Flow Cytometry, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Epigenetics

Article Title: IL-18 serves as a main effector of CAF-derived METTL3 against immunosuppression of NSCLC via driving NF-κB pathway.

doi: 10.1080/15592294.2023.2265625

Figure Lengend Snippet: Figure 2. CAF derived METTL3 alleviated PD-L1-mediated immunosuppression of NSCLC through IL-18. CAFs were transfected with sh-METTL3 or oe-METTL3, CAFs that transfected with sh-NC or oe-NC served as the negative control. a-b, the levels of METTL3 and IL- 18 in CAFs were detected by qRT-PCR and Western blot. c, the levels of IL-18 in CM from CAFs were detected by ELISA. d-e, PD-L1 levels in A549 and H1650 cells were assessed by Western blot and FCM. f, the levels of granzyme B and perforin in CD8+ T cells were detected by ELISA. g, the cytotoxicity of CD8+ T cells was detected by using LDH kit. All data were shown as mean ± SD. n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Firstly, CD8+T were co-cultured with NSCLC cells (A549 and H1650), which pretreated with CAF-CM or METTL3 knockdown or overexpression-CAFCM for 72 h. Subsequently, the cells were collected and homogenized by ultrasonic cell crusher, and the supernatant was collected after centrifugation 10 min. Then the levels of granzyme B and perforin were detected by Human GzmB (Granzyme B) ELISA Kit (E-EL-H1617c, Elabscience, Beijing, China) and Human PRF1 (Perforin 1) ELISA Kit (E-EL-H1123c, Elabscience), the level of IL-18 was detected by Human IL-18 ELISA Kit (E-ELH0253c, Elabscience), according to instructions.

Techniques: Derivative Assay, Transfection, Negative Control, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Epigenetics

Article Title: IL-18 serves as a main effector of CAF-derived METTL3 against immunosuppression of NSCLC via driving NF-κB pathway.

doi: 10.1080/15592294.2023.2265625

Figure Lengend Snippet: Figure 4. IL-18 was main effector of CAF-derived METTL3 against immunosuppression of NSCLC. a-b, IL-18 level was detected by qRT-PCR and Western blot. c-d, PD-L1 level was assessed by Western blot and FCM. e, the levels of granzyme B and perforin were detected by ELISA. F, the cytotoxicity of CD8+ T cells was detected by using LDH kit. All data were shown as mean ± SD. n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Firstly, CD8+T were co-cultured with NSCLC cells (A549 and H1650), which pretreated with CAF-CM or METTL3 knockdown or overexpression-CAFCM for 72 h. Subsequently, the cells were collected and homogenized by ultrasonic cell crusher, and the supernatant was collected after centrifugation 10 min. Then the levels of granzyme B and perforin were detected by Human GzmB (Granzyme B) ELISA Kit (E-EL-H1617c, Elabscience, Beijing, China) and Human PRF1 (Perforin 1) ELISA Kit (E-EL-H1123c, Elabscience), the level of IL-18 was detected by Human IL-18 ELISA Kit (E-ELH0253c, Elabscience), according to instructions.

Techniques: Derivative Assay, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Epigenetics

Article Title: IL-18 serves as a main effector of CAF-derived METTL3 against immunosuppression of NSCLC via driving NF-κB pathway.

doi: 10.1080/15592294.2023.2265625

Figure Lengend Snippet: Figure 5. IL-18 accelerated immunosuppression of NSCLC by driving NF-κB pathway. A-B, IL-18 level was detected by qRT-PCR and Western blot. C, NF-κB pathway related proteins’ expression was measured by Western blot. D-E, PD-L1 level was assessed by Western blot and FCM. F, the levels of granzyme B and perforin were detected by ELISA. G, the cytotoxicity of CD8+ T cells was detected by using LDH kit. All data were shown as mean ± SD. n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Firstly, CD8+T were co-cultured with NSCLC cells (A549 and H1650), which pretreated with CAF-CM or METTL3 knockdown or overexpression-CAFCM for 72 h. Subsequently, the cells were collected and homogenized by ultrasonic cell crusher, and the supernatant was collected after centrifugation 10 min. Then the levels of granzyme B and perforin were detected by Human GzmB (Granzyme B) ELISA Kit (E-EL-H1617c, Elabscience, Beijing, China) and Human PRF1 (Perforin 1) ELISA Kit (E-EL-H1123c, Elabscience), the level of IL-18 was detected by Human IL-18 ELISA Kit (E-ELH0253c, Elabscience), according to instructions.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: CD80-Mediated T-Cell Suppression by Cancer Stem-like Cells in Head and Neck Squamous Cell Carcinoma

