Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: The effect of FGL2 expression on survival and the correlation between FGL2 and immune checkpoints. ( A ) The expression of FGL2 in a human liver tissue microarray was detected by mIHC. Representative images were shown. Scale bar, 20 μm. Kaplan-Meier analysis of overall survival ( B ) and progression-free survival ( C ) of HCC patients based on FGL2 expression ( n = 85). The Gene Expression Profiling Interactive Analysis server was used to analyze the correlation between Fgl2 and Pdcd1 (encoding PD1, ( D ), Cd274 (encoding PD-L1, ( E ), Pdcd1lg2 (encoding PD-L2, ( F ), Ctla4 ( G ), Tigit ( H ), Entpd1 (encoding CD39, ( I ), Havcr2 (encoding TIM3, ( J ), Btla ( K ) and Lag3 ( L ) at the mRNA expression level, respectively. R=Pearson’s correlation coefficient

Article Snippet: After blocking, the TMA was incubated with specific antibodies as follows: FGL2 (1:500) (H00010875-M01, Abnova) and TFEB (1:100) (13372-1-AP, Proteintech).

Techniques: Expressing, Microarray, Gene Expression

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 knockout inhibited tumor growth and downregulated the expression of PD-L1. ( A ) Hepa1-6 cells were inoculated orthotopically into the left liver lobe of WT and Fgl2 -/- mice. The concentration of FGL2 in tumor tissue homogenate was detected by ELISA ( n = 5). ( B ) The livers from C57BL/6 mice bearing orthotopic tumors were shown ( n = 8). ( C ) Representative immunohistochemical images for PD-L1 staining in orthotopic tumors ( n = 4). ( D ) The expression level of PD-L1 on CD45- tumor cells was measured by flow cytometry ( n = 4). Data were represented as median fluorescence intensity (MFI). ( E ) Hepa1-6 cells were inoculated subcutaneously (s.c.) into the right inguinal fold region of WT and Fgl2 -/-mice. The concentration of FGL2 in tumor tissue homogenate was shown ( n = 5). ( F ) Gross images of s.c. tumors of WT or Fgl2 -/- mice ( n = 8). Tumor volume ( G ) and tumor weight ( H ) of the indicated group of C57BL/6 mice ( n = 8). ( I ) The expression level of PD-L1 on tumor cells in the s.c. transplanted hepatomas was detected by flow cytometry ( n = 5). ( J ) WT and Fgl2 -/- mice bearing s.c. tumors were treated with anti-CD3 neutralizing antibodies (200 µg/mice) or IgG1 isotype control (200 µg/mice). Gross images of s.c. tumors in mice treated as indicated ( n = 6). Tumor volume ( K ) and tumor weight ( L ) of the indicated group ( n = 6). Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After blocking, the TMA was incubated with specific antibodies as follows: FGL2 (1:500) (H00010875-M01, Abnova) and TFEB (1:100) (13372-1-AP, Proteintech).

Techniques: Knock-Out, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Flow Cytometry, Fluorescence, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 upregulated PD-L1 expression in HCC cells and attenuated the cytotoxicity of T cells. Protein levels of FGL2 (FLAG) and PD-L1 were analyzed by Western blot (WB) in Huh7 ( A ) and MHCC97H cells ( B ) stably expressing FGL2-FLAG. Lentivirus expressing shRNA against FGL2 (sh-FGL2) was used for FGL2 knockdown, and nontargeting shRNA(sh-control) was added as control. FGL2 and PD-L1 expression in MHCC97H cells were detected by WB ( C ), immunofluorescence (IF) ( D ) and flow cytometry (FCM) ( E ). Scale bar, 25 μm. FGL2 knockdown or nontargeting shRNA-transfected MHCC97H cells were treated with IFN-γ (50 ng/mL) for 24 h. The expression of PD-L1 was detected by WB ( F ) and FCM ( G ). ( H ) T-cell cytotoxicity assay against MHCC97H cells. Apoptotic MHCC97H cells were detected by FCM. Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001

Article Snippet: After blocking, the TMA was incubated with specific antibodies as follows: FGL2 (1:500) (H00010875-M01, Abnova) and TFEB (1:100) (13372-1-AP, Proteintech).

