Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , General morphology of livers 2 days after PHx. b , H&E staining of liver sections 2 days after PHx. c , d , Ki67 + percentages in HNF4α + hepatocytes ( c ) and HNF4α − NPCs ( d ) 2 days after PHx. Means ± SEM are shown, n=3 per group, **P < 0.01 (two-tailed unpaired t test). n.s., not significant. e , EpCAM was highly expressed in biliary epithelial cells (arrow), but not in the colony (dashed line). Stellate cells (GFAP) were not altered around the colony. f , CK19 was highly expressed in biliary epithelial cells (arrow), but not in the colony (dashed line). CD133 was highly expressed in the colony. g , Sox9 and CD44 were highly expressed in biliary epithelial cells (arrowhead), but was not upregulated in the colony (dashed line) compared to surrounding hepatocytes. h , AFP showed no difference between the colony (dashed line) and the surrounding tissue. i , Liver/body weight ratios after PHx. Means ± SEM from 3 or more mice analyzed for each time point and group are shown. j , k , Immunofluorescent staining of SKO liver sections 3 weeks after PHx. Colonies were at various sizes and locations as shown by arrows in j . Macroscopic colonies were found as shown by arrows and dashed lines in k . Immunofluorescent image in k corresponds to the magenta arrow in the liver image. PV, portal vein; CV, central vein. l , Vasculature shown by PECAM did not distinguish the colony (arrow). m , HGF expressed by NPCs was not concentrated in the colony (dashed line). Scale bars, 1 cm ( a ) 100 μm ( b , e - g , i - l ).

Article Snippet: In addition to antibodies used for immunostaining, antibodies for EpCAM (bs-1513R; Bioss), HNF4α (GTX89532; GeneTex), GAPDH (5174; CST), β-actin (A5316; Sigma), Stat3 (9132; CST), Erk1/2 (4695; CST) and Cyclin D1 (sc-20044; Santa Cruz Biotechnology) were used as primary antibodies.

Techniques: Staining, Two Tailed Test

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , b , Immunofluorescence on liver tissue sections 2 days after PHx. HNF4α is a hepatocyte marker and Ki67 is a proliferation marker. Arrow in a points to an area enriched with proliferating hepatocytes. Dashed line in b shows an area with continuous CD133 expression. c , Quantification of proliferating rate in hepatocytes in CD133-positive and -negative areas in SKO livers 2 days after PHx. Each dot indicates one area. Data were collected from 3 mice. Means ± SEM are shown. ****P < 0.0001 (two-tailed unpaired t test). d , While WT hepatocytes proliferated at high frequency everywhere, proliferating hepatocytes in SKO liver were mostly located in patchy areas marked by CD133 expression. e , Immunofluorescence of CD133 on liver tissues at day 0 or 2, and 3 weeks (0d, 2d, 3 wk) after PHx. CD133 + hepatocyte clusters were only found in SKO livers after PHx (light green arrows). In WT livers, CD133 expression was only seen in bile duct epithelial cells (arrowheads). Scale bars, 100 μm ( a , e ).

Article Snippet: In addition to antibodies used for immunostaining, antibodies for EpCAM (bs-1513R; Bioss), HNF4α (GTX89532; GeneTex), GAPDH (5174; CST), β-actin (A5316; Sigma), Stat3 (9132; CST), Erk1/2 (4695; CST) and Cyclin D1 (sc-20044; Santa Cruz Biotechnology) were used as primary antibodies.

