cd8 t cell depletion experiment  (Bio X Cell)

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Iron overload‐associated ZDHHC12 primarily participates in maintaining the activation of CD8+T cells in renal cell carcinoma. (a) UMAP plot showing the subpopulation classification of CD8+ T cells ( GSE121636 ). (b) UMAP visualization of ZDHHC12 expression across different CD8+ T cell subsets. (c) Violin plot depicting ZDHHC12 expression patterns in various CD8+ T cell subpopulations. (d) BALB/c mice fed either a high‐iron‐load diet or a normal diet were subcutaneously inoculated with Renca tumor cells on the dorsum, and starting from the day of inoculation, 200 µg of anti‐CD8 depletion antibody (αCD8) or an isotype control antibody was intraperitoneally administered every three days; tumor volume was measured from day 7 post‐inoculation. Data were presented as mean ± SD. e and f, Western blot (e) and RT‐qPCR (f) analyses were performed on CD8+ T cells sorted from renal cancer tissues of both the normal diet and high‐iron diet groups. (g–i), The figure illustrates schematic diagrams of different treatment groups (g). Specifically, tumor cells were isolated and sorted from the tumor tissues of the renal cancer model. Subsequently, the collected tumor cells were subcutaneously injected into the dorsum of either Zdhhc12 wild‑type or knockout mice. Tumor size was measured regularly, and growth curves were plotted (h). On day 19, the mice were sacrificed, and tumors were excised and weighed (i). (j–m), Portions of tumor tissues were processed for cell extraction, staining, and flow cytometry analysis to evaluate CD8+ T cell function in Zdhhc12 wild‐type and KO mice. Specifically, the proportions of CD3+/CD45+ (j), CD8+/CD3+ (k), GZMB+/CD8+ (l), and IFN‐γ+/CD8+ (m) T cells within tumor masses were quantified. (n,o) Portions of tumor tissues were embedded, sectioned, stained, and subjected to immunofluorescence analysis to assess CD8+ T cell function in Zdhhc12 wild‐type and KO mice. Specifically, multiplex staining was performed for DAPI/CD3/CD8/GZMB, and the proportions of positive cells were quantified (n); similarly, multiplex staining for DAPI/CD3/CD8/IFN‐γ was conducted with subsequent quantification of positive cell ratios (o).

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Activation Assay, Expressing, Control, Western Blot, Quantitative RT-PCR, Isolation, Injection, Knock-Out, Extraction, Staining, Flow Cytometry, Cell Function Assay, Immunofluorescence, Multiplex Assay

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Iron overload promotes the ubiquitin‐proteasome pathway degradation of ZDHHC12 by enhancing its binding with TRIM28. (a) Cells were collected for Western blot analysis after treating sorted CD8+ T cells with FAC at the indicated concentrations for 48 h. (b) Cells were collected for Western blot analysis after treating sorted CD8+ T cells with FAC at the indicated concentration gradients for 48 h. (c) Cells were collected for Western blot analysis after treating 293T cells with FAC at the indicated concentrations for 48 h. (d) Cells were collected for Western blot analysis after treating 293T cells with FAC at the indicated concentration gradients for 48 h. (e) The degradation of ZDHHC12 in 293T cells was assessed by CHX chase assay after treatment with or without FAC (20 µg/mL) for 48 h. (f) HA‐ZDHHC12 was transfected into 293T cells, and co‐immunoprecipitation was performed using an anti‐HA‐ZDHHC12 antibody, followed by mass spectrometry to identify potential ZDHHC12‐interacting proteins. The peptide spectrum of TRIM28 is shown. (g) The degradation of ZDHHC12 in 293T cells was assessed by CHX chase assay with or without TRIM28 knockout. (h) Schematic diagrams depicting the protein domain architecture of TRIM28 and the TRIM28 △RING truncation mutant. (i) Sorted CD8+ T cells were stably transfected with the plasmids illustrated in the figure, followed by cell collection for Western blot analysis and quantitative assessment. (j) After transfection with the indicated plasmids, 293T cells were treated with FAC, followed by MG132 treatment, and then collected for Co‐IP and Western blot analysis. (k) After treatment with FAC (0, 20, 40 µg/mL), 293T cells were treated with MG132, collected, and subjected to Co‐IP and Western blot analysis. (l) After transfection with the indicated plasmids for 24 h, 293T cells were collected for Co‐IP and Western blot analysis.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Ubiquitin Proteomics, Binding Assay, Western Blot, Concentration Assay, Transfection, Immunoprecipitation, Mass Spectrometry, Knock-Out, Mutagenesis, Stable Transfection, Co-Immunoprecipitation Assay

