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: Next, membranes were incubated with the following antibodies: FGL2 (1:500) (sc-100276, Santa Cruz Biotechnology), PD-L1 (1:4000) (66248-1-Ig, Proteintech), DDDDK-Tag (1:5000) (AE063, Abclonal), LAMP1 (1:10000) (67300-1-Ig, Proteintech), CTSD (1:10000) (21327-1-AP, Proteintech), TFEB (1:5000) (68632-1-Ig, Proteintech), phospho-TFEB (1:1000) (37681, CST), TFE3 (1:500) (A7936, Abclonal), MITF (1:1000) (13092-1-AP, Proteintech), GAPDH (1:50000) (60004-1-Ig, Proteintech), Histone H3 (1:5000) (17168-1-AP, Proteintech), p70S6K (1:1000) (2708, CST), phospho-p70S6K (1:1000) (9234, CST), 4EBP1 (1:1000) (9644, CST), phospho-4EBP1 (1:1000) (2855, CST), AKT (1:1000) (9272, CST), phospho-AKT (1:1000) (4060, CST), GSK3β(1:1000) (12456, CST), phospho-GSK3β (1:1000) (5558, CST), PKC (1:4000) (12919-1-AP, Proteintech), phospho-PKC (1:1000) (38938, CST), ERK1/2 (1:1000) (16443-1-AP, Proteintech), phospho-p44/42 MAPK (Erk1/2) (1:2000) (4370, CST), ATG5 (1:2000) (10181-2-AP, Proteintech), ATG7 (1:1000) (10088-2-AP, Proteintech), HRP goat anti-rabbit IgG (1:10000) (A21020, Abbkine) and HRP goat anti-mouse IgG (1:10000) (A21010, Abbkine).

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: Next, membranes were incubated with the following antibodies: FGL2 (1:500) (sc-100276, Santa Cruz Biotechnology), PD-L1 (1:4000) (66248-1-Ig, Proteintech), DDDDK-Tag (1:5000) (AE063, Abclonal), LAMP1 (1:10000) (67300-1-Ig, Proteintech), CTSD (1:10000) (21327-1-AP, Proteintech), TFEB (1:5000) (68632-1-Ig, Proteintech), phospho-TFEB (1:1000) (37681, CST), TFE3 (1:500) (A7936, Abclonal), MITF (1:1000) (13092-1-AP, Proteintech), GAPDH (1:50000) (60004-1-Ig, Proteintech), Histone H3 (1:5000) (17168-1-AP, Proteintech), p70S6K (1:1000) (2708, CST), phospho-p70S6K (1:1000) (9234, CST), 4EBP1 (1:1000) (9644, CST), phospho-4EBP1 (1:1000) (2855, CST), AKT (1:1000) (9272, CST), phospho-AKT (1:1000) (4060, CST), GSK3β(1:1000) (12456, CST), phospho-GSK3β (1:1000) (5558, CST), PKC (1:4000) (12919-1-AP, Proteintech), phospho-PKC (1:1000) (38938, CST), ERK1/2 (1:1000) (16443-1-AP, Proteintech), phospho-p44/42 MAPK (Erk1/2) (1:2000) (4370, CST), ATG5 (1:2000) (10181-2-AP, Proteintech), ATG7 (1:1000) (10088-2-AP, Proteintech), HRP goat anti-rabbit IgG (1:10000) (A21020, Abbkine) and HRP goat anti-mouse IgG (1:10000) (A21010, Abbkine).

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