Techniques: Expressing, Western Blot, Stable Transfection, shRNA, Knockdown, Control, Immunofluorescence, Flow Cytometry, Transfection, Cytotoxicity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 disruption induced lysosome-dependent degradation of PD-L1. HCC cells with FGL2 overexpression ( A ) or knockdown ( B ) were treated with CHX (25 mmol/L) for the indicated time points and then subjected to WB for quantitation of PD-L1 level. The expression of PD-L1 in MHCC97H cells after treatment with proteasome inhibitor (MG132) ( C ) or lysosomal inhibitors, NH4Cl ( D ) and chloroquine ( E ) was detected by WB. ( F ) Images and quantification of LysoTracker red staining in MHCC97H cells were displayed. Scale bar, 100 μm. ( G ) The protein levels of LAMP1 and CTSD were determined by WB. ( H ) The colocalization between PD-L1 and LAMP1 in MHCC97H cells was detected by immunofluorescence assay. The intensity profiles of PD-L1 and LAMP1 along the white line were plotted on the middle panel. The statistical result of the colocalization factor (Pearson’s R value) was shown on the right panel. Scale bar, 25 μm. Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After blocking, the TMA was incubated with specific antibodies as follows: FGL2 (1:500) (H00010875-M01, Abnova) and TFEB (1:100) (13372-1-AP, Proteintech).

Techniques: Disruption, Over Expression, Knockdown, Quantitation Assay, Expressing, Staining, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 disruption promoted the expression of nuclear TFEB. The cytosolic and nuclear distribution of TFEB ( A ) and MITF ( B ) in MHCC97H cells was detected by WB following FGL2 knockdown. Quantification of nuclear TFEB or MITF to Histone-H3 was shown. ( C ) Images and quantification of nuclear TFEB in MHCC97H cells were displayed. Scale bar, 50 μm. The expression levels of TFEB ( D ) and MITF ( E ) were determined by WB in mouse liver cancer tissues ( n = 6). The transcriptional levels of Lamp1, Atp6v0d2 , Ctns and Ctsb were detected by qPCR in mouse liver cancer tissues ( F ) and MHCC97H cells ( G ). TFEB siRNA or control siRNA was transfected to MHCC97H cells pretreated with FGL2 shRNA or control shRNA. The protein levels of LAMP1, CTSD ( H ), and PD-L1 ( I ) were determined by WB. The relative quantification of LAMP1, CTSD or PD-L1 to GAPDH was shown. ( J ) The expression of TFEB in an HCC tissue microarray was detected by mIHC. Representative images were shown. Scale bar, 20 μm. Kaplan-Meier analysis of the progression-free survival ( K ) and overall survival ( L ) of HCC patients based on nuclear TFEB expression ( n = 85). Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: After blocking, the TMA was incubated with specific antibodies as follows: FGL2 (1:500) (H00010875-M01, Abnova) and TFEB (1:100) (13372-1-AP, Proteintech).

Techniques: Disruption, Expressing, Knockdown, Control, Transfection, shRNA, Quantitative Proteomics, Microarray