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: ZDHHC12 inhibits cuproptosis in CD8+T cells through interaction with FDX1. (a) We reviewed the mass spectrometry results and found that FDX1 may interact with ZDHHC12, and the peptide spectrum of FDX1 is shown in the figure. (b) HA‐ZDHHC12 and Flag‐FDX1 were transfected into 293T cells, followed by cell lysis and co‐immunoprecipitation using Flag or HA antibodies, and then analyzed by western blot. (c) 293T cells were lysed and subjected to co‐immunoprecipitation with ZDHHC12 or FDX1 antibodies, followed by western blot detection. (d) Immunofluorescence staining was performed in 293T cells using ZDHHC12 and FDX1 antibodies, and images were acquired and analyzed by confocal microscopy. (e) GST pull‐down assay was performed using the recombinant protein of ZDHHC12. (f) GST pull‐down assay was performed using the recombinant protein of FDX1. (g) Protein structure prediction and molecular docking were conducted for ZDHHC12 and FDX1. (h,i) Western blot (h) and RT‐qPCR (i) were performed in 293T cells after transfection with the indicated plasmids. (j,k) Western blot (j) and RT‐qPCR (k) were performed in 293T cells after transfection with the indicated plasmids. (l) Western blot analysis was conducted in 293T cells 72 h after transfection with the indicated plasmids. (m) Western blot analysis was performed in 293T cells after transfection with the indicated plasmids. (n) CD8+ T cells were isolated from renal cancer tissues of Zdhhc12 WT and Zdhhc12 KO mice and subjected to western blot analysis. (o) After successful transfection with the indicated plasmids in 293T cells, cells were collected and treated with different concentrations of elesclomol‐Cu or disulfiram‐Cu as shown in the figure for 48 h, followed by CCK‐8 assay to measure and quantify cell viability. (p,q) Following successful transfection with the indicated plasmids in 293T cells, cells were collected and immunostained with DLAT and Mito‐tracker, then imaged by confocal microscopy (p) to quantify (q) DLAT oligomerization. (r) After successful transfection with the indicated plasmids in 293T cells, cells were treated with different concentrations of elesclomol‐Cu or disulfiram‐Cu for 48 h in the presence or absence of tetrathiomolybdate (TTM) (1 µ m ) as indicated, followed by CCK‐8 assay to measure and quantify cell viability. (s,t) Following successful transfection with the indicated plasmids in 293T cells, cells were collected after treatment with or without tetrathiomolybdate (TTM) (1 µ m ), then immunostained with DLAT and Mito‐tracker and imaged by confocal microscopy (s) to quantify (t) DLAT oligomerization. (u) After successful transfection with the indicated plasmids in CD8+ T cells, cells were treated with or without tetrathiomolybdate (TTM) (1 µ m ), then collected for western blot analysis. (v) Following successful transfection with the indicated plasmids in 293T cells, cells were collected and treated with different concentrations of elesclomol‐Cu or disulfiram‐Cu as shown in the figure for 48 h, followed by CCK‐8 assay to measure and quantify cell viability. (w) After successful transfection with the indicated plasmids in sorted CD8+ T cells, cells were collected for western blot analysis.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Mass Spectrometry, Transfection, Lysis, Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Confocal Microscopy, Pull Down Assay, Recombinant, Quantitative RT-PCR, Isolation, CCK-8 Assay

