Journal: Bioactive Materials

Article Title: Self-assembled PROTACs enable protein degradation to reprogram the tumor microenvironment for synergistically enhanced colorectal cancer immunotherapy

doi: 10.1016/j.bioactmat.2024.09.022

Figure Lengend Snippet: NP-PROTACs degraded STAT3 and β-catenin, improving the TIME in vivo . ( A ) Treatment regimen diagrams (n = 6). ( B and D ) After the mice were sacrificed, the tumors were photographed, and the tumor weights were recorded. ( C and E ) Both tumor volume and body weight were monitored every day. ( F ) Immunofluorescence staining of tumor tissue revealed the downregulated target protein in each treatment group (scale bars = 100 μm). ( G – J ) Western blot analyses showed that micelle treatment inhibited β-catenin and/or STAT3 levels in vivo . ( K – N ) The levels of CD103 in DCs, the levels of granzyme B and IFN-γ in T cells, and the polarization of M2-type macrophages were analyzed through flow cytometry (n = 3). ( O ) The statistics of the above immune cells were presented as bars, and CD4 + T cell, CD8 + T cell, Th1 infiltration, and polarization of M2-type macrophages were also determined by flow cytometry and were presented. The data were presented as the mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The antibody for CD8a (10122-80-100) was from Biogems.

Techniques: In Vivo, Immunofluorescence, Staining, Western Blot, Flow Cytometry

Journal: Bioactive Materials

Article Title: Self-assembled PROTACs enable protein degradation to reprogram the tumor microenvironment for synergistically enhanced colorectal cancer immunotherapy

doi: 10.1016/j.bioactmat.2024.09.022

Figure Lengend Snippet: Schematic diagram of the anti-tumor mechanism of dual-target-degrading NP-PROTACs. ( A ) β-catenin-targeting LAVTAT and STAT3-targeting SAVTAT were conjugated with DSPE-PEG 2000 -SH through disulfide bonds, jointly self-assembling into NP-PROTACs. ( B ) Upon intravenous injection and accumulation in CRC models, NP-PROTACs released LAVTAT and SAVTAT intracellularly due to cleaved disulfide bonds. Co-degradation of β-catenin and STAT3 triggered cell cycle arrest and apoptosis on CRC cells and improved immune microenvironment. Briefly, the cytotoxic function of CD8 + T cells was activated; an increase in the ratio of M1-type macrophages and a decrease in the ratio of M2-type macrophages were observed; the infiltration of CD103 + DC cells was increased.

Article Snippet: The antibody for CD8a (10122-80-100) was from Biogems.

Techniques: Injection

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: A IOD of ACAT2 expression in CC tissues and adjacent tissues was examined using immunohistochemical staining ( n = 47 biologically independent samples). IOD of DHCR7 B and MSMO1 C expression in CC patients with high ( n = 27 biologically independent samples) or low ( n = 20 biologically independent samples) expression of ACAT2 was examined using immunohistochemical staining. The number of activated CD8 T cells (CD8A + GZMB + ) D or activated NK cells (CD56 + GZMB + ) E infiltrated in the tumor tissues of patients with high ( n = 27 biologically independent samples) and low ACAT2 ( n = 20 biologically independent samples) expression was detected. Data represent mean ± SEM. Statistical analysis was performed using the paired A or unpaired ( B – E ) t-test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: ACAT2 expression in HCeEpiC and CC cell lines was examined using RT-qPCR A and Western blot analysis B ( n = 5 independent experiments). C ACAT2, DHCR7, and MSMO1 expression in CC cells after infection with Scramble-sh, ACAT2-sh #1, and ACAT2-sh #2 was examined using Western blot analysis ( n = 5 independent experiments). D Detection of total cholesterol, free cholesterol, and cholesteryl ester levels in CC cells ( n = 5 independent experiments). The proliferation of CC cells was examined using CCK8 ( E ) and colony formation assays F (n = 5 independent experiments). G CC cells were co-cultured with (E: T = 3:1) with NK cells or CD8 T cells for 6 h, respectively, and the death of CC cells was detected ( n = 5 independent experiments). H IFN-γ and GZMB released from immune cells in a co-culture system with CC cells were examined using ELISA ( n = 5 independent experiments). Data represent mean ± SEM. Statistical analysis was performed using the one-way ( A , B ) or two-way ( C - H ) ANOVA, followed by Tukey’s multiple comparisons test ( A – H ).