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 interacted with mTOR kinase and affected the phosphorylation of mTORC1 pathway. ( A ) Immunoblot analysis of (p-)mTOR, (p-)P70S6K, (p-)4EBP1 and (p-)AKT in MHCC97H cells following FGL2 knockdown. ( B ) Total P70S6K, 4EBP1 and AKT proteins as well as their phosphorylated forms were detected in liver cancer tissues from WT and Fgl2 -/- mouse(n = 6). ( C ) The phosphorylation level of TFEB Ser211 was detected by WB. The expression levels of (p-)P70S6K, (p-)4EBP1 ( D ) and PD-L1 ( E ) were detected in MHCC97H cells treated with FGL2 shRNA or control shRNA in the absence or presence of mTOR signaling activator (MHY1485). ( F ) Co-immunoprecipitation analysis of the interaction between FGL2 and mTOR in MHCC97H cells stably expressing FGL2-FLAG. ( G ) The colocalization of FGL2 and mTOR in MHCC97H cells was detected by immunofluorescence. Scale bar, 25 μm.( H ) 293T cells were co-transfected with HA-mTOR, FLAG-FGL2 or vector plasmids. Cell lysates were subjected to co-immunoprecipitation using anti-HA or anti-FLAG antibodies. ( I ) 293T cells were co-transfected with HA-mTOR truncations and FLAG-FGL2 plasmids. Cell lysates were subjected to co-immunoprecipitation using anti-FLAG antibodies. ( J ) MHCC97H cell lysates were subjected to immunoprecipitation​ with anti-mTOR antibodies, followed by immunoblot analysis. Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: After blocking, the TMA was incubated with specific antibodies as follows: FGL2 (1:500) (H00010875-M01, Abnova) and TFEB (1:100) (13372-1-AP, Proteintech).

Techniques: Phospho-proteomics, Western Blot, Knockdown, Expressing, shRNA, Control, Immunoprecipitation, Stable Transfection, Immunofluorescence, Transfection, Plasmid Preparation

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: FGL2 inhibition enhanced antitumor immune responses of anti-PD1 therapy in HCC. ( A ) WT and Fgl2 -/- mice bearing s.c. tumors were treated with anti-PD1 monoclonal antibodies (αPD1, 100 µg/mice) or IgG isotype control (100 µg/mice). Gross images of tumors in mice treated as indicated ( n = 6). Tumor volume ( B ) and tumor weight ( C ) of the indicated group. ( D ) Representative immunohistochemical images of subcutaneous tumors for PD-L1 staining ( n = 3). Representative flow cytometric plots and quantification of the proportion of IFN-γ+ CD8+ cells ( E ), granzyme B+ CD8+ cells ( F ) and CD107a+ CD8+ cells ( G ) in tumor tissues in response to different treatments. ( H ) Representative flow cytometric plots and quantification of the proportion of Tregs (CD4+ CD25+ Foxp3+) in tumor tissues from the indicated groups. The proportions of Th1 (CD4+ IFN-γ+ T cells) ( I ) and Th2 (CD4+ IL-4+ T cells) cells ( J ) in tumor tissues were shown. Data were presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: After blocking, the TMA was incubated with specific antibodies as follows: FGL2 (1:500) (H00010875-M01, Abnova) and TFEB (1:100) (13372-1-AP, Proteintech).

Techniques: Inhibition, Bioprocessing, Control, Immunohistochemical staining, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Disruption of FGL2 induces TFEB-dependent lysosomal degradation of PD-L1 and enhances the efficacy of anti-PD1 therapy in hepatocellular carcinoma

doi: 10.1186/s12964-026-02704-7

Figure Lengend Snippet: Schematic of FGL2-mediated PD-L1 regulation and therapy potential of combination interference of FGL2 and PD1. In HCC cells, FGL2 stabilizes the mTOR-Raptor interaction and activates mTORC1 signaling. Activated mTORC1 phosphorylates TFEB, thereby suppressing its nuclear translocation and subsequent lysosomal biogenesis. This lysosomal dysfunction​ attenuates PD-L1 degradation, leading to elevated PD-L1 abundance and the inhibition of T cell–mediated cytotoxicity. In HCC mouse models, FGL2 inhibition exerts synergistic antitumor effects with anti-PD1 therapy by downregulating tumorous expression of PD-L1, promoting the activity of tumor-infiltrating lymphocytes, and suppressing the accumulation of Tregs

Article Snippet: After blocking, the TMA was incubated with specific antibodies as follows: FGL2 (1:500) (H00010875-M01, Abnova) and TFEB (1:100) (13372-1-AP, Proteintech).

Techniques: Translocation Assay, Inhibition, Expressing, Activity Assay