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: ZDHHC12‐mediated palmitoylation of FDX1 at Cys152/155 promotes its degradation. (a) In 293T and CD8+T cells, FDX1 was immunoprecipitated using an anti‐FDX1 antibody, followed by the acyl‐biotin exchange (ABE) assay performed either in the presence or absence of hydroxylamine (HAM) treatment, with subsequent streptavidin‐HRP pulldown of biotin‐conjugated proteins to specifically detect palmitoylated FDX1. (b) In 293T cells, FDX1 was immunoprecipitated using an anti‐ FDX1 antibody, followed by the acyl‐biotin exchange (ABE) assay performed with or without hydroxylamine (HAM) treatment, and subsequently enriched with streptavidin‐HRP to isolate biotin‐conjugated proteins, thereby enabling the detection of FDX1 palmitoylation levels in both 2‐BP (25 µ m , 24 h)‐treated and untreated conditions. (c) 293T cells treated with or without palmitic acid azide were collected for Click‐IT reaction and streptavidin pulldown. (d) 293T cells were treated with different concentrations of 2‐BP (+, 20 µ m ; ++, 40 µ m ) for 24 h, then collected for western blot analysis. (e) 293T cells treated with or without 2‐BP (25 µ m , 24 h) were exposed to CHX and collected at different time points for western blot analysis. (f) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (g) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (h) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (i) 293T cells transfected with the indicated plasmids were treated with or without 2‐BP (25 µ m , 24 h) after 24 h, then collected for western blot analysis. (j) The peptide spectrum for FDX1 palmitoylation site identification. (k) Conservation sequences of FDX1 C152 and FDX1 C155 sites across different species. (l) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to Click‐iT pull‐down assay and western blot analysis with or without palmitic acid azide treatment. m, AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (n) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to Click‐iT pull‐down assay and western blot analysis with or without palmitic acid azide treatment, followed by quantitative analysis of FDX1 palmitoylation levels. (o) CD8+ T cells were transfected with the indicated plasmids, then treated with PBS or FAC (20 µg/mL), collected, and stained with the indicated fluorescent dyes for flow cytometry analysis. (p) CD8+ T cells were transfected with the indicated plasmids, then collected and stained with the indicated fluorescent dyes for flow cytometry analysis. (q) CD8+ T cells were transfected with the indicated plasmids, then treated with PBS or FAC (20 µg/mL), collected, and stained with the indicated fluorescent dyes for flow cytometry analysis.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Immunoprecipitation, Western Blot, Knock-Out, Transfection, Pull Down Assay, Staining, Flow Cytometry

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Development of lipid nanoparticles encapsulating Zdhhc12 mRNA (Zdhhc12‐LNP). (a) Schematic diagram of the LNP synthesis process; (b) The average zeta potential and the average particle size; (c) The average PDI and the average encapsulation efficiency; (d) Representative cryo‐EM image showing the morphology of synthesized LNPs. (e) Spectrum showing the purity of the sample. (f) Representative chromatogram showing mRNA capping efficiency. (g) Western blot analysis of Zdhhc12 expression in CD8+ T cells isolated from mice and treated with control, blank LNP, or Zdhhc12‐encapsulated LNP, following a 24 h pre‐stimulation with 2 µg/mL anti‐CD3/CD28 and subsequent 24 h incubation with LNPs. (h) ABE assay and Western blot analysis were performed on CD8+ T cells isolated from mice to assess the palmitoylation level of Fdx1, where cells were first cultured for 24 h post‐infection without anti‐CD3/CD28, restimulated with 2 µg/mL anti‐CD3/CD28 for 24 h, incubated with LNPs for 24 h, and then harvested for analysis. (i) Renca cells were subcutaneously injected into the backs of BALB/c mice, LNPs were administered via intratumoral injection, and when tumors reached an appropriate size, tumors were harvested for sorting of CD8+ T cells, followed by Western blot analysis to assess Zdhhc12 expression level in CD8+ T cells. (j–m) Renca cells were subcutaneously injected into the dorsal region of BALB/c mice. After tumor establishment, mice received intratumoral injection of LNPs or no injection, and serum levels of ALT (j), AST (k), CRE (l), and BUN (m) were measured at appropriate time points. Ns, not significant. (n,o) BALB/c mice were subcutaneously injected with Renca cells, intratumorally administered Zdhhc12‐LNP or control, and intraperitoneally injected with 200 µg anti‐CD8 (αCD8) or control; tumor growth was recorded starting from day 7 post‐tumor inoculation (n), and flow cytometry analysis of CD8+ T cell content in the peripheral blood of mice (n = 5) was performed at the end of the experiment (o), with data presented as mean ± SD.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Zeta Potential Analyzer, Encapsulation, Cryo-EM Sample Prep, Synthesized, Western Blot, Expressing, Isolation, Control, Incubation, Cell Culture, Infection, Injection, Flow Cytometry

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Under normal iron conditions, ZDHHC12‐mediated palmitoylation of FDX1 promotes FDX1 degradation by the mitochondrial matrix protease AFG3L2, ultimately suppressing cuproptosis in CD8+ T cells and maintaining their immune clearance function. Under iron overload conditions, increased binding between ZDHHC12 and TRIM28 leads to ZDHHC12 degradation, consequently inducing cuproptosis in CD8+ T cells and facilitating immune escape.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Histone lactylation increases CXCL1 expression for neutrophil infiltration and immune escape in pancreatic cancer

doi: 10.1038/s41467-026-69311-5

Figure Lengend Snippet: A The expression of neutrophils and CD8 + T cells in low- and high-glycolysis clusters, divided by ssGSEA score of glycolysis-related genes, in TCGA and CPTAC PAAD database. Data are presented as box plots showing the median (center line), the first and third quartiles (box bounds), and the whiskers extend to 1.5 times the interquartile range from the box. B The correlation of glycolysis level and neutrophils or CD8 + T cells expression in TCGA ( n = 179 patients) and CPTAC ( n = 140 patients) PAAD database. C Kaplan–Meier plots representing survival probabilities in TCGA-PAAD patients according to the relative level of glycolysis-related or neutrophil-related gene expression. D The diagram illustrating the targets of 2DG and shRNA in the glycolysis process. E CyTOF analysis of tumor-infiltrating immunocytes in subcutaneous tumor models with or without 2DG treatment: tSNE plots showing 12 meta-clusters based on the expression of 41 markers for the immunocytes. F Bar chart of the frequencies of the immune cell subsets in two experimental groups. G A schematic diagram showing the orthotopic PDAC model ( n = 6 mice per group in one experiment) with or without 2DG treatment. H , I Image and weights of the orthotopic tumors in the experimental groups from ( G ) at the end of the experiments. ( n = 6 mice per group). ( J – M ) Tumor-infiltrating neutrophils, CD8 + T cells, GZMB + CD8 + T cells, and PD-1 + CD8 + T cells isolated from the orthotopic tumors were analyzed using flow cytometry: representative graphs of flow cytometry (left panel) and statistical analysis of the cell ratio (right panel) ( n = 6 mice per group). N A schematic diagram showing the orthotopic tumor model in C57BL/6 J mice ( n = 6 mice per group in one experiment) treated with or without anti-Ly6G/anti-CD8 antibody. O , P Image and weights of the orthotopic tumors in the experimental groups from ( N ) at the end of the experiments ( n = 6 mice per group). Q – S Tumor-infiltrating neutrophils, CD8 + T cells, and GZMB + CD8 + T cells isolated from the orthotopic tumors were analyzed using flow cytometry: Representative graphs of flow cytometry (left panel) and statistical analysis of the cell ratio (right panel) ( n = 6 mice per group). T , U Representative luminescence images of the mouse model in ( N ) and statistical analysis of the total flux ( n = 6 mice per group). Data represent mean ± SEM. Statistical analysis was conducted using the two-tailed unpaired Student’s t test ( A, I–M, P–S, and U ), two-tailed Pearson’s correlation analysis ( B ), and log-rank test ( C ). Source data are provided as a Source Data file.

Article Snippet: After 11 consecutive days of antibody administration, the mice were euthanized, the peripheral blood was harvested for flow cytometry analysis to assess the efficiency of neutrophil depletion; (3) For CD8 + T cells depletion, mice received an intraperitoneal injection of anti-IgG2a or anti-CD8 antibody (Selleck, #A2102) treatment (0.2 mg/mouse, i.p.) 2 days before implantation.

Techniques: Expressing, Gene Expression, shRNA, Isolation, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Histone lactylation increases CXCL1 expression for neutrophil infiltration and immune escape in pancreatic cancer

doi: 10.1038/s41467-026-69311-5

Figure Lengend Snippet: A The flowchart illustrates that RNA sequencing was conducted using two PDAC cell lines with or without 2DG treatment. Subsequently, the intersection of detected genes within the chemokine family in both cell lines is identified, and a heatmap is generated to display the log 2 fold change (2DG versus vehicle) of these genes. B , C Relative mRNA levels of Cxcl1 in PDAC cell lines following glycolysis inhibition, either by using 2DG or silencing LDH, were analyzed by qRT-PCR ( n = 3 independent experiments). D , E Relative protein levels of CXCL1 in PDAC cell lines following glycolysis inhibition, either by using 2DG or silencing LDH, were determined using ELISA assay ( n = 3 independent experiments). F Representative IHC images of subcutaneous tumors ( n = 6 mice for 2DG treatment, n = 5 mice for shLDH/shNTC groups) stained by CXCL1 antibody (scale bars = 100 μm). G Relative serum CXCL1 levels in mice treated with or without 2DG were measured by ELISA ( n = 6 mice per group). H Relative serum CXCL1 levels in healthy donors and PDAC patients were measured by ELISA (22 healthy samples and 27 PDAC samples). I Schematic diagram showing the in vitro migration assay: human/mouse neutrophils were co-incubated with the culture medium supernatant from PDAC cells treated with 2DG or LDH-knockdown. J Neutrophils were co-cultured with CD8 + T cells in different proportions, and the proliferation of CD8 + T cells was detected with the CFSE assay ( n = 6 biologically independent samples). K The migratory activity of neutrophils co-incubated with the culture medium supernatant from PDAC cells treated with shLDH and recombinant CXCL1 was analyzed by counting the penetrated cell numbers ( n = 3 biologically independent samples). L The neutrophils were treated with SX-682 or Navarixin for 1.5 h in advance, and the migratory activity of neutrophils co-incubated with the culture medium supernatant from PDAC cell lines treated with shLDH was analyzed by counting the penetrated cell numbers ( n = 3 biologically independent samples). M Representative luminescence images of the orthotopic tumor in mice and statistical analysis of MFI ( n = 5 mice per group). N Tumor-infiltrating neutrophils isolated from the orthotopic tumors were analyzed using flow cytometry ( n = 5 mice per group). Data represent mean ± SEM. Statistical analysis was conducted using the two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Article Snippet: After 11 consecutive days of antibody administration, the mice were euthanized, the peripheral blood was harvested for flow cytometry analysis to assess the efficiency of neutrophil depletion; (3) For CD8 + T cells depletion, mice received an intraperitoneal injection of anti-IgG2a or anti-CD8 antibody (Selleck, #A2102) treatment (0.2 mg/mouse, i.p.) 2 days before implantation.