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Infection, Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: A ACAT2 knockdown efficiency in U14 cells was examined using western blot analysis ( n = 10 independent experiments). B Volume changes of transplanted tumors in mice subcutaneously inoculated with U14 cells (n = 10 animals). C The images and weight of the tumors harvested on day 21 ( n = 10 animals). D Protein expression of ACAT2, MSMO1, DHCR7, and PCNA in transplanted tumors was examined using western blot analysis ( n = 10 animals). E Detection of total cholesterol, free cholesterol, and cholesteryl ester levels in transplanted tumors ( n = 10 animals). The gating strategy for GZMB + NK cells and CD8 + T cells F and quantification G were analyzed using flow cytometry ( n = 10 animals). H Survival of mice over 60 days after subcutaneous inoculation of U14 cells was analyzed using the log-rank test ( n = 20 animals). Data represent mean ± SEM. Statistical analysis was performed using the one-way ( A , C , E , G ) or two-way ( B , D ) ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Knockdown, Western Blot, Expressing, Flow Cytometry

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: The proliferation of CC cells was examined using CCK8 A and colony formation assays B ( n = 5 independent experiments). C CC cells were co-cultured with (E: T = 3:1) with NK cells or CD8 + T cells, and the death of CC cells was detected ( n = 5 independent experiments). D IFN-γ and GZMB released from immune cells in a co-culture system with CC cells were examined using ELISA ( n = 5 independent experiments). E TGF-β1 released by CC cells was examined using ELISA ( n = 5 independent experiments). F PD-L1 expression levels in CC cells were observed using immunofluorescence staining ( n = 5 independent experiments). Data represent mean ± SEM. Statistical analysis was performed using the two-way ( A – F ) ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Staining

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: A Volume changes of transplanted tumors in mice subcutaneously inoculated with U14 cells ( n = 5 animals). B The images and weight of the tumors harvested on day 21 ( n = 5 animals). The gating strategy for GZMB + NK cells and CD8 + T cells C and quantification D were analyzed using flow cytometry ( n = 5 animals). Data represent mean ± SEM. Statistical analysis was performed using the one-way ( B , D ) or two-way A ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Flow Cytometry

Journal: Pharmacological research

Article Title: USP19 deficiency enhances T-cell-mediated antitumor immunity by promoting PD-L1 degradation in colorectal cancer.

doi: 10.1016/j.phrs.2025.107668

Figure Lengend Snippet: Fig. 5. PD-L1 reconstitution reverses USP19 deficiency-induced antitumor immunity. A, B PD-L1 reconstituted in USP19 depletion RKO cells was co-cultured with activated T cells for 48 hours, crystal violet (A) and CCK-8 (B) showed that PD-L1 reconstituted promoted RKO cell viability. C, D Annexin V-FITC and pro- pidium iodide (PI) apoptosis assays showed that PD-L1 reconstituted reversed the apoptosis ratio mediated by USP19 deficiency. E Western blot analysis showed that PD-L1 reconstituted reversed the cleaved caspase-3 protein expression mediated by USP19 deficiency. F-H Flow cytometry revealed that PD-L1 reconstitution reversed the USP19 deficiency-induced increase in the secretion of IFN-γ and GzmB by CD8+ T cells. Data in (B, D, G, H) are shown as mean ± SD. Statistical significance was assessed using one-way and two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: GAPDH (cat# AC002, ABclonal), USP19 (cat# 25768–1-AP, Proteinch), Myc (cat# M192–3, MBL), PD-L1 (cat# 28076–1-AP, Proteinch), HA (cat# M180–3, MBL), Flag (cat #M185–3L, MBL), Lymphocyte separation medium (cat# 7111012, Dakewe), PE/Cyanine7 anti-human CD8a (cat# 300914, BioLegend), FITC anti-human CD3 (cat# 300306, BioLegend), APC anti-human IFN-γ (cat# 502512, BioLegend), Brilliant Violet 421TM anti-human/mouse Granzyme B Recombinant (cat# 396414, BioLegend), Zombie AquaTM Fixable Viability Kit (cat# 423101,BioLegend), Brefeldin A Solution (1,000X) (cat# 420601, BioLegend), Fixation Buffer (cat# 420801, BioLegend), Intracellular Staining Perm Wash Buffer (10X) (cat# 421002, BioLegend), Rat IgG2b isotype control-InVivo (cat# A2116, selleck), InVivcMAb anti-mouse PD-L1 (cat# 10 F.9G2, BioXCell), FITC Anti-Mouse CD4 Antibody [GK1.5] (cat# E-AB-F1097C, Elabscience), PerCP/Cyanine5.5 Anti-Mouse CD3 Antibody (cat# E-AB-F1013J, Elabscience), PE AntiMouse CD3 Antibody [17A2] (cat# E-AB-F1013D, Elabscience), APC Anti-Mouse CD8a Antibody [53–6.7] (cat# E-AB-F1104E, Elabscience), PE/Cyanine7 Anti-Mouse CD45 Antibody (cat# E-AB-F1136H, Elabscience), Caspase-8 Rabbit mAb (cat# 4790, CST), Cleaved Caspase-8 Rabbit mAb (cat# 9496, CST), Cleaved Caspase-9 Rabbit mAb (cat# 7237, CST), Caspase-9 Rabbit mAb (cat# A119654, ABclonal), Caspase3 Rabbit pAb (cat# A0214, ABclonal), Cleaved Caspase-3 (5A1E) Rabbit mAb (cat# 9664, CST), PARP (46D11) Rabbit mAb (cat# 9532, CST).

Techniques: Cell Culture, CCK-8 Assay, Western Blot, Expressing, Flow Cytometry

Journal: Nature immunology

Article Title: Cytotoxic innate lymphoid cells sense cancer cell-expressed interleukin-15 to suppress human and murine malignancies.

doi: 10.1038/s41590-022-01213-2

Figure Lengend Snippet: Fig. 1 | chRCC and ccRCC tumors exhibit differential immune cell infiltration and CD8 T cell phenotypes. a, t-Distributed stochastic neighbor embedding (tSNE) of transcriptional profiles from leukocytes isolated from one chRCC tumor and one ccRCC tumor. Each dot represents a single CD45+ cell, and colors represent clusters denoted by cell type inferred from lineage markers and differential gene expression. b, tSNE plot as in a, colored by histology (chromophobe or clear cell). c, For each CD8 cluster, the frequency out of all CD8α+ clusters at which it was found in chRCC (n = 1) and ccRCC (n = 1) tumors. d, Violin plots showing log-normalized expression of selected differentially expressed genes (DEGs) among three CD8 clusters. e, Representative plots of flow cytometric analysis of the percentage of CD3+CD8α+ T cells out of the lymphocyte gate (CD45+SSCLow) in blood, adjacent normal kidney and tumor samples from one patient of the indicated histology. Quantification is CD3+CD8α+ T cells out of total CD45+ cells. f, Representative histograms of PD-1 expression in CD3+CD8α+ T cells from blood, adjacent normal kidney and tumor tissues from a single patient of the indicated histology. Quantification of flow cytometric analysis of percentage of PD-1+CD3+CD8α+ T cells in blood, adjacent normal kidney and tumor samples from the indicated histology. e,f, Each pair of symbols connected by a line denotes an individual patient (chRCC blood n = 6 (e and f), kidney n = 9 (e) and 10 (f), tumor n = 9 (e) and 10 (f); ccRCC blood n = 14, kidney n = 15 and tumor n = 16). One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test was used for statistical analysis (NS, not significant; **P < 0.01, ****P < 0.0001.

Article Snippet: Antihuman CD8a (RPA-T8, #25-0088-T100) and biotinylated anti-human CD3 (UCHT1, #30-0038-U100) were purchased from Tonbo Biosciences.

Techniques: Isolation, Gene Expression, Expressing, Comparison

Journal: Aging (Albany NY)

Article Title: EGFR-specific CAR-T cells trigger cell lysis in EGFR-positive TNBC

doi: 10.18632/aging.102510

Figure Lengend Snippet: Characterization of T lymphocytes from PBMCs. ( A – B ) T cell phenotypes and subsets were examined by flow cytometry after labeling with anti-CD3-PE-Cy7, anti-CD4-PE, and anti-CD8-APC-Cy7.

Article Snippet: Anti-Human CD3 (PE-Cy7), anti-Human CD4 (PE), and anti-Human CD8 (APC-Cy7) antibodies, as well as corresponding mouse IgG controls, were purchased from BioGems.

Techniques: Flow Cytometry, Labeling

Journal: iScience

Article Title: Hypoxic-immune axis orchestrates metastatic dissemination via HIF isoform imbalance in pancreatic neuroendocrine tumors

doi: 10.1016/j.isci.2025.114340

Figure Lengend Snippet: Immune cell infiltration in KRAS G12C mutant PNETs (A) Quantitative analysis using Image Plus 6.0 software reveals a significant increase in Tregs (CD4 + , CD25 + , and FoxP3 + ) within KRAS- G12C mutant PNETs compared to wild-type KRAS PNET tissues, suggesting KRAS G12C -driven immunosuppressive cell recruitment. (B) Fluorescence intensity analysis demonstrates reduced infiltration of CD8 + cytotoxic T cells and HLA-DR + activated cells in KRAS G12C mutant tumors, indicative of impaired antitumor immunity. (C) Elevated MDSCs counts in KRAS G12C mutant PNETs correlate with enhanced immune evasion.

Article Snippet: APC Anti-Human CD8a Antibody [OKT-8] , Elabscience , E-AB-F1110E.

Techniques: Mutagenesis, Software, Fluorescence

Journal: iScience

Article Title: Hypoxic-immune axis orchestrates metastatic dissemination via HIF isoform imbalance in pancreatic neuroendocrine tumors

doi: 10.1016/j.isci.2025.114340

Figure Lengend Snippet: Tregs, CD8 + T cells, and HLA-DR + cells in KRAS G12C -mutated PNETs (A) Flow cytometry plots and fluorescence intensity histograms demonstrate elevated CD4 + T cell proportions in KRAS G12C patient blood samples compared to wild-type KRAS tumors and healthy controls. (B) Quantification shows a significant enrichment of CD25 + T cells in KRAS G12C patients, surpassing both wild-type KRAS tumors and normal controls. (C) Quantitative data and histogram overlays confirm a substantial increase in FoxP3 + T cells frequency in KRAS G12C patients, with levels moderately elevated compared to wild-type KRAS and significantly higher than healthy individuals. (D) A slight decrease in CD8 + T cell frequency in KRAS G12C samples relative to wild-type KRAS, with levels significantly lower than those in healthy individuals (E) A moderate reduction in HLA-DR + cell frequency in KRAS G12C patients compared to wild-type KRAS, alongside a marked suppression relative to healthy controls.

Article Snippet: APC Anti-Human CD8a Antibody [OKT-8] , Elabscience , E-AB-F1110E.

Techniques: Flow Cytometry, Fluorescence