Journal: Cancer Research

Article Title: Asialoglycoprotein Receptor 1 Functions as a Tumor Suppressor in Liver Cancer via Inhibition of STAT3

doi: 10.1158/0008-5472.can-21-4337

Figure Lengend Snippet: Figure 1. ASGR1 is critical in the progression of liver cancer. A, ASGR1 screening process based on the GEO and TCGA databases. B, IHC score of ASGR1 in matched tumor and nontumor tissues from 113 patients with liver cancer (left; the median and upper and lower quartiles of tumor areas are plotted in a box-and-whisker plot, P < 0.0001, paired Student t test); comparison of tumor volume between high and low IHC scores (middle, means SD, P ¼ 0.0469, unpaired Student t test); IHC scores of liver cancer tissues with and without vascular invasion (right; P ¼ 0.0366, unpaired Student t test). C, Left, IHC scores of tumor tissues at different pathological grades (P ¼ 0.0187, ANOVA). Right, survival analysis of patients with liver cancer with high and low IHC scores of ASGR1 (P ¼ 0.0022, log-rank test). D, Construction of ASGR1 knockout mice and establishment of the HDI model. The coding regions in ASGR1 transcript (ENSMUST00000146411.8) were precisely knocked out by CRISPR/Cas9 technology to construct ASGR1/ mice. Six-week ASGR1/ mice and wild-type (ASGR1wt/wt) mice were rapidly injected via tail vein with 2 mL of PBS containing 10 mg sleeping beauty (SB) transposon, 25 mg C-MYC, and 25 mg N-Ras plasmids to establish the HDI model. E, PET/CT results and livers of mice 2 weeks after the establishment of HDI model. F, Survival analysis of ASGR1wt/wt mice and ASGR1/ mice in HDI model (P ¼ 0.03, Log-rank test).

Article Snippet: One week after xenograft tumor formation, mice in the ASGR1 knockdown group were intraperitoneally injected with PBS or cryptotanshinone (50 mg/kg, 0.1 mL/10g, MCE) every two days.

Techniques: Whisker Assay, Comparison, Knock-Out, CRISPR, Construct, Injection, Positron Emission Tomography-Computed Tomography

Journal: Cancer Research

Article Title: Asialoglycoprotein Receptor 1 Functions as a Tumor Suppressor in Liver Cancer via Inhibition of STAT3

doi: 10.1158/0008-5472.can-21-4337

Figure Lengend Snippet: Figure 2. ASGR1 inhibits liver cancer growth. A, CCK-8 assay of liver cancer cells (means SD, unpaired Student t test). B and C, Quantitative results of the clone formation assay (means SD, unpaired Student t test) and EdU experiment on liver cancer cells (box-and-whisker plot, unpaired Student t test). D, SK-Hep-1 cells overexpressing ASGR1 and control cells were used to construct subcutaneous xenograft tumor models in nude mice. The volume and weight of the xenograft tumor were measured (means SD, unpaired Student t test). E, IHC was used to detect the expression of ASGR1 and Ki-67 in subcutaneous xenograft tumors. F, Left, orthotopic liver tumor model in nude mice with ASGR1 stable knockdown cells or ASGR1 stable overexpression cells and control cells. Right, the volume and weight of xenograft in each group were measured (means SD, unpaired Student t test). G, Left, PET/CT results and livers of the HDI model constructed by ASGR1 knockout mice. Middle, LW/BW before and after combined injection of PT3-EF1a-ASGR1 (E_ASGR1) or PT3-EF1a (E_control) plasmid (means SD, unpaired Student t test). Right, standard uptake value (SUV) of liver in PET/CT (means SD, unpaired Student t test). H, Detection of the liver function indices AST, ALT, and LDH levels in the serum of HDI model mice (means SD, unpaired Student t test).

Article Snippet: One week after xenograft tumor formation, mice in the ASGR1 knockdown group were intraperitoneally injected with PBS or cryptotanshinone (50 mg/kg, 0.1 mL/10g, MCE) every two days.

Techniques: CCK-8 Assay, Tube Formation Assay, Whisker Assay, Control, Construct, Expressing, Knockdown, Over Expression, Positron Emission Tomography-Computed Tomography, Knock-Out, Injection, Plasmid Preparation

Journal: Cancer Research

Article Title: Asialoglycoprotein Receptor 1 Functions as a Tumor Suppressor in Liver Cancer via Inhibition of STAT3

doi: 10.1158/0008-5472.can-21-4337

Figure Lengend Snippet: Figure 3. ASGR1 suppresses liver cancer progression through inhibition of STAT3 phosphorylation. A, Changes in the transcription levels of ASGR1-overexpressing and negative control SNU449 cells were detected by RNA sequencing (n ¼ 6). R studio was used for differential analysis, and 2,843 differential genes were screened out (left; R package: DESeq2;FDR<0.05, |LogFC|>0.6). Differential geneswere enriched with respect to each transcription factor by R studio in the regulatory target gene sets of the molecular signatures database (right; R package: clusterprofiler). B, After overexpression of ASGR1 in SNU449 and SK-Hep-1 cells and knockdown of ASGR1 in HCCLM3 cells, the phosphorylation levels of STAT3 at tyrosine 705 and serine 727 sites were detected by immunoblotting. C, The expression of Tyr705STAT3 was investigated by immunofluorescence assay. D, Changes in Tyr705STAT3 and Ser727STAT3 in the cytoplasm and nucleus of SNU449 cells were detected. E, Changes in STAT3 target genes in SNU449 cells were detected by RT-qPCR (means SD, unpaired Student t test). F, IHC results of the HDI model. G, STAT3 target genes in the livers of ASGR1/ and ASGR1wt/wt mice were detected by RT-qPCR (means SD, unpaired Student t test). H, The expression of Tyr705STAT3 in SNU449 cells stimulated with IL6 (5 ng/mL) was detected. I, Tyr705STAT3 expression in HCCLM3 cells was detected after treatment with CTS (1 mg/mL). J, HCCLM3 cells were injected subcutaneously in 6-week-old nude mice to form xenograft tumors. One week after xenograft tumor formation, mice were intraperitoneally injected with PBS or CTS (50 mg/kg, 0.1 mL/10g) every two days. The volume of the xenograft tumor of the mice was measured every two days (means SD, unpaired Student t test). ns, nonsignificant.

Article Snippet: One week after xenograft tumor formation, mice in the ASGR1 knockdown group were intraperitoneally injected with PBS or cryptotanshinone (50 mg/kg, 0.1 mL/10g, MCE) every two days.

Techniques: Inhibition, Phospho-proteomics, Negative Control, RNA Sequencing, Over Expression, Knockdown, Western Blot, Expressing, Quantitative RT-PCR, Injection

Journal: Cancer Research

Article Title: Asialoglycoprotein Receptor 1 Functions as a Tumor Suppressor in Liver Cancer via Inhibition of STAT3

doi: 10.1158/0008-5472.can-21-4337

Figure Lengend Snippet: Figure 4. NLK interacts with ASGR1 and binds to the SH2 domain of STAT3. A, NLK was identified as a potential ASGR1-binding protein by IP/MS. B and C, Immunoblot analysis of the endogenous interaction between ASGR1 and NLK after coimmunoprecipitation in SK-Hep-1 and SNU449 cells. D, Immunoblot analysis of endogenous IP between STAT3 and NLK in SK-Hep-1 and SNU449 cells. E, Exogenous coimmunoprecipitation experiment of Flag-tagged NLK with HA-tagged STAT3 or STAT3-D580–670aa mutant was performed in HEK293T cells. F, Immunoblot analysis of Tyr705STAT3 and Ser727STAT3 expression after transfection with the NLK-Flag plasmid.

Article Snippet: One week after xenograft tumor formation, mice in the ASGR1 knockdown group were intraperitoneally injected with PBS or cryptotanshinone (50 mg/kg, 0.1 mL/10g, MCE) every two days.

Techniques: Binding Assay, Protein-Protein interactions, Western Blot, Mutagenesis, Expressing, Transfection, Plasmid Preparation

Journal: Cancer Research

Article Title: Asialoglycoprotein Receptor 1 Functions as a Tumor Suppressor in Liver Cancer via Inhibition of STAT3

doi: 10.1158/0008-5472.can-21-4337

Figure Lengend Snippet: Figure 5. NLK inhibits JAK1-mediated STAT3 phosphorylation and STAT3 dimerization. A, HCCLM3 cells with or without ASGR1 knockdown were transduced with NLK plasmid. Tyr705STAT3 expression was determined by immunoblotting. B, ASGR1 was overexpressed in NLK knockdown SK-Hep-1 cells, and Tyr705STAT3 expression was detected by immunoblot. C and D, Left, HA-IP experiments with indicated plasmids were performed in HEK293T cells. Right, relative quantification of Flag relative to HA in the IP protein (means SD, unpairedStudentt test).E, Immunoblot of NLK affecting endogenous interactions between STAT3and JAK1. Relative quantification of JAK1 relative to STAT3 in IP protein (means SD, unpaired Student t test). F, Immunoblot of ASGR1 affecting endogenous interactions between STAT3 and NLK. Relative quantification of STAT3 relative to NLK in IP protein (means SD, unpaired Student t test). G, After transfection of the indicated plasmids, Tyr705STAT3 expression was detected by immunoblotting. H, Effects of the K167M mutation of NLK on STAT3 phosphorylation. I, Effects of the D264A and T298A mutation of NLK on STAT3 phosphorylation. J, Flag-IP experiment with indicated that plasmids were performed in HEK293T cells. Relative quantification of HA relative to Flag in IP protein (means SD, unpaired Student t test). ns, nonsignificant.

Article Snippet: One week after xenograft tumor formation, mice in the ASGR1 knockdown group were intraperitoneally injected with PBS or cryptotanshinone (50 mg/kg, 0.1 mL/10g, MCE) every two days.

Techniques: Phospho-proteomics, Knockdown, Transduction, Plasmid Preparation, Expressing, Western Blot, Transfection, Mutagenesis

Journal: Cancer Research

Article Title: Asialoglycoprotein Receptor 1 Functions as a Tumor Suppressor in Liver Cancer via Inhibition of STAT3

doi: 10.1158/0008-5472.can-21-4337

Figure Lengend Snippet: Figure 6. NLK competes with GP130 for binding to STAT3. A, Immunoblot of NLK affecting endogenous interactions between STAT3 and GP130. Relative quantification of GP130 relative to STAT3 in IP protein (means SD, unpaired Student t test). B, Immunoblot analysis of endogenous interaction between GP130 and NLK. C, Immunoblot analysis of endogenous interaction between GP130 and ASGR1. D, Immunoblot of ASGR1 affecting endogenous interactions between NLK and GP130. Quantification of NLK relative to GP130 in IP protein (means SD, unpaired Student t test). E, GP130-IP experiments with indicated plasmid were performed in SK- Hep-1 cells. Relative quantification of STAT3 relative to GP130 in IP protein (means SD, unpaired Student t test). F, PT3-EF1A-NLK–wild-type (E_NLK-WT) and E_ASGR1 plasmids were used for combined injection in the HDI model, and PET/CT was used to observe the tumorigenesis. Top, standard uptake value (SUV) of liver of HDI mice in PET/CT (means SD, unpaired Student t test). Bottom, ratio of liver weight to body weight of HDI mice (means SD, unpaired Student t test).

Article Snippet: One week after xenograft tumor formation, mice in the ASGR1 knockdown group were intraperitoneally injected with PBS or cryptotanshinone (50 mg/kg, 0.1 mL/10g, MCE) every two days.

Techniques: Binding Assay, Western Blot, Plasmid Preparation, Injection, Positron Emission Tomography-Computed Tomography

Journal: Cancer Research

Article Title: Asialoglycoprotein Receptor 1 Functions as a Tumor Suppressor in Liver Cancer via Inhibition of STAT3

doi: 10.1158/0008-5472.can-21-4337

Figure Lengend Snippet: Figure 7. NLK combines with and silences STAT3 depending on the NLK domain region. A, According to the domain sequence of NLK in UniProt, six NLK truncations were constructed. These truncations are NLK-domain (Flag-NLK-D), NLK-N-terminal (Flag-NLK-N), NLK-C-terminal (Flag-NLK-C), NLK-domain deletion mutation (Flag-NLK-DD), NLK-C-terminal deletion mutation (Flag-NLK-DC), and NLK-N-terminal deletion mutation (Flag-NLK-DN). B and E, Flag-IP experiments with indicated NLK truncations with HA-tagged STAT3 were performed in HEK293T cells. Relative quantification of HA relative to Flag in IP protein (means SD, unpaired Student t test). C and D, The protein level of Tyr705STAT3 was determined by immunoblotting in ASGR1 knockdown HCCLM3 cells transfected with the indicated plasmids. F, ASGR1 knockout mice were used to construct HDI models. PT3-EF1A-NLK-domain (E_NLK-D), PT3-EF1A-NLK–C-terminal deletion mutation (E_NLK-DC), PT3-EF1A-NLK–N-terminal deletion mutation (E_NLK-DN), and E_control plasmids were used for combined injection, and PET/CT was used to observe the formation of liver cancer. G, Top, ratio of liver weight to body weight of HDI mice (means SD, unpaired Student t test). Bottom, standard uptake value (SUV) of liver of HDI mice in PET/CT (means SD, unpaired Student t test). H, IHC results of liver cancer in HDI mice. ns, nonsignificant.

Article Snippet: One week after xenograft tumor formation, mice in the ASGR1 knockdown group were intraperitoneally injected with PBS or cryptotanshinone (50 mg/kg, 0.1 mL/10g, MCE) every two days.

Techniques: Sequencing, Construct, Mutagenesis, Western Blot, Knockdown, Transfection, Knock-Out, Control, Injection, Positron Emission Tomography-Computed Tomography

Journal: BMJ Open Diabetes Research & Care

Article Title: Mouse model of metformin-induced diarrhea

doi: 10.1136/bmjdrc-2019-000898

Figure Lengend Snippet: Effects of wood creosote on diabetic obese mice treated with metformin. Wood creosote does not affect efficacy of metformin. Weight change (A) and blood glucose levels (fed condition) (B) were examined during the experiment in . Data are shown as means±SEM. No significant differences between the metformin group and the metformin with wood creosote (M+C) group were observed. Mice were fasted for 3 hours after the final administration of drugs. (C) One g/kg glucose was administrated via oral gavage. One to 2 µL blood was collected via tail vein at indicated time points, and blood glucose was measured by using GlucoSensor. (D) At day 4 of , mice were fasted for 2 hours and then injected intraperitoneally with 36 µg/kg insulin. Blood glucose levels are shown as % initial. At day 5 in , blood was collected and measured serum bile acid levels (E) and alanine aminotransferase (F) were measured. (G) Western blots were carried out using liver protein (50 µg) to detect levels of phospho-AMPK and phoshopho-AKT. AKT, Protein Kinase B; AMPK, AMP-activated kinase.

Article Snippet: Antibodies are as follows: Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1E6D9, Proteintech, Rosemont, Illinois, USA), anti-AMPK (ABV10739, ABGENT, San Diego, California, USA), anti-phospho AMPK (pT172) (40H9, Cell Signaling Technology, Danvers, Massachusetts, USA), anti-total Protein Kinase B (AKT) (200–401 N98, Rockland, Limerick, Pennsylvania, USA), and anti-phosho AKT (pS473) (D9E, Cell Signaling Technology).

Techniques: Injection, Western Blot

Journal: Cell

Article Title: Inflammation Improves Glucose Homeostasis Through IKKβ-XBP1s Interaction

doi: 10.1016/j.cell.2016.10.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-phospho-XBP1s Thr48 , Covance , Raised against CRAAGSEASGT(p)PQARKRQR.

Techniques: Recombinant, Plasmid Preparation, Expressing, Sequencing, Software