Techniques: RNA Sequencing, Generated, Inhibition, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, In Vitro, Migration, Incubation, Knockdown, Cell Culture, CFSE Assay, Activity Assay, Recombinant, Isolation, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Histone lactylation increases CXCL1 expression for neutrophil infiltration and immune escape in pancreatic cancer

doi: 10.1038/s41467-026-69311-5

Figure Lengend Snippet: A Molecular docking simulated the binding affinities of PCAF for acetyl-CoA and lactyl-CoA. B Co-IP was performed to confirm the interaction between H3K18la and PCAF in PANC1 and KPC cells. PCAF and H3K18la were detected on the same gel. C, D Western blot analysis demonstrates the level of H3K18la in PDAC cells with or without the treatment of PCAF inhibitors (bromosporine or Embelin). Histone H3 and H3K18la blots are from parallel-processed separate gels (size conflict) with samples from the same experiment. E Western blot analysis demonstrates the level of H3K18la in PDAC cells following the knockdown of Pcaf . Histone H3, PCAF, β-actin and H3K18la blots are from parallel-processed separate gels with samples from the same experiment. F Western blots of in vitro histone acetylation or lactylation assay, the samples as indicated. Blots are from parallel-processed separate gels with samples from the same experiment. G , H Relative Cxcl1 RNA and protein levels with the vehicle or bromosporine treatment were analyzed by qRT-PCR and ELISA ( n = 3 independent experiments), respectively. I Relative Cxcl1 RNA levels after Pcaf knockdown were analyzed by qRT-PCR ( n = 3 independent experiments). J A schematic diagram showing the orthotopic tumor construction in C57BL/6 J mice ( n = 5 mice per group in one experiment, 2 × 10 6 KPC-luc cells per mouse) with or without bromosporine treatment. K , L Image and weights of the orthotopic tumors in the experimental groups from ( J ) at the end of the experiments ( n = 5 mice per group). M Lactylation levels of H3K18 and PanK in orthotopic tumors with or without bromosporine treatment were detected by western blot ( n = 4 biologically independent samples per group). Histone H3, H3K18la, and PanKla blots are from parallel-processed separate gels (size conflict) with samples from the same experiment. N Serum CXCL1 levels in mice with or without bromosporine treatment were measured by ELISA ( n = 5 mice per group). O – R Tumor-infiltrating neutrophils, CD8 + T cells, GZMB + CD8 + T cells, and PD-1 + CD8 + T cells isolated from the orthotopic tumors were analyzed using flow cytometry. Representative graphs of flow cytometry (left panel) and statistical analysis of the cell ratio (right panel) ( n = 5 mice per group). S The migratory abilities of neutrophils co-incubated with the culture medium supernatant from shNTC/ Pcaf PDAC cells treated with recombinant CXCL1 were analyzed by counting the penetrated cell numbers ( n = 3 biologically independent samples). T Representative IHC images stained by CXCL1 or H3K18la antibody (scale bar = 100 μm, left panel), correlation analysis of H-scores of CXCL1 and H3K18la in PDAC samples ( n = 21 patients, right panel). Data represent mean ± SEM. Statistical analysis was conducted using the two-tailed unpaired Student’s t test ( G – I , L , and N – S ) and two-tailed Pearson’s correlation analysis ( T ). Unless otherwise indicated, all western blots had three independent experimental repetitions with consistent results. Source data are provided as a Source Data file.

