Journal: Advanced Science

Article Title: GLS1‐RNA Polymerase II Axis Mediates Glutamine‐Dependent Hepatoprotective Effects on Alcoholic Liver Disease in High‐Protein Diets

doi: 10.1002/advs.202502738

Figure Lengend Snippet: GLS1 interacts with POLR2E or POLR2H. A) Schematic of immunoprecipitation using mass spectrometry (IP–MS) to identify proteins associated with GLS1 in LO2 cells. B) CoIP analysis of the interaction of endogenous GLS1 with endogenous POLR2E or POLR2H in the primary hepatocytes. β‐actin served as the loading control. C–F) CoIP analysis of the interaction of GAC–Myc and POLR2H‐HA, KGA‐Flag and POLR2H‐HA, GAC–Myc and POLR2E‐HA and KGA‐Flag and POLR2E‐HA in HEK293T cells. β‐actin served as the loading control. G) CoIP analysis of the interaction of endogenous GLS1 with endogenous POLR2E or POLR2H in the primary hepatocytes treated with ethanol (600 m m ) for 24 h. β‐actin served as the loading control. H,I) CoIP analysis of the interaction of GAC–Myc or KGA‐Flag with endogenous POLR2H, GAC–Myc or KGA‐Flag with endogenous POLR2E treated with ethanol (400 m m ) for 24 h in HEK293T cells. β‐actin served as the loading control.

Article Snippet: For analysis of endogenous proteins, the primary hepatocytes lysed by cell lysis IP buffer were incubated overnight at 4 °C with GLS1 (12855‐1; Proteintech) or POLR2E (sc‐390902; santa cruz) or POLR2H (sc‐398512; santa cruz) antibody and protein A+G Agarose beads.

Techniques: Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Control

Journal: Advanced Science

Article Title: GLS1‐RNA Polymerase II Axis Mediates Glutamine‐Dependent Hepatoprotective Effects on Alcoholic Liver Disease in High‐Protein Diets

doi: 10.1002/advs.202502738

Figure Lengend Snippet: GLS1 interacts with POLR2H or POLR2E to reduce the activity of RNA pol II. A,B) The protein complex structure of GLS1, POLR2H and POLR2E performed on the GROMACS platform. The corresponding location of the putative binding motif on GLS1 and POLR2H protein, GLS1 and POLR2E protein. (C) The truncated GLS1 variants. D,E) CoIP analysis of the interaction of truncated GLS1 proteins and POLR2E‐HA or POLR2H‐HA in lysates of HEK293T cells. β‐actin served as the loading control. F) RNA pol II activity of AML12 cells expressing pGL3‐Basic or pGL3‐Promoter with KGA WT or ΔKGA‐3. Cells were serum starved overnight followed by ethanol (400 m m ) stimulation for 1 h. n = 10. G,H) The truncated POLR2E or POLR2H variants. I,J) CoIP analysis of the interaction of truncated POLR2E or POLR2H proteins and KGA‐Flag in lysates of HEK293T cells. β‐actin served as the loading control. K) RNA pol II activity of AML12 cells expressing pGL3‐Basic or pGL3‐Promoter with GLS1 and POLR2E WT or ΔPOLR2E‐3 or POLR2H WT or ΔPOLR2H‐1. Cells were serum starved overnight followed by ethanol (400 m m ) stimulation for 1 h. n = 7–10. Data in (F) and (K) are presented as the mean ± SEM, determined by one‐way ANOVA and Fisher's LSD test.

Article Snippet: For analysis of endogenous proteins, the primary hepatocytes lysed by cell lysis IP buffer were incubated overnight at 4 °C with GLS1 (12855‐1; Proteintech) or POLR2E (sc‐390902; santa cruz) or POLR2H (sc‐398512; santa cruz) antibody and protein A+G Agarose beads.

