Journal: Nature Metabolism

Article Title: Prolonged breastfeeding protects from obesity by hypothalamic action of hepatic FGF21

doi: 10.1038/s42255-022-00602-z

Figure Lengend Snippet: Effect of injecting an adenoviral vector encoding a scrambled RNA (Ad-hSyn-DIO-EGFP) or an shRNA against D2R (Ad-hSyn-DIO-shD2R-EGFP) in a Cre-dependent manner followed by ICV injection of vehicle or FGF21 into D2R-Cre mice on body weight ( a ), effect of injecting an adenoviral vector encoding a scrambled RNA (AAV-EF1A-EGFP-floxed) or an shRNA against FGFR1 (AAV8-EGFP-sh fgfr1 -floxed) in a Cre-dependent manner, followed by ICV injection of vehicle or FGF21 in D2R-Cre mice on body weight ( b ) and quantification of immunolabeling for UCP1 in BAT ( c ).Values are represented as means ± SEM, n per group indicated in each figure. Exact P values are shown. Statistical differences according to a two-sided Student’s t-test (normal data) ( a , b and c ).

Article Snippet: Real-time PCR was performed on the Applied Biosystems 7900HT Fast Real-Time PCR system using exon-boundary-specific TaqMan Gene Expression Assays (Applied Biosystem): FGFR1 ( Fgfr1- Mm00438930_m1).

Techniques: Plasmid Preparation, shRNA, Injection, Immunolabeling

Journal: Nature Metabolism

Article Title: Prolonged breastfeeding protects from obesity by hypothalamic action of hepatic FGF21

doi: 10.1038/s42255-022-00602-z

Figure Lengend Snippet: a , Photomicrographs showing the colocalization of GFP and FGFR1 in the LHA/ZI of D2r -Cre mice. b – e , Effect of injecting an adenoviral vector encoding a scrambled RNA (AAV-EF1A-EGFP-floxed) or an shRNA against Fgfr1 (AAV8-EGFP-sh Fgfr1 -floxed) in a Cre-dependent manner, followed by ICV injection of vehicle or FGF21 in D2r -Cre mice on body weight change ( b ); cumulative food intake at 24 h and 8 h ( c ); infrared thermal images and quantification of BAT interscapular temperature ( n = 11–13; d ); and BAT protein levels of PGC1α and UCP1 ( e ). f , Photomicrographs showing the colocalization of GFP and Vgat in the LHA/ZI of D2r -Cre mice. g – j , Effect of injecting an adenoviral vector encoding a scrambled RNA (Ad-hSyn-DIO-EGFP) or an shRNA against D2r (Ad-hSyn-DIO-sh D2r -EGFP) in a Cre-dependent manner, followed by ICV injection of vehicle or FGF21 in Vgat-ires-Cre mice on body weight change ( g ), cumulative food intake ( h ); infrared thermal images and quantification of BAT interscapular temperature ( i ); and BAT protein levels of PGC1α and UCP1 ( j ). Protein data were expressed as percentages in relation to control (GFP vehicle) animals. β-actin was used to normalize protein levels. Dividing lines indicate splicing within the same gel. Values are represented as means ± s.e.m., n per group. Exact P values are shown. Statistical differences were determined by a two-sided Student’s t -test (normal data; b – d , g and h ), or a two-sided Mann–Whitney U test (non-normal data and non-homogeneous variance; e , i and j ).

Article Snippet: Real-time PCR was performed on the Applied Biosystems 7900HT Fast Real-Time PCR system using exon-boundary-specific TaqMan Gene Expression Assays (Applied Biosystem): FGFR1 ( Fgfr1- Mm00438930_m1).

Techniques: Plasmid Preparation, shRNA, Injection, MANN-WHITNEY

Journal: Nature Metabolism

Article Title: Prolonged breastfeeding protects from obesity by hypothalamic action of hepatic FGF21

doi: 10.1038/s42255-022-00602-z

Figure Lengend Snippet: a , b , Fluorescent activated cell sorting (FACS) to isolate D2R-expressing neurons (GFP-positive) in the LHA/ZI ( a ); FGF21 receptor (FGFR1) mRNA is expressed in isolated D2R cells from the LHA/ZI ( b ). c , d , Effect of injecting an adenoviral vector encoding a scrambled RNA (Ad-hSyn-DIO-EGFP) or an shRNA against D2R (Ad-hSyn-DIO-sh D2r -EGFP) in a Cre-dependent manner, followed by ICV injection of vehicle or FGF21 in Vgat-ires-Cre mice on body weight ( c ) and quantification of immunolabeling for UCP1 in BAT ( d ). e , Photomicrograph showing the colocalization of Vimentin and GFP in the lateral ventricle.