Article Snippet: After 11 consecutive days of antibody administration, the mice were euthanized, the peripheral blood was harvested for flow cytometry analysis to assess the efficiency of neutrophil depletion; (3) For CD8 + T cells depletion, mice received an intraperitoneal injection of anti-IgG2a or anti-CD8 antibody (Selleck, #A2102) treatment (0.2 mg/mouse, i.p.) 2 days before implantation.

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Western Blot, Knockdown, In Vitro, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Isolation, Flow Cytometry, Incubation, Recombinant, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Histone lactylation increases CXCL1 expression for neutrophil infiltration and immune escape in pancreatic cancer

doi: 10.1038/s41467-026-69311-5

Figure Lengend Snippet: A A schematic diagram showing the subcutaneous tumor model ( n = 6 mice per group in one experiment, 6 × 10 6 PANC02 cells per mouse) treated with bromosporine and anti-PD-1 antibody. B. The tumor volume growth curves of subcutaneous tumors from ( A ). C , D Image and weights of the subcutaneous tumors at the end point of experiments ( n = 6 mice per group). E The statistical analysis of the cell ratio of tumor-infiltrating neutrophils and CD8 + T cells isolated from subcutaneous tumors ( n = 3 mice per group). F Western blot analysis demonstrates H3K18la levels in the subcutaneous tumors ( n = 3 biologically independent samples per group) from ( A ). Histone H3 and H3K18la blots are from parallel-processed separate gels (size conflict) with samples from the same experiment. G Relative serum CXCL1 levels in mice treated with or without bromosporine/anti-PD-1 antibody were measured by ELISA ( n = 6 mice per group). H A schematic diagram showing the combinational treatment schedule for the orthotopic KPC-luc tumor model ( n = 21 mice per group in one experiment, 2 × 10 6 KPC-luc cells per mouse). I , J Image and weights of the orthotopic tumors at the end point of experiments ( n = 6 mice per group). K – N Tumor-infiltrating neutrophils, CD8 + T cells, GZMB + CD8 + T cells, and PD-1 + CD8 + T cells were analyzed using flow cytometry. Representative graphs of flow cytometry (left panel) and statistical analysis of the cell ratio (right panel) ( n = 6 mice per group). O Western blot analysis showing the H3K18la levels in the orthotopic tumors ( n = 3 biologically independent samples per group) from ( H ). Histone H3 and H3K18la blots are from parallel-processed separate gels (size conflict) with samples from the same experiment. P Representative luminescence images and Quantification of radiance intensity of the mouse model in ( H ). Q Survival probability of mice with orthotopically transplanted PDAC ( n = 15 mice per group). R A working model displaying the signaling pathway through which the aerobic glycolysis-mediated Lactate-PCAF-H3K18la-CXCL1 axis modulates the tumor microenvironment in pancreatic cancer, and the scientific basis for the development of a novel therapeutic strategy for PDAC. Data represent mean ± SEM. Statistical analysis was conducted using the two-tailed unpaired Student’s t test ( B , D , E , G , J – N , P ) and the log-rank test ( Q ). Source data are provided as a Source Data file.

Article Snippet: After 11 consecutive days of antibody administration, the mice were euthanized, the peripheral blood was harvested for flow cytometry analysis to assess the efficiency of neutrophil depletion; (3) For CD8 + T cells depletion, mice received an intraperitoneal injection of anti-IgG2a or anti-CD8 antibody (Selleck, #A2102) treatment (0.2 mg/mouse, i.p.) 2 days before implantation.

Techniques: Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Two Tailed Test

Journal: Cancer immunology research

Article Title: RAS(ON) multi-selective inhibition drives antitumor immunity in preclinical models of NRAS -mutant melanoma