Techniques: Activity Assay, Binding Assay, Control, Expressing

Journal: Advanced Science

Article Title: GLS1‐RNA Polymerase II Axis Mediates Glutamine‐Dependent Hepatoprotective Effects on Alcoholic Liver Disease in High‐Protein Diets

doi: 10.1002/advs.202502738

Figure Lengend Snippet: GLS1 reduces hepatic alcoholic steatosis through interacting with POLR2E or POLR2H. A) TG levels of the primary hepatocytes expressing with KGA WT or ΔKGA‐3. Cells were serum starved overnight followed by ethanol (600 m m ) stimulation for 24 h. The amount was normalized to the protein content. n = 5‐6. B) Western blots of ACLY, ACC, FASN levels in the primary hepatocytes described in (A); β‐actin serves as a loading control. C) TG levels of the primary hepatocytes expressing with GLS1 and POLR2E WT or ΔPOLR2E‐3 or POLR2H WT or ΔPOLR2H‐1. Cells were serum starved overnight followed by ethanol (600 m m ) stimulation for 24 h. The amount was normalized to the protein content. n = 4–6. D) Western blots of ACLY, ACC, FASN levels in the primary hepatocytes described in (C); β‐actin serves as a loading control. E) Schematic illustrating the groups and procedures of the mouse model. C57BL/6J mice were injected with AAV8‐TBG‐GLS1 with POLR2E WT or POLR2H WT or their truncated variants (ΔPOLR2E‐3 or ΔPOLR2H‐1) via the tail vein for additional 4‐week pair or ethanol feeding. n = 8–10 biologically independent mice per group. F) Change curves of body weight of mice. G) Liver TG levels of mice in the indicated group in (E); n = 6‐7. H) Representative H&E staining of liver sections in the indicated group in (E). Scale bars, 100 and 200 µm. I) Western blots of ACLY, ACC, FASN levels in the primary hepatocytes described in (E); β‐actin serves as a loading control. Data in (A), (C), (G) are presented as the mean ± SEM, determined by one‐way ANOVA and Fisher's LSD test.

Article Snippet: For analysis of endogenous proteins, the primary hepatocytes lysed by cell lysis IP buffer were incubated overnight at 4 °C with GLS1 (12855‐1; Proteintech) or POLR2E (sc‐390902; santa cruz) or POLR2H (sc‐398512; santa cruz) antibody and protein A+G Agarose beads.

Techniques: Expressing, Western Blot, Control, Injection, Staining

Journal: Advanced Science

Article Title: GLS1‐RNA Polymerase II Axis Mediates Glutamine‐Dependent Hepatoprotective Effects on Alcoholic Liver Disease in High‐Protein Diets

doi: 10.1002/advs.202502738

Figure Lengend Snippet: Schematic diagram of glutamine‐regulated GLS1 coordinates RNA polymerase II manipulates AFLD. High‐protein diet reduces alcoholic hepatic steatosis through glutamine stabilizing GLS1 to regulate RNA pol II activity by interacting with POLR2E or POLR2H.

Article Snippet: For analysis of endogenous proteins, the primary hepatocytes lysed by cell lysis IP buffer were incubated overnight at 4 °C with GLS1 (12855‐1; Proteintech) or POLR2E (sc‐390902; santa cruz) or POLR2H (sc‐398512; santa cruz) antibody and protein A+G Agarose beads.

Techniques: Activity Assay

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 6. Anti-MICA/B 7C6 mAb boosts anti-tumor cytotoxic function of peripheral NK cells from iCCA patients against the HuCCT-1 cell line. A): peripheral NK cell degranulation, evaluated as frequency of CD107a+ NK cells, in iCCA patients (n = 13) and HC (n = 16) in the presence of 7C6 mAb or IgG1-Fc. Parametric paired and unpaired t tests were used to compare data. B): dot plots showing the frequency of CD3-CD56+ CD107a+ NK cells in a HC and a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc. C): the proportion of circulating IFNγ+ NK cells in patients (n = 10) and HC (n = 11) in the presence of 7C6 mAb compared with IgG1-Fc. To compare paired data, the parametric t test and the non-parametric Wilcoxon t test were used. To compare unpaired data, the parametric t test and the non-parametric Mann-Whitney U test were used. D): representative dot plots showing the frequency of CD3-CD56+ IFNγ+ NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: MANN-WHITNEY

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 7. Anti-MICA/B 7C6 mAb boosts anti-tumor cytotoxic function of peripheral NK cells from iCCA patients against patient-derived iCCA cell lines. A): peripheral NK cell degranulation, evaluated as CD107a+NK frequency, in iCCA patients (n = 12) and HC (n = 8) in the presence of 7C6 mAb or IgG1-Fc using patient- derived primary tumor cell lines as targets. Parametric paired and unpaired t tests were used to compare data. B): dot plots showing the frequency of CD3-CD56 + CD107a+ NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc. C): proportion of circulating IFNγ+NK cells in patients (n = 10) and in HC (n = 8) in the presence of 7C6 mAb compared with IgG1-Fc. To compare paired data, we used the parametric t test and the non-parametric Wilcoxon t test. To compare unpaired data, the parametric t test and the non-parametric Mann-Whitney U test were used. D): representative dot plots showing the frequency of CD3-CD56+ IFNγ +NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: Derivative Assay, MANN-WHITNEY