Article Snippet: Real-time PCR was performed on the Applied Biosystems 7900HT Fast Real-Time PCR system using exon-boundary-specific TaqMan Gene Expression Assays (Applied Biosystem): FGFR1 ( Fgfr1- Mm00438930_m1).

Techniques: FACS, Expressing, Isolation, Plasmid Preparation, shRNA, Injection, Immunolabeling

Journal: Nature Metabolism

Article Title: Prolonged breastfeeding protects from obesity by hypothalamic action of hepatic FGF21

doi: 10.1038/s42255-022-00602-z

Figure Lengend Snippet: a , Representative photomicrographs of the tuberal region of the hypothalamus of a wild-type mouse showing tanycytic processes (arrows) and cell bodies (arrowheads) labelled by fluorescent FGF21 in the median eminence (inset), 1 min after injection into the jugular vein or after the ICV injection. b , c , Immunoreactivity of FGF21 is increased in tanycytes of SW-HFD and DW-HFD rats ( n = 3; b ); mRNA expression of FGF21 in SW-HFD and DW-HFD rats ( n = 3; c ). d – f , Effect of injecting an AAV1/2 into the lateral ventricle expressing Cre recombinase under transcriptional control of the hDio2 promoter (AAV1/2-hDio2-iCre) together with an AAV-GFP-floxed or an shRNA against Fgfr1 (AAV-GFP-sh Fgfr1 -floxed) in wild-type (WT) mice on infrared thermal images and quantification of BAT interscapular temperature ( n = 4–6; d ); quantification of immunolabelling for UCP1 in BAT ( n = 4–6; e ); BAT protein levels of PGC1α and UCP1 ( n = 4–6; f ). Protein data were expressed as percentages in relation to control (GFP) animals. β-actin was used to normalize protein levels. Dividing lines indicate splicing within the same gel. Values are represented as means ± s.e.m., n per group. Exact P values are shown. Statistical differences were determined by a two-sided Student’s t -test (normal data; b – e ), or a two-sided Mann–Whitney U test (non-normal data and non-homogeneous variance; f ).

Article Snippet: Real-time PCR was performed on the Applied Biosystems 7900HT Fast Real-Time PCR system using exon-boundary-specific TaqMan Gene Expression Assays (Applied Biosystem): FGFR1 ( Fgfr1- Mm00438930_m1).

Techniques: Injection, Expressing, shRNA, MANN-WHITNEY

Journal: Journal of neuroinflammation

Article Title: The regulation of NFKB1 on CD200R1 expression and their potential roles in Parkinson's disease.

doi: 10.1186/s12974-024-03231-3

Figure Lengend Snippet: Fig. 2 NFKB1 is a potential transcription factor of CD200R1 and regulates its transcriptional activity. (A) Schematic representation of the CD200R1 core promoter sequence. The region covers the nucleotides from − 482 to -146 based on the CD200R1 translation initiation site. (B) In silico prediction of the possible transcription factors. (C) Dual-luciferase assay results in human HEK293T cells transfected with the reporter construct containing the core pro moter and overexpressing indicated transcription factors. (D) Dual-luciferase assay results in human U937 cells after overexpressing the CD200R1 core promoter and RELA, CREB1 and NFKB1, respectively. (E) Dual-luciferase assay results in microglia cell line BV2 transfected with the core promoter vector and NFKB1. One-way ANOVA was used for comparison. **P < 0.01, **** P < 0.0001

Article Snippet: The validity of the antibody for ELISA assay was confirmed by the manufacturer with recombinant CD200R1 protein (https://www.rndsystems.com/cn/ products/human-cd200r1-antibody_af3414).

Techniques: Activity Assay, Sequencing, In Silico, Luciferase, Transfection, Construct, Plasmid Preparation, Comparison

Journal: Journal of neuroinflammation

Article Title: The regulation of NFKB1 on CD200R1 expression and their potential roles in Parkinson's disease.

doi: 10.1186/s12974-024-03231-3

Figure Lengend Snippet: Fig. 4 Expression levels of NFKB1 and CD200R1 in PD patients. (A, B) CD200R1 (A) and NFKB1 (B) mRNA levels were assessed in PBMCs from PD patients (n = 44) with age- and sex-matched healthy controls (n = 45) by RT-PCR. Non-parameter analysis was used for between-group comparison. (C) Spearman correlation analysis between CD200R1 and NFKB1 mRNA expressions in PBMCs from PD population

Article Snippet: The validity of the antibody for ELISA assay was confirmed by the manufacturer with recombinant CD200R1 protein (https://www.rndsystems.com/cn/ products/human-cd200r1-antibody_af3414).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Comparison

Journal: Cell reports

Article Title: Molecular motor protein KIF5C mediates structural plasticity and long-term memory by constraining local translation

doi: 10.1016/j.celrep.2021.109369

Figure Lengend Snippet: (A) Experimental strategy for puromycin labeling experiment. (B) WB images of Shank2, Syngap, GluR1, CamKIIβ-1, eIF3g, and actin in synaptoneurosomes prepared from KIF5C OE- or eGFP-expressing primary HP neurons treated with puromycin. (C) Shank2, Syngap, GluR1, CamKIIβ-1, and eIF3g expression levels normalized to actin in synaptoneurosomes prepared from KIF5C OE or eGFP neurons treated with puromycin. (D) Experimental strategy for detecting newly synthesized proteins in KIF5C OE neurons by proximity labeling assay (PLA). (E) Confocal images showing proximity labeling respectively for eIF3G, GluR1, and CamKIIβ-1 in KIF5C OE-GFP- or eGFP-expressing primary HP neurons treated with puromycin. Newly synthesized proteins are shown in red. (F–H) Quantification of normalized positive puncta counts in labeled dendrites. (I–K) Quantification of normalized positive puncta counts in labeled soma. (L) Experimental strategy. (M) WB images of pS6K (T-389), S6K, pS6 (S235/236), S6, pCREB (S133), CREB, eIF3g, Kinesin, and actin. Primary neurons transduced with eGFP control and KIF5C OE-expressing lentiviruses. Cells lysed at DIV 16 and subjected to immunoblotting. (N) Normalized pS6K to S6K, pS6 to S6, pCREB to CREB, eIF3g to actin, and KIF5C to actin in total lysate of KIF5C OE- or eGFP-expressing neurons. (O) Experimental plan for EIF3G KD and KIF5C OE in same neuron for confocal live imaging. (P) Confocal projection images showing changes in spine, digitally enlarged image in inset. (Q and R) Quantitative analyses of image data shown in (P) completed by selecting dendrites with length of 100 μm. Bar graphs show change in spine density or morphology. Changes compared between neurons in control and KIF5C OE groups. Unpaired, two-tailed Student’s t test or one-way ANOVA followed by Tukey’s or Dunnett’s post hoc test. Error bars represent SEM. *p < 0.05, *p < 0.005, and ***p < 0.0005. Scale bar: 1, 2, or 20 μm.

Article Snippet: After 2 more rounds of PBS washing, and blocking in 5% horse serum in 0.1% Triton X-100 PBS, neurons were then incubated with mouse anti-puromyin antibody (1:2000, Sigma, MABE342) and one of rabbit antibody for C terminus eIF3g (1:1000, Novus Biologicals, NB100–93298), GluR1 (1:1000, CST, 13185), or CamKIIβ-1 (1:1000, Sigma, 13–9800) overnight.

Techniques: Labeling, Expressing, Synthesized, Transduction, Control, Western Blot, Imaging, Two Tailed Test