doi: 10.1158/2326-6066.CIR-25-0744

Figure Lengend Snippet: (A) Flow cytometry analysis of melanoma antigen panel for MHC-I, MHC-II and PD-L1 in control, responder, and treatment escape groups. Tumor cells were normalized to SOX10 + cells, n=3/group. (B) Immunohistochemistry (IHC) staining and quantification of MHC-I and PD-L1 expression in control, responder, and treatment escape groups, n=3/group. H-score was used to access MHC-I expression and the percentage of positive cells for PD-L1. (C) Flow cytometry analysis of T cell panel (number of infiltrated CD4 + and CD8 + T cells, activated CD4 + /CD69 + and CD8 + /CD69 + T cells, and memory CD44 + /CD62L − /CD4 + and CD44 + /CD62L − /CD8 + T cells) of control, responder, and treatment escape groups. Cells were normalized to the total number of live cells. (D) Flow cytometry analysis of Foxp3 + CD4 + T cells (Tregs) in control, responder, and treatment escape groups. Tregs were normalized to the total number of CD4 + cells. (E) Immunohistochemistry staining and quantification of CD8, CD69 and PD-1 expression in control, responder, and treatment escape groups. n=3/group. H-score was used to access CD8 and CD69 expressions and the percentage of positive cells for PD1, according to the best fit suggested by the software. (F) Flow cytometry analysis of exhaustion markers (PD-1, TIM-3, LAG-3 and CTLA-4) in control, responder, and treatment escape groups. T cells were normalized to the total number of live cells. Scale bar = 200 μm (G) Multiplex immunofluorescence (IF) staining of control, responder, and treatment escape tumors for DAPI, CD4, CD8, CD11b, CD11c, Ly6G, Ly6C, CD34 and SOX10 expression (H) Scoring of the multiplex IF images of the whole slides. (I) Degrees of clustering of CD4 + or CD8 + T cells versus M-MDSCs (CD11b + Ly6C + Ly6G − ) and PMN-MDSCs (CD11b + Ly6C − Ly6G + ) of control, responder, and treatment escape groups, n=3/group. Cells were normalized by the number of total cells. The graphs are represented as average ± SEM. One-way ANOVA was performed followed by Tukey’s multiple comparisons test: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ns: non-significant; C: control (not treated cells); R: responder; TE: treatment escape.

Article Snippet: For CD4 + and CD8 + T-cell depletion, specific antibodies (anti-CD4, clone YTS191; cat. #BE0003–1; BioXCell, RRID:AB_1107636 and anti-CD8a, clone YTS169.4, cat. #BE0117, BioXCell, RRID:AB_10950145) were used.

Techniques: Flow Cytometry, Control, Immunohistochemistry, Expressing, Staining, Software, Multiplex Assay, Immunofluorescence

Journal: Cancer immunology research

Article Title: RAS(ON) multi-selective inhibition drives antitumor immunity in preclinical models of NRAS -mutant melanoma

doi: 10.1158/2326-6066.CIR-25-0744

Figure Lengend Snippet: (A-B) Tumor growth and survival curves following pre-treatment with IgG control (n=5) or anti-CD4 + anti-CD8a +/− RMC-7977 (all n=15). Vehicle or RMC-7977 treatment was initiated when tumors reached around 100mm 3 . For T cell depletion, mice were treated 3 days before the injection of the tumor with the Anti-CD4 and Anti-CD8a antibodies (1µg/µL, I.P.) and then every 4 days thereafter. The T cell depleted mice were treated with Anti-CD4 and Anti-CD8a antibodies (1µg/µL, I.P., every 4 days), Anti-CD4 + Anti-CD8a + RMC-7977 was treated with Anti-CD4 and Anti-CD8a antibodies (1µg/µL, I.P., every 4 days) and RMC-7977 (25 mg/kg, P.O., q.d.); and RMC-7977 group was treated with only RMC-7977 (25 mg/kg, P.O., q.d.) for 7 weeks. Plots correspond to each individual mouse. Dotted lines indicate when treatment started and stopped.

Article Snippet: For CD4 + and CD8 + T-cell depletion, specific antibodies (anti-CD4, clone YTS191; cat. #BE0003–1; BioXCell, RRID:AB_1107636 and anti-CD8a, clone YTS169.4, cat. #BE0117, BioXCell, RRID:AB_10950145) were used.

Techniques: Control, Injection

Journal: Cancer immunology research

Article Title: RAS(ON) multi-selective inhibition drives antitumor immunity in preclinical models of NRAS -mutant melanoma

doi: 10.1158/2326-6066.CIR-25-0744

Figure Lengend Snippet: (A-B) The effects of IgG control, anti-PD-1, RMC-7977 or anti-PD-1+RMC-7977 on the growth of OSUMMER.13 tumors. Treatment was initiated when tumors reached around 100mm 3 . The IgG group was treated with IgG control (2µg/µL, I.P., every 5 days: n=5), the vehicle group was treated with vehicle (P.O., q.d.: n=5), the vehicle + IgG group was treated with IgG control (2µg/µL, I.P., every 5 days: n=5) and vehicle (P.O., q.d.: n=5), total of n=15M for controls, the anti-PD-1 group was treated with Anti-PD-1 (2µg/µL, I.P., every 5 days: n=7), the RMC-7977 group was treated with RMC-7977 (25 mg/kg, P.O., q.d.: n=7), and the RMC-7977 + anti-PD-1 group (n=7) was treated with a combination of RMC-7977 and Anti-PD1 as indicated for 7 weeks. Plots correspond to each individual mouse. (C) Immunohistochemistry and quantification of pERK, Ki67, Melan-A, CD8, CD69, PD-1 and MHC-I of control (vehicle), anti-PD-1, RMC-7977 and RMC-7977 + anti-PD-1 group, n=3/group. H-score was used to access pERK, Ki67 and MHC-I expressions and the percentage of positive cells for melan-A and PD1, according to the best fit suggested by the software. Scale bar = 200 μm (D) Flow cytometry analysis of the T cell panel (number of infiltrated CD4 + and CD8 + T cells, activated CD4 + /CD69 + and CD8 + /CD69 + T cells, and memory CD44 + /CD62L − /CD4 + and CD44 + /CD62L − /CD8 + T cells) of vehicle, anti-PD1, RMC-7977 and the RMC-7977 + anti-PD-1 groups. The graphs are represented as average ± SEM. One-way ANOVA was performed followed by Tukey’s multiple comparisons test: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ns: non-significant. Dotted lines indicate when treatment started, stopped and when rechallenge was carried out.