Techniques: Immunofluorescence, Marker, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , Schematic illustration of mosaic rescue experiment of Shp2 in Shp2-deficient liver. b , Immunofluorescence showing Shp2-rescued hepatocytes also labeled with GFP. c , GFP + hepatocytes were proliferating (Ki67 + ; arrows), while other proliferating cells were not hepatocytes (arrowheads). Areas with colonies were not included here. d , Colonies were frequently constituted by GFP-negative hepatocytes (dashed line). Orange dots: non-specific stains of debris caused by HTVi. e , Some GFP + hepatocytes showed expression of CD133 (arrowheads). Dashed lines show CD133-positive areas. f , qRT-PCR analysis of WT and SKO liver lysates 2 days after PHx. n=3, means ± SEM are shown, *P < 0.05, **P < 0.01 (two-tailed unpaired t test). g , Immunofluorescence on SKO liver section 2 days after PHx. White dashed lines mark Porcupine + cells enriched in CD133 + colonies, rarely detected in non-colony area (arrowheads). Pink dashed lines: vasculature. h , Immunofluorescence on the colonies (arrows) in Shp2-deficient hepatocyte culture in vitro. Arrowhead, dying cells with high autofluorescence. i , Immunoblot analysis of WT and SKO liver lysates 2 days after PHx. j , qRT-PCR analysis of SKO liver tissue lysates without PHx (0d, 3 mice) and with PHx (2d, 3 mice). Means ± SEM are shown. **P < 0.01 (two-tailed unpaired t test). k , qRT-PCR analysis of hepatocyte and NPC fractions from SKO livers (3 mice) 2 days after PHx. HNF4α and CD45 were used as positive controls for the fractionation. β-actin was used for normalization, because GAPDH was highly expressed by hepatocytes. Means ± SEM are shown. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired t test). l , qPCR analyses of PLC cells treated with Mek inhibitor and Porcn inhibitor. Means ± SD from 4 wells are shown. *P < 0.05, **P < 0.01, (two-tailed unpaired t test). n.s., not significant. Scale bars, 100 μm ( b - e , g , h ).

Article Snippet: In addition to antibodies used for immunostaining, antibodies for EpCAM (bs-1513R; Bioss), HNF4α (GTX89532; GeneTex), GAPDH (5174; CST), β-actin (A5316; Sigma), Stat3 (9132; CST), Erk1/2 (4695; CST) and Cyclin D1 (sc-20044; Santa Cruz Biotechnology) were used as primary antibodies.

Techniques: Immunofluorescence, Labeling, Expressing, Quantitative RT-PCR, Two Tailed Test, In Vitro, Western Blot, Fractionation

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , b , Immunofluorescence of liver sections 2 days after CCl 4 injection. Proliferating hepatocytes were scattered in WT livers, whereas they were highly concentrated in CD133 + colonies (white dashed line) in SKO livers as shown in a . CD133 + /EpCAM + /HNF4α - bile duct epithelial cells (arrowheads) were not associated with the colonies. Pink dashed lines, injured areas. PV, portal vein. c , Immunofluorescence of Met hep-/- liver sections 2 days after PHx or CCl4 injection. White dashed lines, CD133 + colonies. Pink dashed line, injured area. d , qRT-PCR analysis of PLC cell lysates treated with Shp2 or MEK inhibitors (Shp2i and MEKi). **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-tailed unpaired t test, each compared with DMSO treatment). Means ± SD from 3 replicates are shown. e , Immunoblotting of PLC cell lysates treated with inhibitors or transfected with Shp2 targeting CRISPR vector. Guide RNA targeting the AAVS1 safe harbor site was used as a control (sgCtrl). f , qRT-PCR analysis of MCF10A cell lysates treated with the inhibitors. *P < 0.05, ***P < 0.001, ****P < 0.0001 (two-tailed unpaired t test, each compared with DMSO treatment). Means ± SD from 3 replicates are shown. g , Immunoblotting of MCF10A cell lysates treated with inhibitors. h , qRT-PCR analysis of lysates from various cell lines treated with Shp2 or MEK inhibitors. *P < 0.05, ***P < 0.001, ****P < 0.0001 (two-tailed unpaired t test, each compared with DMSO treatment). Means ± SD from 3 replicates are shown. Scale bars, 100 μm ( a - c ).

Article Snippet: In addition to antibodies used for immunostaining, antibodies for EpCAM (bs-1513R; Bioss), HNF4α (GTX89532; GeneTex), GAPDH (5174; CST), β-actin (A5316; Sigma), Stat3 (9132; CST), Erk1/2 (4695; CST) and Cyclin D1 (sc-20044; Santa Cruz Biotechnology) were used as primary antibodies.

Techniques: Immunofluorescence, Injection, Quantitative RT-PCR, Two Tailed Test, Western Blot, Transfection, CRISPR, Plasmid Preparation

Journal: bioRxiv

Article Title: CD133 + Intercellsome Mediates Direct Cell-Cell Communication to Offset Intracellular Signal Deficit

doi: 10.1101/2022.05.16.492226

Figure Lengend Snippet: a , Immunoblotting of HuR in the CD133 + vesicles from SKO liver after PHx. GAPDH and HNF4α were used as controls for cytoplasmic and nuclear fractions, respectively. b , Immunofluorescence on PLC cells. Arrows, peri-nuclear areas enriched with HuR in the cytoplasm; Arrowheads show colocalization of HuR on the CD133 + filament bridging two cells. c , Immunofluorescence on PLC cells treated with the Shp2 inhibitor (SHP099). Arrowheads show strong localization of HuR on the CD133 + filaments. d , Immunofluorescence on PLC cells transfected with CD133 expression vector treated with a Shp2 inhibitor (SHP099). Arrowheads show strong localization of HuR on the CD133 + filaments. e , Immunoblotting of CD133 + vesicles isolated from MEK inhibitor-treated PLC cells. f , qRT-PCR analysis of PLC cell lysates after treatment with CD133 + vesicles isolated from MEK inhibitor-treated PLC cells. RNase and Triton X-100 were used to digest the RNA content of the vesicles. *P < 0.05 (two-tailed unpaired t test). n.s., not statistically significant. Means ± SD from 3 replicates are shown. g , A model and predictions for single cell RNA-seq data analysis. h , Total IEG expression levels. Cyclin D1-positive and -negative cells were analyzed separately to evaluate the influence of cell cycle on the IEG analysis. See Methods section for what the bars and dots represent. i , j , Box plots (Tukey’s) of IEG diversity within each cell (calculated as entropy) and IEG variations among cells. Analyses were focused on cyclin D1 + cells in all groups for fair comparison. k , l , Plot of intracellular IEG diversity against total IEG expression levels in SKO hepatocytes 2 days after PHx. Blue color gradient indicates cyclin D1 expression levels. For the simulation in l , the parameters used were: Group of 5 cells, X=1/12, model 3 (see also and Methods). Green and Black arrows show typical profiles of CD133-positive and -negative cells, respectively. m , Box plot (Tukey’s) of intracellular IEG diversity after simulation. The analysis was not limited to cyclin D1-positive or -negative cells. Note the simulation of the IEG exchange attracted the cells from cyclin D1-low profile to cyclin D1-high profile ( k - m ). Statistics was performed by Wilcoxon rank sum test adjusted by FDR in i , j and m . Scale bars, 50 μm ( b , c ), 5 μm ( d ).

Article Snippet: In addition to antibodies used for immunostaining, antibodies for EpCAM (bs-1513R; Bioss), HNF4α (GTX89532; GeneTex), GAPDH (5174; CST), β-actin (A5316; Sigma), Stat3 (9132; CST), Erk1/2 (4695; CST) and Cyclin D1 (sc-20044; Santa Cruz Biotechnology) were used as primary antibodies.

Techniques: Western Blot, Immunofluorescence, Transfection, Expressing, Plasmid Preparation, Isolation, Quantitative RT-PCR, Two Tailed Test, RNA Sequencing Assay

Journal: Scientific Reports

Article Title: Identification of HNF-4α as a key transcription factor to promote ChREBP expression in response to glucose

doi: 10.1038/srep23944

Figure Lengend Snippet: ( a ). Gene structure of human ChREBP-α and ChREBP-β with indication of splice sites and translational start sites (ATG). Note that ChREBP-α and ChREBP-β are transcribed from different promoters separated by 23 kb. ( b ). Real-time PCR analysis for mRNA levels of ChREBP-α at 48 hours after expression plasmids containing control (GFP) or other cDNAs are transfected in 293T cells. *indicates p < 0.05 when compared with the GFP-transfected sample. ( c ). Real-time PCR analysis for mRNA levels of ChREBP-β at 48 hours after expression plasmids containing control (GFP) or other cDNAs are transfected in 293T cells. *indicates p < 0.05 when compared with the GFP-transfected sample. ( d , e ). Real-time PCR analysis for mRNA levels of ChREBP-α, ChREBP-β and total ChREBP ( d ) and western blot analysis for endogenous ChREBP expression ( e ) at 48 hours after HA-GFP (vector) or HA-HNF-4α expression plasmids are transfected in HepG2 cells. *indicates p < 0.05 when compared with the vector-transfected sample. Tubulin serves as the loading control. ( f ).Real-time PCR analysis for mRNA levels of HNF-4α, ChREBP-α, ChREBP-β and total ChREBP at 72 hours after control (NC) or two HNF-4α siRNAs (HNF-4α-1 and HNF-4α-2) are transfected in HepG2 cells. *indicates p < 0.05 when compared with the corresponding NC-transfected sample. ( g , h ). Real-time PCR analysis for mRNA levels of HNF-4α, ChREBP-α, ChREBP-β and total ChREBP ( g ) and western blot analysis for endogenous ChREBP expression ( h ) at 48 hours after control (NC) or HNF-4α shRNAs are transfected in mouse primary hepatocytes. *indicates p < 0.05 when compared with the corresponding NC-transfected sample. Actin serves as the loading control.

Article Snippet: The following reagents were used: Dulbecco’s Modified Eagle’s Medium (DMEM) (Hyclone, USA), Opti-MEM and no glucose DMEM (Invitrogen, USA), Fetal bovine serum (FBS) (Biochrom, Germany), Triton X-100 and Nonidet P40 (NP-40) (Sigma, USA), anti-ChREBP (Novus Biologicals, USA), anti-HNF-4α (Absci, USA), anti-actin (Cell Signaling Technology, USA), anti-FLAG (Sigma, USA), anti-HA and anti-Myc (Santa Cruz Biotechnology, USA) antibody.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Transfection, Western Blot, Plasmid Preparation

Journal: Scientific Reports

Article Title: Identification of HNF-4α as a key transcription factor to promote ChREBP expression in response to glucose

doi: 10.1038/srep23944

Figure Lengend Snippet: ( a , b ). Real-time PCR analysis for mRNA levels of HNF-4α (A) and total ChREBP (B) in mouse primary hepatocytes treated with 0 (G0), 5.6 (G5.6) or 25 mM glucose (G25) for 18 hours. *indicates p < 0.05 when compared with the corresponding 0 mM glucose-treated sample. ( c ). Western blot analysis for endogenous ChREBP and HNF-4α protein expression in mouse primary hepatocytes treated with 0, 5.6 or 25 mM glucose for 18 hours. Tubulin serves as the loading control. ( d ). Real-time PCR analysis for mRNA levels of ChREBP-α, ChREBP-β and total ChREBP in mouse liver after being fasted or fed for 18 hours. *indicates p < 0.05. ( e ). Real-time PCR analysis for mRNA levels of FAS, L-PK, SCD1, G6Pase and PEPCK in mouse liver after being fasted or fed for 18 hours. *indicates p < 0.05. ( f ). Western blot analysis for endogenous ChREBP and HNF-4α protein expression in mouse liver after being fasted or fed for 18 hours. Tubulin serves as the loading control. ( g ). Immunohistochemistry analysis for mouse liver after being fasted or fed for 18 hours using anti-ChREBP and anti-HNF-4α antibodies. Bars represent 100 μm.

Article Snippet: The following reagents were used: Dulbecco’s Modified Eagle’s Medium (DMEM) (Hyclone, USA), Opti-MEM and no glucose DMEM (Invitrogen, USA), Fetal bovine serum (FBS) (Biochrom, Germany), Triton X-100 and Nonidet P40 (NP-40) (Sigma, USA), anti-ChREBP (Novus Biologicals, USA), anti-HNF-4α (Absci, USA), anti-actin (Cell Signaling Technology, USA), anti-FLAG (Sigma, USA), anti-HA and anti-Myc (Santa Cruz Biotechnology, USA) antibody.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunohistochemistry

Journal: Scientific Reports

Article Title: Identification of HNF-4α as a key transcription factor to promote ChREBP expression in response to glucose

doi: 10.1038/srep23944

Figure Lengend Snippet: ( a ). Luciferase activity assay shows that HA-HNF-4α fails to increase transcriptional activity of the 4 kb ChREBP-α promoter compared with the HA-GFP-transfected sample. 24 hours after the 4 kb ChREBP-α promoter in pGL3-Basic plasmid and the HA-GFP, HA-LXR or HA-HNF-4α expression plasmid are transfected in 293T cells, luciferase activity is analyzed. *indicates p < 0.05 when compared with the HA-GFP-transfected sample. ( b ). Luciferase activity assay shows that HNF-4α enhances transcriptional activity of the pGL3-Promoter plasmid containing the 140 bp sequence in intron 12 of ChREBP-α (IV) compared with the control. I, II, III and IV are the pGL3-Promoter plasmids containing the 164 bp, 174 bp, 378 bp and 140 bp sequences located in intron 2, intron 6, intron 7 and intron 12 of ChREBP-α. 24 hours after I, II, III or IV and the HA-GFP or HA-HNF-4α expression plasmid are transfected in 293T cells, luciferase activity is analyzed. *indicates p < 0.05 when compared with the HA-GFP-transfected sample. ( c ). Luciferase activity assay shows that deletion of the E-box in IV reduced induction of the transcriptional activity of IV by HNF-4α compared with the control. 24 hours after the pGL3-Promoter plasmid, IV or IVΔE-box and the HA-GFP, HA-LXR or HA-HNF-4α expression plasmid are transfected in 293T cells, luciferase activity is analyzed. *indicates p < 0.05 when compared with the HA-GFP-transfected sample. Western blot analysis using the anti-HA antibody shows protein levels of ectopically expressed HA-HNF-4α and HA-LXR. ( d ). ChIP analysis for HepG2 cells using an anti-HNF-4α antibody, nonspecific IgG or anti-histone antibody shows that HNF-4α binds the 140 bp region in intron 12 of the ChREBP-α gene.

Article Snippet: The following reagents were used: Dulbecco’s Modified Eagle’s Medium (DMEM) (Hyclone, USA), Opti-MEM and no glucose DMEM (Invitrogen, USA), Fetal bovine serum (FBS) (Biochrom, Germany), Triton X-100 and Nonidet P40 (NP-40) (Sigma, USA), anti-ChREBP (Novus Biologicals, USA), anti-HNF-4α (Absci, USA), anti-actin (Cell Signaling Technology, USA), anti-FLAG (Sigma, USA), anti-HA and anti-Myc (Santa Cruz Biotechnology, USA) antibody.

Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing, Sequencing, Western Blot

Journal: Scientific Reports

Article Title: Identification of HNF-4α as a key transcription factor to promote ChREBP expression in response to glucose

doi: 10.1038/srep23944

Figure Lengend Snippet: ( a ). Schematic diagrams of the ChREBP-β promoter fragments in the pGL4-Basic plasmid. ( b ). Luciferase activity analysis for the ChREBP-β promoter fragments in the pGL4-Basic plasmid at 24 hours after the ChREBP-β promoter fragment plasmids and empty vector or HA-HNF-4α expression plasmid are transfected in 293T cells. Western blot analysis using the anti-HA antibody shows protein levels of ectopically expressed HA-HNF-4α. Actin serves as the loading control. *indicates p < 0.05 when compared with the other four black bars. ( c ). Real-time PCR analysis for mRNA levels of HNF-4α at 72 hours after control (NC) or two HNF-4α siRNAs (HNF-4α-1 and HNF-4α-2) are transfected in 293T cells. ( d ). Luciferase activity analysis for the 2.9 kb ChREBP-β promoter in the pGL4-Basic plasmid at 72 hours after the ChREBP-β promoter plasmid and control or two HNF-4α siRNAs are transfected in 293T cells. ( e ). DNA sequences of the three DR1 sites in the nucleotides −139 to −38 region of the ChREBP-β promoter. ( f ). Luciferase activity analysis for the 2.9 kb (WT), ΔDR1-A, ΔDR1-B and ΔDR1-C ChREBP-β promoter in the pGL4-Basic plasmids at 24 hours after the ChREBP-β promoter plasmids and empty vector or FLAG-HNF-4α expression plasmid are transfected in 293T cells. ( g ). Luciferase activity analysis for the 2.9 kb (WT), Δ(−57~−53) and Δ(−42~−38) ChREBP-β promoter in the pGL4-Basic plasmids at 24 hours after the ChREBP-β promoter plasmids and empty vector or FLAG-HNF-4α expression plasmid are transfected in 293T cells. ( h ). Luciferase activity analysis for the pGL4, 2.9 kb (WT), 0.3 kb, ΔDR1-C and DR1-C mutant ChREBP-β promoter in the pGL4-Basic plasmids at 24 hours after the ChREBP-β promoter plasmids and empty vector or HA-HNF-4α expression plasmid are transfected in 293T cells. The DR1-C mutant contains 7 underlined point mutations (G C GG ACTCT GA A A). (i). ChIP analysis for HepG2 cells using an anti-HNF-4α antibody, nonspecific IgG or anti-histone antibody shows that HNF-4α binds ChREBP-β promoter. *in C-D and F-H indicates p < 0.05 when compared with the corresponding NC or empty vector -transfected sample.

Article Snippet: The following reagents were used: Dulbecco’s Modified Eagle’s Medium (DMEM) (Hyclone, USA), Opti-MEM and no glucose DMEM (Invitrogen, USA), Fetal bovine serum (FBS) (Biochrom, Germany), Triton X-100 and Nonidet P40 (NP-40) (Sigma, USA), anti-ChREBP (Novus Biologicals, USA), anti-HNF-4α (Absci, USA), anti-actin (Cell Signaling Technology, USA), anti-FLAG (Sigma, USA), anti-HA and anti-Myc (Santa Cruz Biotechnology, USA) antibody.

Techniques: Plasmid Preparation, Luciferase, Activity Assay, Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Mutagenesis

Journal: Scientific Reports

Article Title: Identification of HNF-4α as a key transcription factor to promote ChREBP expression in response to glucose

doi: 10.1038/srep23944

Figure Lengend Snippet: (a). Luciferase activity analysis for the 2.9 kb ChREBP-β promoter in the pGL4-Basic plasmid at 24 hours after the ChREBP-β promoter plasmid and empty vector or HA-HNF-4α or HA-ChREBP-α or HA-ChREBP-α and FLAG-Mlx or HA-HNF-4α and HA-ChREBP-α or HA-HNF-4α, HA-ChREBP-α and FLAG-Mlx expression plasmids are transfected in 293T cells. *indicates p < 0.05. (b). Luciferase activity analysis for the 2.9 kb (WT), ΔE-box1, ΔE-box2, and ΔChoRE ChREBP-β promoter in the pGL4-Basic plasmids at 24 hours after the ChREBP-β promoter plasmids and empty vector or HA-HNF-4α expression plasmid are transfected in 293T cells. *indicates p < 0.05 when compared with the empty vector-transfected sample. (c). Co-IP analysis for ectopically expressed ChREBP-α, HNF-4α and Mlx at 48 hours after transfection in 293T cells. Tubulin serves as the loading control. (d). Co-IP analysis for ectopically expressed ChREBP-α, HNF-4α and Mlx in 293T cells treated with 0 (G0), 5.6 (G5.6) or 25 mM glucose (G25) for 18 hours. Actin serves as the loading control. The intensity of bands has been measured and the ratio between immunoprecipitated FLAG-ChREBP-α and HA-HNF-4α is shown as fold of induction at G5.6 or G25 compared with G0.

Article Snippet: The following reagents were used: Dulbecco’s Modified Eagle’s Medium (DMEM) (Hyclone, USA), Opti-MEM and no glucose DMEM (Invitrogen, USA), Fetal bovine serum (FBS) (Biochrom, Germany), Triton X-100 and Nonidet P40 (NP-40) (Sigma, USA), anti-ChREBP (Novus Biologicals, USA), anti-HNF-4α (Absci, USA), anti-actin (Cell Signaling Technology, USA), anti-FLAG (Sigma, USA), anti-HA and anti-Myc (Santa Cruz Biotechnology, USA) antibody.

Techniques: Luciferase, Activity Assay, Plasmid Preparation, Expressing, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: Scientific Reports

Article Title: Identification of HNF-4α as a key transcription factor to promote ChREBP expression in response to glucose

doi: 10.1038/srep23944

Figure Lengend Snippet: (a). Real-time PCR analysis for mRNA levels of ChREBP-α, ChREBP-β and total ChREBP in HepG2 cells treated with 0 or 25 mM glucose for 18 hours after control (NC) or two HNF-4α siRNAs (HNF-4α-1 and HNF-4α-2) are transfected. *indicates p < 0.05. (b). Western blot analysis for endogenous ChREBP expression in HepG2 cells treated with 0 or 25 mM glucose for 18 hours after control (NC) or two HNF-4α siRNAs (HNF-4α-1 and HNF-4α-2) are transfected. Tubulin serves as the loading control. (c). Western blot analysis for subcellular localization of endogenous HNF-4α in HepG2 cells after 0, 2.5, 5.6 and 25 mM glucose treatment for 18 hours. PARP and tubulin serve as loading controls and cell fractionation controls. ( d , e ). ChIP analysis for HepG2 cells using an anti-HNF-4α antibody, nonspecific IgG or anti-histone antibody shows that binding capacity of HNF-4α for ChREBP-α ( d ) and ChREBP-β ( e ) increases under the 25 mM glucose condition (G25) when compared to the 0 mM glucose condition (G0). (f). Diagram showing that glucose promotes ChREBP transcription by increasing HNF-4α nuclear localization and binding to the cis -elements in ChREBP-α and ChREBP-β.

Article Snippet: The following reagents were used: Dulbecco’s Modified Eagle’s Medium (DMEM) (Hyclone, USA), Opti-MEM and no glucose DMEM (Invitrogen, USA), Fetal bovine serum (FBS) (Biochrom, Germany), Triton X-100 and Nonidet P40 (NP-40) (Sigma, USA), anti-ChREBP (Novus Biologicals, USA), anti-HNF-4α (Absci, USA), anti-actin (Cell Signaling Technology, USA), anti-FLAG (Sigma, USA), anti-HA and anti-Myc (Santa Cruz Biotechnology, USA) antibody.

Techniques: Real-time Polymerase Chain Reaction, Transfection, Western Blot, Expressing, Cell Fractionation, Binding Assay