Journal: Nature Communications

Article Title: Ceramide disrupts TM9SF2-PGK1 axis to redirect PD-L1 trafficking and enhance antitumor immunity

doi: 10.1038/s41467-026-70764-x

Figure Lengend Snippet: A An outline of the CRISPR-Cas9-based deletion library screen targeting genes related to sphingolipid synthesis or transport essential for cell surface PD-L1 expression. H460 cells expressing Cas9 were mutagenized with a focused lentiviral sgRNA library and PD-L1 low cells enriched by FACS sorting. B Significant hits identified from cellular screens are shown. LFC: The log2 fold change of sgRNA. C PD-L1 expression in sgNTC and sg TM9SF1-4 H460 cells was analyzed by Western blot. NTC: No Template Control. D Western blot assessment to evaluate PD-L1 expression in H460 cells overexpressing FLAG-tagged TM9SF1-4 versus empty vector (EV). FLAG antibody is used for the detection of TM9SF1/3/4. TM9SF2 antibody is used for the detection of TM9SF2. E The lifetime of PD-L1 was assessed in sgNTC and sg TM9SF1-4 H460 cells administered cycloheximide (CHX, 25 µg/mL) over time. PD-L1 levels were standardized to GAPDH. F A schematic overview of the T-cell-reliant cytotoxicity is shown. H460 cells transduced using TM9SF2 -targeting sgRNA or control sgRNA were co-cultured with activated T cells. Cytotoxicity was evaluated through clonogenic assays, survival analyses, and apoptosis measurements. G Cell viability showing T-cell cytotoxicity in H460 cells with TM9SF2 depletion following activated T cells co-culture. H , I Quantitative analysis of IFN-γ and CD107a expression in CD8 + T cells derived from co-culture are shown ( n = 5 independent experiments). Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Source data are provided as a file.

Article Snippet: HEK293T cells (Procell Corporation, #CL-0005), B16-F10 cells (Procell Corporation, #CL-0319), H460 cells (Procell Corporation, #CL-0299), and mouse AML cells (C1498) (ATCC, #TIB-49) were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM, containing 10% fetal bovine serum (FBS) as well as 1% penicillin-streptomycin) (37 °C, 5% CO 2 ).

Techniques: CRISPR, Expressing, Western Blot, Control, Plasmid Preparation, Cell Culture, Co-Culture Assay, Derivative Assay

Journal: Nature Communications

Article Title: Ceramide disrupts TM9SF2-PGK1 axis to redirect PD-L1 trafficking and enhance antitumor immunity

doi: 10.1038/s41467-026-70764-x

Figure Lengend Snippet: A Top five identified positive regulators ranked by enrichment scores from the immunoprecipitation-mass spectrometry (IP-MS) screen. Immunoprecipitation was performed using anti-FLAG beads in HEK293T cells transfected using TM9SF2 -FLAG. For B – E , G , HA antibody is used for the detection of HA-tagged PGK1. B Co-immunoprecipitation assays validating the interactions between TM9SF2 and PGK1 in H460 cells after 48 h of IFN-γ stimulation (100 ng/mL). C Western blot analysis of PGK1 and PD-L1 in H460 cells transduced with PGK1 sgRNAs versus sgNTC or H460 cells overexpressing HA-tagged PGK1 ( n = 3 independent experiments). D Co-immunoprecipitation assays demonstrating the association between TM9SF2 and PGK1 wild-type, S203D (phosphorylation-mimetic mutant), or S203A (phosphorylation-deficient mutant). E Western blot analysis of PD-L1 levels in PGK1 -depleted H460 cells transfected with PGK1 wild type or phospho-mutants. F , G The half-life of PD-L1 was assessed in H460 cells with PGK1 depletion or overexpression following treatment with CHX (25 µg/mL). For H – J , C57BL/6 J mice underwent subcutaneous injection with B16 cells transduced with Pgk1 shRNA or control shRNA. H Representative images and volumes of B16 tumors. I Proportions of tumor-permeating CD8 + T cells in tumors ( n = 5 mice). J Flow cytometry analysis of IFN-γ and CD107a levels in tumor-permeating CD8 + T cells in tumors ( n = 5 mice). K Schematic of the experimental design: For L-N, C57BL/6 J mice were subcutaneously inoculated with B16 cells followed by daily intraperitoneal administration of DC-PGKI (a PGK1 inhibitor, 5 and 10 mg/kg) for one week. L Representative image of B16 tumors. M Proportions of tumor-permeating CD8 + T cells in tumors ( n = 5 mice). N The proportions of IFN-γ + and CD107a + tumor-permeating CD8 + T cells in tumors ( n = 5 mice). For O – Q , C57BL/6 J mice were injected with B16 cells overexpressing Pgk1 or EV. O Representative image of B16 tumors. P Proportions of tumor-permeating CD8 + T cells in tumors ( n = 5 mice). Q Flow cytometry analysis of IFN-γ and CD107a levels in tumor-permeating CD8 + T cells in tumors ( n = 5 mice). Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Source data are provided as a file.