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 8. 7C6 mAb enhances the anti-tumor effect of liver- and tumor-infiltrating NK cells in iCCA patients. A): Frequency of degranulating CD107a+NK cells in LIL (n = 13) and TIL (n = 10) of iCCA patients in the presence of anti-MICA/B 7C6 mAb or IgG1-Fc using autologous tumor-derived cell lines as targets. Parametric paired and unpaired t tests were used to compare data. B): representative dot plots showing the frequency of CD3-CD56+ CD107a+ LIL- and TIL-NK cells in the presence of 7C6 mAb or IgG1-Fc. C): proportion of IFNγ+ NK cells in LIL (n = 10) and TIL (n = 8) of iCCA patients in the presence of 7C6 mAb compared with IgG1-Fc using autologous tumor-derived cell lines as targets. The parametric t test and non-parametric Wilcoxon t test were used to compare paired data. The parametric t test was used to compare unpaired data.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: Derivative Assay

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 9. Cytotoxicity assay of HC PBMC, patient PBMC, LIL and TIL cells. A, B): Frequency of CFSE+LIVE/DEAD (LD)+ HuCCT-1 cell line targets when HC PBMC (n = 5), patient PBMC (n = 10), LIL (n = 8) and TIL (n = 5) were used as effector cells in the presence of 7C6 mAb and isotype control (IgG1). The parametric paired t tests were used to compare data. C, D): Frequency of CFSE+LD+ patient-derived cell line targets when HC PBMC (n = 5), patient PBMC (n = 8), LIL (n = 8) and TIL (n = 4) were used as effectors in the presence of 7C6 and isotype control. The parametric paired t test was used to compare data in panel C. The non-parametric Wilcoxon t test was used to compare paired data in panel D. Target cell death was determined as frequency of CFSE+LD+ cells.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: Cytotoxicity Assay, Control, Derivative Assay

Journal: Frontiers in Pharmacology

Article Title: Effect of gentiopicroside on endogenous formaldehyde homocysteine–pathway related proteins in rats with non-alcoholic steatohepatitis

doi: 10.3389/fphar.2025.1700101

Figure Lengend Snippet: Expression levels of CBS, ALDH2, AHCY, MAT1A and MTHFR proteins in liver tissues of rats in each group. (A) Expression levels of MAT1A protein in liver tissues of rats in each group (n = 3); (B) Expression levels of AHCY protein in liver tissues of rats in each group (n = 3); (C) Expression levels of MYHFR protein in liver tissues of rats in each group (n = 3); (D) Expression levels of ALDH2 protein in liver tissues of rats in each group (n = 3); (E) Expression levels of CBS protein in liver tissues of rats in each group (n = 3). Note: Data are expressed as the mean ± SD. “#” indicates that P < 0.05 compared with the normal group; “*” indicates that P < 0.05 compared with the model group.

Article Snippet: ROS, GSH, MDA, SOD, SAH, SAM, GST, CAT, GSH, HCY were obtained from Jiangsu Enzyme Immunoassay Biotechnology Co., LTD. Their item numbers are MM-88564O1, MM-20251R1, MM-2037H1, MM-20387R1, MM-50456H1, MM-0248H1, MM-21254R1, MM-20447R1, MM-20251R1 and MM-50456H1. β-actin antibody (Proteintech, 20536-1-AP); ALDH2 antibody (Proteintech, 15310-1-AP); MAT1A antibody (Proteintech, 12395-1-AP); MTHFR antibody (Proteintech, 26591-1-AP); CBS antibody (Proteintech, 14787-1-AP); AHCY antibody (Proteintech, 10757-2-AP); PVDF membrane with a thickness of 0.45 μm (IPVH00010, Millipore); Universal total RNA extraction reagent (U7431, Suzhou Youyi Landi Biotechnology Co., LTD.) Taq SYBR® Green qPCR Premix (Universal) (EG0117M, Jiangsu Bishi Mei Biotechnology Co., LTD.) All-in-One First-Strand Synthesis MasterMix (with dsDNase) was obtained from Jiangsu Bestmate Biotechnology Co., LTD. (item number (EG15133S).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Effect of gentiopicroside on endogenous formaldehyde homocysteine–pathway related proteins in rats with non-alcoholic steatohepatitis