Article Snippet: For CD4 + and CD8 + T-cell depletion, specific antibodies (anti-CD4, clone YTS191; cat. #BE0003–1; BioXCell, RRID:AB_1107636 and anti-CD8a, clone YTS169.4, cat. #BE0117, BioXCell, RRID:AB_10950145) were used.

Techniques: Control, Immunohistochemistry, Software, Flow Cytometry

Journal: Redox biology

Article Title: Radiation-induced upregulation of itaconate in macrophages promotes the radioresistance of non-small cell lung cancer by stabilizing NRF2 protein and suppressing immune response.

doi: 10.1016/j.redox.2025.103711

Figure Lengend Snippet: Fig. 5. Knockout of Acod1 in macrophages further enhance the activation of tumor immune microenvironment by radiotherapy (A) Gating strategy for detection of NK, CD4+ and CD8+ T cells by flow cytometry. (B–G) Flow cytometry analysis of CD45+ immune cells (B), CD3+ (C), CD4+ (D), CD8+ (E) T cells, the ratio of CD4+/CD8+ T cells (F), and NK cells (G) in the TME of LLC subcutaneous tumor model in mice treated with radiation (8 Gy x 3) in Acod1f/f Lyz2cre− (HO−) and Acod1f/f Lyz2cre+ (HO+) mice (n = 6). (H) Representative immunofluorescence staining of CD8 (green) in the TME at the end of experiment in Acod1f/f Lyz2cre−(HO−) and Acod1f/f Lyz2cre+ (HO+) mice. (I–J) Flow cytometry analysis of CD107a+ (I), and IFN-γ+ CD8+ T cells (J) in the TME of LLC subcutaneous tumor model described above (n = 6). *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.

Article Snippet: CD8+ T cell depletion Anti-mouse CD8 monoclonal antibody (clone 2.43) (#A2102, Selleck, China) was intraperitoneally injected at a dose of 200 μg per mouse, and 150 μg per mouse was injected 3 days later.

Techniques: Knock-Out, Activation Assay, Flow Cytometry, Immunofluorescence, Staining

Journal: Redox biology

Article Title: Radiation-induced upregulation of itaconate in macrophages promotes the radioresistance of non-small cell lung cancer by stabilizing NRF2 protein and suppressing immune response.

doi: 10.1016/j.redox.2025.103711

Figure Lengend Snippet: Fig. 6. The antitumor effect of Acod1 knockout in macrophages combined with radiotherapy partially depends on CD8+ T cells (A) CD8+ T cells clearance efficiency in mouse spleen detected by flow cytometry (n = 3). (B) CD8+ T cells clearance efficiency in mouse peripheral blood detected by flow cytometry (n = 3). (C) Tumor growth curves of LLC subcutaneous tumor in Acod1f/f Lyz2cre−(HO−) and Acod1f/f Lyz2cre+ (HO+) mice treated with 8 Gy x 3 radiotherapy and IgG or neutralizing CD8 antibody (α-CD8) (n = 6). (D–E) Tumor weight (D) and tumor images (E) on day 16 after treatment in different groups (n = 6). (F) The schematic diagram depicting that macrophages up-regulate the expression of Acod1 through activating NF-κB pathway after radiotherapy, thereby regulating the radiosensitivity of tumor cells and immune cell infiltration. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.

Article Snippet: CD8+ T cell depletion Anti-mouse CD8 monoclonal antibody (clone 2.43) (#A2102, Selleck, China) was intraperitoneally injected at a dose of 200 μg per mouse, and 150 μg per mouse was injected 3 days later.

Techniques: Knock-Out, Flow Cytometry, Expressing