Article Snippet: HEK293T cells (Procell Corporation, #CL-0005), B16-F10 cells (Procell Corporation, #CL-0319), H460 cells (Procell Corporation, #CL-0299), and mouse AML cells (C1498) (ATCC, #TIB-49) were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM, containing 10% fetal bovine serum (FBS) as well as 1% penicillin-streptomycin) (37 °C, 5% CO 2 ).

Techniques: Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Transfection, Western Blot, Transduction, Phospho-proteomics, Mutagenesis, Over Expression, Injection, shRNA, Control, Flow Cytometry

Journal: Nature Communications

Article Title: Ceramide disrupts TM9SF2-PGK1 axis to redirect PD-L1 trafficking and enhance antitumor immunity

doi: 10.1038/s41467-026-70764-x

Figure Lengend Snippet: A Bar plot showing altered cellular processes and signaling pathways in shTM9SF2 H460 cells compared to shNC cells. B A schematic of the PD-L1 surface internalization assay in H460 cells transduced with sgRNAs is shown. C Surface PD-L1 retention in H460 cells was measured by flow cytometry after genetic depletion of PGK1 or TM9SF2 ( n = 3 independent experiments). D PD-L1 recycling was quantified by flow cytometry in H460 cells following PGK1 or TM9SF2 knockout ( n = 3 independent experiments). E Immunofluorescence showing PD-L1 colocalization with RAB11 in sg PGK1 or sg TM9SF2 H460 cells versus sgNTC (PD-L1 in green, RAB11 in red, DAPI in blue). Scale bar: 5 μm. The right panels display intensity profiles of PD-L1 versus RAB11 following the white line. For F , G , HA antibody is used for the detection of HA-tagged PGK1. F Co-immunoprecipitation assay demonstrating TM9SF2 interaction with PGK1, RAB11, and PD-L1 in H460 cells, with or without IFN-γ treatment (100 ng/mL, 48 h). G Co-immunoprecipitation assay evaluating interactions of PGK1 wild-type and its phospho-mutants (S203A and S203D) using TM9SF2, PD-L1, and RAB11 in H460 cells. Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Source data are provided as a file.

Article Snippet: HEK293T cells (Procell Corporation, #CL-0005), B16-F10 cells (Procell Corporation, #CL-0319), H460 cells (Procell Corporation, #CL-0299), and mouse AML cells (C1498) (ATCC, #TIB-49) were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM, containing 10% fetal bovine serum (FBS) as well as 1% penicillin-streptomycin) (37 °C, 5% CO 2 ).

Techniques: Protein-Protein interactions, Transduction, Flow Cytometry, Knock-Out, Immunofluorescence, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: Ceramide disrupts TM9SF2-PGK1 axis to redirect PD-L1 trafficking and enhance antitumor immunity