doi: 10.3390/cells15030266

Figure Lengend Snippet: Blockade of CD80 inhibits HNSCC progression and enhances CD8 + T-cell function. ( A ) MOC1 and MOC2 cells were injected subcutaneously into C57BL/6 mice. Representative images show the tumors 18 days after implantation. ( B ) Tumor volume and tumor weight were significantly reduced in C57BL/6 mice treated with anti-CD80 antibody. Values are mean ± SD. ** p < 0.01; *** p < 0.001; Student’s t test; n = 6. ( C ) Representative H&E staining of tumors from mice injected with MOC2 cells. Scale bar, 25 μm. ( D – F ) Representative immunohistochemical staining and quantitative analysis of Caspase3, CD8, GZMB, Perforin, and IFN-γ from MOC2-bearing mice treated with isotype IgG or anti-CD80 antibody. Scale bar, 40 μm. Values are mean ± SD. ** p < 0.01; *** p < 0.001; Student’s t test; n = 6.

Article Snippet: The primary antibodies used were anti-caspase3 antibody (1:100, Absin Bioscience Inc., Shanghai, China, Cat#abs119676), anti-CD8 antibody (1:100, Santa Cruz Biotechnology, Dallas, TX, USA, Cat#sc-1177), anti-GZMB antibody (1:100, Proteintech, Wuhan, China, Cat# 13588-1-AP), anti-perforin antibody (1:100, Proteintech, Wuhan, China, Cat# 14580-1-AP), and anti-IFN- γ antibody (1:100, Proteintech, Wuhan, China, Cat#15365-1-AP).

Techniques: Cell Function Assay, Injection, Staining, Immunohistochemical staining

Journal: Journal of Immunology Research

Article Title: Unveiling the Interplay Between Dendritic Cells and Natural Killer Cells as Key Players in Leishmania Infection

doi: 10.1155/jimr/3176927

Figure Lengend Snippet: Degranulation of pNK cells induced by loaded moDCs. The release of perforin (A) and granzyme B (B) by pNK cells stimulated by moDCs infected with L. infantum (moDC+Li) or L. amazonensis (moDC+La) parasites, or primed by EVs (moDC+LaEVs or moDC+LiEVs) were evaluated using colorimetric immunoassays. Perforin and granzyme B released by pNK cells induced by unloaded moDCs (moDC) were also assessed. Data from six canine samples ( n = 6) with two replicates per sample are represented by box plots showing interquartile range, median, maximum, and minimum values. Statistically significant differences are indicated by ⁣ ∗ p < 0.05 and ⁣ ∗∗ p < 0.01. EVs, extracellular vesicles; moDCs, monocyte-derived DCs.

Article Snippet: To assess NK cell cytotoxicity, perforin, and granzyme B concentrations were estimated in the coculture supernatants using the dog/canine perforin 1 PRF1 ELISA Kit (Novatein Bioscience, USA) and Human granzyme B ELISA Kit (Quimigen Unipessoal LDA, Portugal), respectively, following the manufacturer's instructions.

Techniques: Infection, Derivative Assay

Journal: Journal of Immunology Research

Article Title: Unveiling the Interplay Between Dendritic Cells and Natural Killer Cells as Key Players in Leishmania Infection

doi: 10.1155/jimr/3176927

Figure Lengend Snippet: Proposed model illustrating the in vitro crosstalk between DCs and NK cells during Leishmania infection. DCs infected with L. infantum (Li) or L. amazonensis (La), or primed with EVs derived from L. infantum (LiEVs) or L. amazonensis (LaEVs) activate innate immune cells through distinct pathways. Loaded DCs induce NK cells to generate chemokines, which can attract other immune cells to the site of infection. The interplay between L. infantum infected moDCs and NK cells promotes the generation of the immunosuppressive cytokine IL-10, which can favor active infection. In contrast, L. amazonensis drives a mix of anti- and pro-inflammatory cytokines associated with perforin release, promoting a balanced immune response, that may limit infection and favor the establishment of chronic disease. DCs primed with parasite derived EVs stimulate the upregulation of anti-inflammatory cytokines, highlighting the role of EVs in shaping the early immune response. EVs, extracellular vesicles; moDCs, monocyte-derived DCs.

Article Snippet: To assess NK cell cytotoxicity, perforin, and granzyme B concentrations were estimated in the coculture supernatants using the dog/canine perforin 1 PRF1 ELISA Kit (Novatein Bioscience, USA) and Human granzyme B ELISA Kit (Quimigen Unipessoal LDA, Portugal), respectively, following the manufacturer's instructions.

Techniques: In Vitro, Infection, Derivative Assay