Journal: Cell reports

Article Title: Molecular motor protein KIF5C mediates structural plasticity and long-term memory by constraining local translation

doi: 10.1016/j.celrep.2021.109369

Figure Lengend Snippet: (A) Experimental strategy for in vivo manipulation and mouse behavior using lentivirus to overexpress KIF5C . (B) MWM test strategy. (C) Heatmap showing mouse tracking during MWM. (D–I) MWM performance. (D) Time spent in Q1 during probe test. (E) Total visits to Q1 during probe test. (F) Time spent every 30 s in Q1 during probe test. (G) Time spent in Q4 during probe test. (H) Total distance traveled during probe test. (I) Swimming velocity during test. (J) Contextual fear memory test strategy. (K) Contextual freezing responses during test at 24 h after conditioning. (L) Total distance traveled during test at 24 h after conditioning. (M) Mean percentage freezing time-averaged every minute during 5 min test at 24 h after conditioning. (N) Experimental strategy for assessing consolidation, extinction, and recall of contextual fear in CA1 KIF5C OE mice using reduced-intensity foot shocks during training. (O) Extinction training shows significant reduction in percent freezing at 30 min. Control, n = 11; KIF5C -O/E, n = 9. For all statistical analyses, data expressed as means ± SEM (*p < 0.05, **p < 0.05; unpaired, two-tailed Student’s t test). (P) KIF5C functions as critical regulator of structural plasticity associated with LTM, linking transcription, transport, and local translation. KIF5C mediates long-distance transport of RNA substrates for local translation for learning-related synaptic remodeling. EIF3G , regulator of eukaryotic translational initiation, and several key components determining plasticity and function of excitatory synapses such as GLUR1 , SYNGAP , SHANK2 , and CaMKII-β , are transported as RNA cargo of KIF5C .

Article Snippet: After 2 more rounds of PBS washing, and blocking in 5% horse serum in 0.1% Triton X-100 PBS, neurons were then incubated with mouse anti-puromyin antibody (1:2000, Sigma, MABE342) and one of rabbit antibody for C terminus eIF3g (1:1000, Novus Biologicals, NB100–93298), GluR1 (1:1000, CST, 13185), or CamKIIβ-1 (1:1000, Sigma, 13–9800) overnight.

Techniques: In Vivo, Control, Two Tailed Test

Journal: Cell reports

Article Title: Molecular motor protein KIF5C mediates structural plasticity and long-term memory by constraining local translation

doi: 10.1016/j.celrep.2021.109369

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: After 2 more rounds of PBS washing, and blocking in 5% horse serum in 0.1% Triton X-100 PBS, neurons were then incubated with mouse anti-puromyin antibody (1:2000, Sigma, MABE342) and one of rabbit antibody for C terminus eIF3g (1:1000, Novus Biologicals, NB100–93298), GluR1 (1:1000, CST, 13185), or CamKIIβ-1 (1:1000, Sigma, 13–9800) overnight.

Techniques: Virus, Recombinant, Cell Culture, Saline, Sterility, Live Cell Imaging, Transfection, Protein Extraction, Protease Inhibitor, Labeling, SYBR Green Assay, Ointment, Adhesive, Gel Extraction, Isolation, TA Cloning, Sequencing, Plasmid Preparation, shRNA, Software, Control

Journal: Cell Reports Medicine

Article Title: MYC activation impairs cell-intrinsic IFNγ signaling and confers resistance to anti-PD1/PD-L1 therapy in lung cancer

doi: 10.1016/j.xcrm.2023.101006

Figure Lengend Snippet: Genetic inactivation and expression of IFNγ receptor 1 (IFNGR1) in LC (A) Chromatograms depicting, at the genomic level, the p.Q212X mutation at IFNGR1 in PDC11 cells and the matched normal DNA. The nucleotide change is indicated by an arrow. (B) Western blot of IFNGR1 and PD-L1 in the indicated LC cells. ACTIN and TUBULIN, protein-loading controls. (C) Western blot showing changes in levels of the indicated proteins in the PCD11 cells expressing ectopic wild-type IFNGR1 (IFNGR1-PDC11) on treatment with IFNγ (30 nM), as compared with the empty vector (EV-PDC11) control. Experiments were independently repeated at least twice with similar results. (D) mRNA levels assessed by real-time qPCR of the indicated transcripts (relative to ACTIN ) under basal conditions and after administering IFNγ (30 nM), in H596 cells, in PCD11 parental cells (PDC11), in cells expressing ectopic wild-type IFNGR1 and in EV-PDC11 controls. Results are presented as the median of independent biological triplicates. Asterisks denote statistical significance (∗p < 0.05; ∗∗∗p < 0.005; ∗∗∗∗p < 0.001) from two-tailed Student’s t tests. Data are presented as the mean ± SD. (E) Representative weak and strong immunostaining of IFNGR1 in lung primary tumors. Scale bars, 50 μm. (F) Distribution of the immunostaining of IFNGR1 among lung tumors, by histopathology and levels of HLA-I/B2M and PD-L1. Fisher’s exact test. ns, not significant, LuAD, lung adenocarcinoma; LuSCC, lung squamous cell carcinoma; ns, not significant.

Article Snippet: To determine IFNGR1 levels by immunohistochemistry we used the polyclonal anti-IFNGR1 antibody (10808-1-AP, Proteintech; 1:200 dilution.

Techniques: Expressing, Mutagenesis, Western Blot, Plasmid Preparation, Control, Two Tailed Test, Immunostaining, Histopathology

Journal: Cell Reports Medicine

Article Title: MYC activation impairs cell-intrinsic IFNγ signaling and confers resistance to anti-PD1/PD-L1 therapy in lung cancer

doi: 10.1016/j.xcrm.2023.101006

Figure Lengend Snippet: Oncogenic MYC and low level of constitutive expression of IFNγsign genes (A) Oncoplot of the presence of alterations in the indicated genes. Immunoresponse indicates the presence of mutations of genes involved in immunorecognition (e.g., B2M , TAP1/2 , CALR ) or response to IFNγ ( JAK2 and IFNGR1 ) , and CCLE at cBioPortal. (B) Graphs showing gene expression values in transcripts per million (TPMs) for the transcripts within the IFNγ signature from RNA-seq and at GEO: GSE109720) in (lo)MAX-, (hi)MAX-, and (hi)MYC/MAX-expressing cells. Bars indicate means; differences assessed by two-sided Student’s t test. ns, not significant; S, SCLC. (C) Gene set enrichment analysis (GSEA) showing an inverse correlation in gene expression between the IFNγsign and datasets, including from lungs of mice overexpressing Nmyc (GEO: GSE6077). FDR, false discovery rate; NES, normalized enrichment score.

Article Snippet: To determine IFNGR1 levels by immunohistochemistry we used the polyclonal anti-IFNGR1 antibody (10808-1-AP, Proteintech; 1:200 dilution.

Techniques: Expressing, Gene Expression, RNA Sequencing

Journal: Cell Reports Medicine

Article Title: MYC activation impairs cell-intrinsic IFNγ signaling and confers resistance to anti-PD1/PD-L1 therapy in lung cancer

doi: 10.1016/j.xcrm.2023.101006

Figure Lengend Snippet:

Article Snippet: To determine IFNGR1 levels by immunohistochemistry we used the polyclonal anti-IFNGR1 antibody (10808-1-AP, Proteintech; 1:200 dilution.

Techniques: Virus, Recombinant, Modification, DC Protein Assay, Plasmid Preparation, Sequencing, SYBR Green Assay, Magnetic Beads, Gene Expression, Software

Journal: Biology

Article Title: KDM6B Variants May Contribute to the Pathophysiology of Human Cerebral Folate Deficiency

doi: 10.3390/biology12010074

Figure Lengend Snippet: Overexpression of KDM6B mutants affected FOLR1 protein level and folate uptake ability of HeLa cells. ( A ) HeLa cells were transfected with mutated and wild-type constructs of Flag-tagged KDM6B and pcDNA3.1 backbone vector for 48 h, and Western blotting was performed to quantify FOLR1 protein level in each group. GAPDH was used as loading control. ( B ) Western blotting was repeated three times and a Student t -test was performed to compare the protein levels between wild-type and mutant. Significance compared to WT: “a” represents p < 0.001; “c” represents p < 0.05.

Article Snippet: 48 h after transfection, cells were fixed with 4% paraformaldehyde for 30 min and cell membranns were permeabilized with 0.3% Triton X-100 in TBST for 20 min, and then blocked with 10% normal goat serum (NGS) in Tris-buffered saline with 0.1% Tween ® 20 Detergent (TBST) for 1 h. Cells were then incubated with FOLR1 antibody (1:100, Proteintech, Rosemont, IL, USA, 23355-1-AP) and flag antibody (1:1000, ThermoFisher Scientific, MA1-91878) overnight at 4 °C.

Techniques: Over Expression, Transfection, Construct, Plasmid Preparation, Western Blot, Control, 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 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