doi: 10.1038/s41467-026-70764-x

Figure Lengend Snippet: A Heatmap showing increased proteins involved in PD-L1 lysosomal degradation in sgPGK1 H460 cells versus sgNTC cells. Western blot of HIP1R levels in sg PGK1 ( B ) and sg TM9SF2 ( C ) H460 cells versus sgNTC cells ( n = 3 independent experiments). For D – F , HA antibody is used for the detection of HA-tagged PGK1. D Half-life analysis of HIP1R abundance in CHX (25 μg/mL)-treated H460 cells with PGK1 depletion or HA- PGK1 overexpression. E Western blot analyses of HIP1R ubiquitination in H460 cells reconstituted with MYC- HIP1R and HA- PGK1 for 36 h, followed by treatment with MG132 (10 μM, 8 h) and with or without IFN-γ administration (100 ng/mL, 8 h). MYC antibody is used for the detection of MYC-tagged HIP1R. F Western blot analyses of HIP1R ubiquitination in HEK293T cells reconstituted using MYC- HIP1R and HA- PGK1 wild-type and phospho-mutants for 36 h, followed by MG132 treatment (10 μM, 8 h). G Co-immunoprecipitation analysis examining IFN-γ effects on PGK1 association with GFP-HIP1R Δ966-979 and GFP-HIP1R. H460 cells transfected with FLAG- PGK1 and either GFP- HIP1R or GFP- HIP1R Δ966-979 were treated with IFN-γ (100 ng/mL, 8 h). Flag antibody is used for the detection of FLAG-tagged PGK1 and GFP antibody is used for the detection of GFP-tagged HIP1R. H A schematic of GFP fusion constructs is shown, including HIP1R lysosomal sorting signal (LL/LI, GFP-S1) and its corresponding mutant (AA, GFP-S2). I CHX-chase assay showing GFP-S1 and GFP-S2 protein levels over time in sg PGK1 or sgNTC H460 cells treated with CHX (25 μg/mL). Source data are provided as a file.

Article Snippet: HEK293T cells (Procell Corporation, #CL-0005), B16-F10 cells (Procell Corporation, #CL-0319), H460 cells (Procell Corporation, #CL-0299), and mouse AML cells (C1498) (ATCC, #TIB-49) were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM, containing 10% fetal bovine serum (FBS) as well as 1% penicillin-streptomycin) (37 °C, 5% CO 2 ).

Techniques: Western Blot, Over Expression, Ubiquitin Proteomics, Immunoprecipitation, Transfection, Construct, Mutagenesis

Journal: Nature Communications

Article Title: Ceramide disrupts TM9SF2-PGK1 axis to redirect PD-L1 trafficking and enhance antitumor immunity

doi: 10.1038/s41467-026-70764-x

Figure Lengend Snippet: A Summary of ceramides that were up- or down-regulated following TM9SF2 depletion in H460 cells from lipidomics analysis. B PD-L1 levels were analyzed using Western blot in H460 cells administered cholesteryl:phosphatidylcholine (CholPC) alone or CholPC loaded with ceramides and derivatives (31.25 μM, 40 h). C PD-L1 surface levels in H460 cells given cholPC alone or cholPC loaded with ceramides and derivatives (31.25 μM, 40 h) ( n = 3 independent experiments). D , E Half-life analysis of PD-L1 or HIP1R concentrations in H460 cells overexpressing TM9SF2 for the indicated time points following CHX (25 µg/mL) treatment. Cells underwent pre-treated using 31.25 μM cholPC loaded with Cer(d18:1/26:0) for 40 h. F Co-immunoprecipitation analysis investigating the effect of Cer(d18:1/26:0) on the interaction between PGK1 and TM9SF2. HEK293T cells pre-transfected with HA- PGK1 or FLAG- TM9SF2 were treated with or without Cer(d18:1/26:0) (31.25 μM, 40 h). HA antibody is used for the detection of HA-tagged PGK1. G Immunofluorescence showing colocalization of PD-L1 with RAB11 in H460 cells with or without Cer(d18:1/26:0) treatment (31.25 μM, 40 h). (PD-L1 in green, RAB11 in red, DAPI in blue.) Scale bars: 4 μm. Intensity of PD-L1 and RAB11 following the white line are shown in right panels. H Schematic of the experimental protocol: For I – K , C57BL/6 J mice were subcutaneously inoculated with B16 cells and then administered intraperitoneal injections of either CholPC or Cer(d18:1/26:0) (10 and 20 mg/kg/day) for one week. All statistical comparisons were performed against the CholPC-treated control group. I Representative image of B16 tumors. J Proportion of tumor-permeating CD8 + T cells in B16 tumors ( n = 5 mice). K IFN-γ and CD107a levels in tumor-permeating CD8 + T cells in B16 tumors ( n = 5 mice). Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Source data are provided as a file.

Article Snippet: HEK293T cells (Procell Corporation, #CL-0005), B16-F10 cells (Procell Corporation, #CL-0319), H460 cells (Procell Corporation, #CL-0299), and mouse AML cells (C1498) (ATCC, #TIB-49) were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM, containing 10% fetal bovine serum (FBS) as well as 1% penicillin-streptomycin) (37 °C, 5% CO 2 ).

Techniques: Western Blot, Immunoprecipitation, Transfection, Immunofluorescence, Control

Journal: Cell Communication and Signaling : CCS

Article Title: The p53 tumor suppressor modulates the expression of proteins that control natural killer cell activity

doi: 10.1186/s12964-026-02772-9

Figure Lengend Snippet: SLAM family member 7 expression is reduced in p53-deficient cells. A . Western blot analysis of indicated proteins in p53-proficient and p53-deficient A549 cells treated with the specified compounds for 30 hours. B . Relative SLAM family member 7 (SLAMF7) mRNA levels measured by semi-quantitative real-time polymerase chain reaction in p53-proficient and p53-deficient A549 and U-2 OS cells under the same treatment conditions. Statistical significance was calculated using a Student’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001). C . Protein expression in p53-proficient (+) and p53-deficient (-) U-2 OS. D . Protein expression in p53-proficient (+) and p53-deficient (-) NCI-H460 cells. The SLAMF7 panel is shown with both short and long exposure times to visualize the protein bands in actinomycin D (ActD) and nutlin-3a (Nut3a) (A+N)-treated cells better

Article Snippet: NCI-H460 cells (RRID: CVCL_0459, lung cancer, ATCC) and WM793B cells (RRID: CVCL_8787, melanoma) were cultured in RPMI-1640 supplemented with 2 mM L-glutamine, 4.5 g/L glucose, 1 mM sodium pyruvate, and 10% heat-inactivated FBS.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cell Communication and Signaling : CCS

Article Title: The p53 tumor suppressor modulates the expression of proteins that control natural killer cell activity

doi: 10.1186/s12964-026-02772-9

Figure Lengend Snippet: Co-treatment with actinomycin D and nutlin-3a sensitizes cancer cells to killing by NK-92 cells. A . Viability of A549 cells measured by MTS assay. Cells were either mock-treated (Con) or treated with actinomycin D (ActD) and nutlin-3a (Nut3a) combined (A+N) for 48 hours, then trypsinized, counted, and incubated for 24 hours in RPMI1640 medium alone or in co-culture with NK-92 cells at two effector-to-target (E: T) ratios (NK-92: A549 – 1:1 and 5:1) for 24 hours. After co-incubation, the medium was replaced with DMEM, and the A549 cells were allowed to recover for 72 hours before assessing metabolic activity via the MTS assay. Five biological replicates were performed. Viability of mock-control cells and drug-treated cells without NK-92 exposure was set to 100%. Statistical analysis: ordinary one-way ANOVA and multiple comparisons test (** p < 0.01, **** p < 0.0001). B . MTS assay of A549 cells pre-treated for 48 hours with ActD, Nut3a, or A+N, followed by 24 hours of co-incubation with NK-92 cells. Viability was assessed as in A . Four biological replicates were analyzed. Statistical analysis: ordinary one-way ANOVA and multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). C . Viability of NCI-H460 cells pre-exposed to A+N or Con for 48 hours, followed by 24 hours of co-incubation with the NK-92 cell line at a 1:1 ratio. Viability was assessed as in A . Statistical significance: unpaired, two-tailed t-test (*** p < 0.001). D . Macroscopic visualization of A549 cells surviving NK-92-mediated killing. Cells were pre-treated for 48 hours with A+N or Con, then seeded on a 6-well plate with or without NK-92 cells at E: T ratios of 1:1 or 5:1 (NK-92:A549). After a 24-hour co-incubation, the RPMI medium for NK-92 cells was replaced with fresh DMEM, and the A549 cells were allowed to recover for 5-7 days. Remaining adherent cells were fixed with methanol and stained with 0.01% crystal violet. Representative results from three biological replicates are shown

Article Snippet: NCI-H460 cells (RRID: CVCL_0459, lung cancer, ATCC) and WM793B cells (RRID: CVCL_8787, melanoma) were cultured in RPMI-1640 supplemented with 2 mM L-glutamine, 4.5 g/L glucose, 1 mM sodium pyruvate, and 10% heat-inactivated FBS.

Techniques: MTS Assay, Incubation, Co-Culture Assay, Activity Assay, Control, Two Tailed Test, Staining