doi: 10.3389/fphar.2025.1700101

Figure Lengend Snippet: Expression levels of CBS, ALDH2, AHCY, MAT1A and MTHFRmRNA in liver tissues of rats in each group (A) Expression levels of ALDH2 mRNA in liver tissues of rats in each group (n = 3); (B) Expression levels of MTHFRmRNA in liver tissues of rats in each group (n = 3); (C) Expression levels of CBS mRNA in liver tissues of rats in each group (n = 3); (D) Expression levels of AHCY mRNA in liver tissues of rats in each group (n = 3); (E) Expression levels of MAT1A mRNA in liver tissues of rats in each group (n = 3). Note: “#” indicates that P < 0.05 compared with the normal group; “*” indicates that P < 0.05 compared with the model group.

Article Snippet: ROS, GSH, MDA, SOD, SAH, SAM, GST, CAT, GSH, HCY were obtained from Jiangsu Enzyme Immunoassay Biotechnology Co., LTD. Their item numbers are MM-88564O1, MM-20251R1, MM-2037H1, MM-20387R1, MM-50456H1, MM-0248H1, MM-21254R1, MM-20447R1, MM-20251R1 and MM-50456H1. β-actin antibody (Proteintech, 20536-1-AP); ALDH2 antibody (Proteintech, 15310-1-AP); MAT1A antibody (Proteintech, 12395-1-AP); MTHFR antibody (Proteintech, 26591-1-AP); CBS antibody (Proteintech, 14787-1-AP); AHCY antibody (Proteintech, 10757-2-AP); PVDF membrane with a thickness of 0.45 μm (IPVH00010, Millipore); Universal total RNA extraction reagent (U7431, Suzhou Youyi Landi Biotechnology Co., LTD.) Taq SYBR® Green qPCR Premix (Universal) (EG0117M, Jiangsu Bishi Mei Biotechnology Co., LTD.) All-in-One First-Strand Synthesis MasterMix (with dsDNase) was obtained from Jiangsu Bestmate Biotechnology Co., LTD. (item number (EG15133S).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Effect of gentiopicroside on endogenous formaldehyde homocysteine–pathway related proteins in rats with non-alcoholic steatohepatitis

doi: 10.3389/fphar.2025.1700101

Figure Lengend Snippet: The mechanism diagram of endogenous formaldehyde participating in one-carbon metabolism leading to homocysteine accumulation and causing NASH. Note: formaldehyde (Endogenous FA); Glutathione (GSH) Mitochondrial aldehyde dehydrogenase 2 (ALDH2); Formate; Tetrahydrofolate (THF); 5, 10-methylenetetrahydrofolate (5, 10-CH2-THF); Methylenetetrahydrofolate reductase (MTHFR); Homocysteine (HCY); S-adenosine homocysteine (SAH); Reactive oxygen species (ROS); Non-alcoholic steatohepatitis (NASH); Methionine Cystathionine -β -synthase (CBS); 5-methyltetrahydrofolate (5-CH3-THF); S-adenosylmethionine (SAM); Methionine adenosyltransferase 1A (MAT1A); S-adenosine homocysteine hydrolase (AHCY).

Article Snippet: ROS, GSH, MDA, SOD, SAH, SAM, GST, CAT, GSH, HCY were obtained from Jiangsu Enzyme Immunoassay Biotechnology Co., LTD. Their item numbers are MM-88564O1, MM-20251R1, MM-2037H1, MM-20387R1, MM-50456H1, MM-0248H1, MM-21254R1, MM-20447R1, MM-20251R1 and MM-50456H1. β-actin antibody (Proteintech, 20536-1-AP); ALDH2 antibody (Proteintech, 15310-1-AP); MAT1A antibody (Proteintech, 12395-1-AP); MTHFR antibody (Proteintech, 26591-1-AP); CBS antibody (Proteintech, 14787-1-AP); AHCY antibody (Proteintech, 10757-2-AP); PVDF membrane with a thickness of 0.45 μm (IPVH00010, Millipore); Universal total RNA extraction reagent (U7431, Suzhou Youyi Landi Biotechnology Co., LTD.) Taq SYBR® Green qPCR Premix (Universal) (EG0117M, Jiangsu Bishi Mei Biotechnology Co., LTD.) All-in-One First-Strand Synthesis MasterMix (with dsDNase) was obtained from Jiangsu Bestmate Biotechnology Co., LTD. (item number (EG15133S).

Techniques: