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Hnf4a

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HNF4A is a transcription factor that plays a crucial role in the regulation of gene expression in various tissues, particularly in the liver, pancreas, and kidneys. It is involved in the control of metabolic pathways and cellular differentiation. This product is a laboratory reagent for research purposes only.

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Lab products found in correlation

Hoechst 3342, by Thermo Fisher Scientific (2 mentions) Mitotracker deep red fm, by Thermo Fisher Scientific (2 mentions) Lsm 710 live duo scan confocal microscope, by Zeiss (2 mentions) Anti aat, by Santa Cruz Biotechnology (2 mentions) Triton x, by Merck Group (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Occludin, by Proteintech (1 mentions) Trpv6, by Proteintech (1 mentions) Actin, by Proteintech (1 mentions) Protease inhibitor, by Beyotime (1 mentions) Hrp conjugated secondary antibody, by Proteintech (1 mentions) Foxa2, by Abcam (1 mentions) β catenin antibody, by Proteintech (1 mentions) Mf52 n, by Mshot (1 mentions) Donkey serum, by Thermo Fisher Scientific (1 mentions) Alexa 568, by Thermo Fisher Scientific (1 mentions) Pan cadherin, by Merck Group (1 mentions) Col x, by Abcam (1 mentions) Alexa 647, by Thermo Fisher Scientific (1 mentions) Phosphor smad2 3, by Cell Signaling Technology (1 mentions)

12 protocols using hnf4a

1

Quantification of Intestinal Tight Junctions

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Ileal tissue or cell samples were added to RIPA lysate buffer containing 1% protease inhibitor (Beyotime) to extract total protein. The primary antibodies against Occludin (Proteintech, dilution ratio 1:1000), ZO-1 (Proteintech, dilution ratio 1:1000), TRPV6 (Proteintech, dilution ratio 1:1000), KLB (Aviva Systems, dilution ratio 1:1000), FXR1 (Proteintech cat#13194-1-AP, RRID: AB_2110702, dilution ratio 1:1000), HNF4A (Abcam cat#ab181604, RRID: AB_2890918, dilution ratio 1:1000), Actin (Proteintech, dilution ratio 1:2000) were used. The appropriate HRP-conjugated secondary antibodies (Proteintech, dilution ratio 1:4000) were utilized after overnight incubation of the primary antibodies at 4°C. The antibody validation was provided in the supplemental materials.
Hou Z., Ding Q., Li Y., Zhao Z., Yan F., Li Y., Wang X., Xu J., Chen W., Wu G., Ruan X, & Zhao L. (2022). Intestinal epithelial β Klotho is a critical protective factor in alcohol-induced intestinal barrier dysfunction and liver injury. eBioMedicine, 82, 104181.
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2

Immunofluorescence Staining of Hepatic Markers

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Cells cultured on 6-well plates (1000 μL/well)/12-well plates (400 μL/well) were divided into different groups. After washing with phosphate-buffered saline + 0.05% Tween (PBST) three times (5 min each time), the cells were fixed with precooled methyl alcohol for 20 min at − 20 °C. Triton X-100 (0.5%) was added to the cell slide and incubated for 20 min for transparency. After that, the cells were washed with PBST and blocked with bovine serum albumin (BSA) (Abcone) for 30 min. Finally, the cells were incubated overnight with SOX17 (Rabbit, Proteintech, 1:100)/FOXA2 (Mouse, Abcam, 1:200), HNF4A (Rabbit, Abcam, 1:200)/AFP (Mouse, ABclonal, 1:100), CK19 (Rabbit, Proteintech, 1:100)/ALB (Mouse, Proteintech, 1:100), or β-catenin antibody (Rabbit, Proteintech, 1:100) at 4 °C. After washing 3 times, the cells were incubated with goat anti-mouse/rabbit IgG-FITC or goat anti-mouse/rabbit IgG-RBITC fluorescent secondary antibodies (Earthox, 1:500) for 60 min. Then, the cells were mixed with DAPI reagent for 10 min. Cell slides were observed under an optical microscope (MF52-N, Mshot) or with Axio Imager M2 software (Nikon) (×100, ×200 magnification).
Bai F., Duan J., Yang D., Lai X., Zhu X., He X, & Hu A. (2022). Integrative network analysis of circular RNAs reveals regulatory mechanisms for hepatic specification of human iPSC-derived endoderm. Stem Cell Research & Therapy, 13, 468.
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3

Multiparametric Immunophenotyping of Hepatocyte-like Cells

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After fixation with 4% paraformaldehyde, cells were blocked and permeabilized in 1% w/v bovine serum albumin (BSA, Sigma-Aldrich), 10% donkey serum (Life Technologies) and 0.1% Triton) for 30 min at RT. For nuclear antigens, cells were treated with 0.5% Triton (Sigma-Aldrich), then primary antibodies, CTNNB (mouse, 1:100; bdbiosciences), CK19 (rabbit, 1:500; abcam), HNF4a (rabbit, 1:100; abcam), AFP (mouse, 1:100; abcam) and ALB (goat, 1:100; Bethyl), ASGR1 (mouse, 1:100; Thermo Fisher Scientific), COL1 (rabbit, 1:500; abcam), Vimentin (rabbit, 1:200; abcam); CD44 (mouse, 1:200 abcam), CD31 (rabbit, 1:50; abcam); Col-X (mouse, 1:250; abcam), MRP2 (mouse, 1:200; abcam), ZO-1 (mouse, 1:100; abcam), CD26 (rabbit, 1:100; abcam), Pan-Cadherin (mouse, 1:100; Sigma), Claudin 1 (rabbit, 1:100; abcam), Occludin (mouse, 1:100; abcam), Stem121 (mouse, 1:100; Cellartis), phosphor-SMAD2/3 (rabbit, 1:100; Cell signalling technology) and GLI1 (rabbit, 1:100; abcam) were applied for overnight at 4 °C. After washes, cells were then incubated with Alexa 647, Alexa 568, Alexa 488 conjugated secondary antibodies or 488-Phalloidin (1:250; Life Technologies). Samples were counterstained with DAPI (NucBlue, Life Technologies). Confocal micrographs were captured using Nikon Ti spinning disk confocal microscope equipped with Andor Neo camera and images were processed by NIS-element software.
Ng S.S., Saeb-Parsy K., Blackford S.J., Segal J.M., Serra M.P., Horcas-Lopez M., No D.Y., Mastoridis S., Jassem W., Frank C.W., Cho N.J., Nakauchi H., Glenn J.S, & Rashid S.T. (2018). Human iPS derived progenitors bioengineered into liver organoids using an inverted colloidal crystal poly (ethylene glycol) scaffold. Biomaterials, 182, 299-311.
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4

Quantification of EGFR Phosphorylation in Liver

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Liver tissue was homogenized in RIPA Buffer, supplemented with protease, and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was determined by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Protein (30 mg) was separated on a 4–15% gradient SDS Gel (BioRad, Hercules, CA), transferred, blocked and incubated with primary antibody according to the manufacturer’s instructions. The membranes were incubated in Pierce™ ECL agents (Thermo Fisher Scientific, Waltham, MA) and luminescent signals were captured with BioRad Chemidoc Imaging System. Western blot bands were quantified using BioRad Image Software. Primary antibodies for phosphorylated epidermal growth factor receptor (EGFR) Y1173 and Actin were obtained from Cell Signaling Technology (Danvers, MA, USA), total EGFR from Santa Cruz (Santa Cruz, CA, USA) and hepatocyte nuclear factor 4 alpha (HNF4A) from Abcam (Cambridge, MA, USA).
Wahlang B., Jin J., Hardesty J.E., Head K.Z., Shi H., Falkner K.C., Prough R.A., Klinge C.M, & Cave M.C. (2019). Identifying sex differences arising from polychlorinated biphenyl exposures in toxicant-associated liver disease. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association, 129, 64-76.
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5

Immunoprecipitation and Western Blot

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Cells were incubated with pre-cold IP lysis buffer for 5 min and then transferred to a 1.5 mL tube for centrifuged at 13,000 g for 10 min. The supernatant was collected and completed the IP process following the protocol of A Dynabeads protein G IP kit (Thermofisher, US). The WB was performed as previously described [21 (link)]. MARC2 (#ab224097), p27 (#ab32034), CCND1 (#ab16663) and SKP2 (#ab183039) were purchased from Abcam; YAP (#14074S), HNF4A (#3113S), pRB (#9301T), pLATS1 (#9157), LATS1 (#9153), pYAP(127) (#13008) and pYAP(397) (#13619) were purchased from Cell Signaling Technology; RNF123 (#sc-101122) used for co-IP and RB (#sc-74562) was purchased from Santa [22 (link), 23 (link)].
Wu D., Wang Y., Yang G., Zhang S., Liu Y., Zhou S., Guo H., Liang S., Cui Y., Zhang B., Ma K., Zhang C., Liu Y., Sun L., Wang J, & Liu L. (2020). A novel mitochondrial amidoxime reducing component 2 is a favorable indicator of cancer and suppresses the progression of hepatocellular carcinoma by regulating the expression of p27. Oncogene, 39(38), 6099-6112.
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6

Hepatocyte Differentiation and Mitochondrial Imaging

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For immunohistochemistry analysis, cells were passaged at day 5 of differentiation into 2-well chamber slides for the remainder of hepatic directed differentiation (ThermoFisher Scientific). For mitochondrial morphology analysis MitoTracker Deep Red FM (ThermoFisher Scientific) was applied at 200nM for 45 min prior to fixation. Cells were fixed using 4% paraformaldehyde for 10 min at room temperatures. For antibody labeling, cells were permeabilized with 0.3% Triton X-(MilliporeSigma) and blocked with 4% normal donkey or goat serum prior to incubation overnight with primary antibodies. Cells were probed using anti-AAT (Santa Cruz Biotechnologies), HNF4A (Abcam), or 2C1 (a kind gift from Elena Miranda and David Lomas) antibodies. Following incubation with primary antibody, cells were washed in PBS with 0.05% Tween20 (MilliporeSigma) and then incubated with secondary antibodies, anti-rabbit IgG-AlexaFluor647 and anti-mouse IgG-AlexaFluor488, for 1h at room temperature. Finally, cells were again washed with PBS containing 0.05% Tween20 and then counterstained with Hoechst 3342 (ThermoFisher Scientific). Cells were imaged using the Zeiss LSM 710-Live Duo scan confocal microscope and images were processed with ImageJ software.
Kaserman J.E., Werder R.B., Wang F., Matte T., Higgins M.I., Dodge M., Lindstrom-Vautrin J., Bawa P., Hinds A., Bullitt E., Caballero I.S., Shi X., Gerszten R.E., Brunetti-Pierri N., Liesa M., Villacorta-Martin C., Hollenberg A.N., Kotton D.N, & Wilson A.A. (2022). Human iPSC-hepatocyte modeling of alpha-1 antitrypsin heterozygosity reveals metabolic dysregulation and cellular heterogeneity. Cell reports, 41(10), 111775.
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7

Hepatocyte Growth Factor Regeneration

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HPF (>98% purity) was purchased from Chengdu ConBon Biotech Co., Ltd. (Chengdu, China). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), SOD, GSH, MDA, ALB and TBA test kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). CCl4 was provided by Shanghai MACKLIN Biochemical Technology Co., Ltd. (Shanghai, China). PAS and Oil-Red-O were provided by Beijing Solarbio Science Tenchnology Co., Ltd. (Beijing, China). hepatocyte growth factor (HGF) was provided by PeproTech (Rocky Hill, NJ, USA).
The following antibodies were utilized in this study: AFP (ab213328, Abcam, London, UK), CK18 (ab133263, Abcam, London, UK), HNF4A (ab199431, Abcam, London, UK), ALB (16475-1-AP, Proteintech, Chicago, IL, USA), GAPDH (10494-1-AP, Proteintech, Chicago, IL, USA), and CYP3A4 (1742R, Bioss, Beijing, China).
Yang B., Luo Q.L., Wang N., Hu Y.T., Zheng W.X., Li H., Maierziya M., Gu J, & Wang Q. (2023). HPF Modulates the Differentiation of BMSCs into HLCs and Promotes the Recovery of Acute Liver Injury in Mice. International Journal of Molecular Sciences, 24(6), 5686.
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8

Comprehensive Protein Analysis Using Western Blot

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Total protein extracts and western blot procedures were carried out as previously described25 (link). Antibodies specific for the following targets were used: HNF4A (Abcam, USA); Sp1, c-Myc, and CREB (Cell Signaling Technology, USA); p-HNF4A (Ser313), p-Sp1 (1:500, Thr739), p-c-Myc (Ser62), p-CREB (Ser133), p-JNK (Thr183+Tyr185), and p-p38 (Tyr323) (Biosynthesis Biotechnology, Beijing, China); extracellular signal–regulated kinase (ERK; Proteintech, Wuhan, China); p-ERK (Thr202+Tyr204, Bioworld, Nanjing, China); and c-Jun N-terminal kinase (JNK), p38, and GAPDH (Proteintech, Wuhan, China). The antibodies specific for Cav-1, Pofut1, NICD, HES1, and HEY1 were identical to those used in the immunohistochemical analysis described above. Chemiluminescence detection was performed using an ECL Kit (GE Healthcare, USA). GAPDH was detected on the same membrane as a loading control.
Zhang C., Huang H., Zhang J., Wu Q., Chen X., Huang T., Li W., Liu Y, & Zhang J. (2019). Caveolin-1 promotes invasion and metastasis by upregulating Pofut1 expression in mouse hepatocellular carcinoma. Cell Death & Disease, 10(7), 477.
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9

Quantifying Hepatocyte-Like Cell Differentiation

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Before intracellular staining, differentiated HLCs were dissociated using TrypLE (Thermo Fisher Scientific) reagent. Cells and then fixed and permeabilized with Cytofix/Cytoperm™ (Becton Dickinson, Franklin Lakes, NJ, USA) following the manufacturer’s guidance and instructions. Antibodies against ALB and hepatocyte nuclear factor 4α (HNF4A, Abcam, Cambridge, UK) were used to quantify the differentiation efficiency. Labeled primary antibodies were indirectly visualized using fluorescence-conjugated secondary antibodies. Data were acquired for each sample using a NovoCyte flow cytometer (ACEA Biosciences, San Diego, CA, USA).
Kim D., Kim S.B., Ryu J.L., Hong H., Chang J.H., Yoo T.J., Jin X., Park H.J., Han C., Lee B.H., Choi J.H., Yoo H.W., Kim J.H, & Woo D.H. (2020). Human Embryonic Stem Cell-Derived Wilson’s Disease Model for Screening Drug Efficacy. Cells, 9(4), 872.
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10

Hepatocyte Differentiation and Mitochondrial Imaging

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For immunohistochemistry analysis, cells were passaged at day 5 of differentiation into 2-well chamber slides for the remainder of hepatic directed differentiation (ThermoFisher Scientific). For mitochondrial morphology analysis MitoTracker Deep Red FM (ThermoFisher Scientific) was applied at 200nM for 45 min prior to fixation. Cells were fixed using 4% paraformaldehyde for 10 min at room temperatures. For antibody labeling, cells were permeabilized with 0.3% Triton X-(MilliporeSigma) and blocked with 4% normal donkey or goat serum prior to incubation overnight with primary antibodies. Cells were probed using anti-AAT (Santa Cruz Biotechnologies), HNF4A (Abcam), or 2C1 (a kind gift from Elena Miranda and David Lomas) antibodies. Following incubation with primary antibody, cells were washed in PBS with 0.05% Tween20 (MilliporeSigma) and then incubated with secondary antibodies, anti-rabbit IgG-AlexaFluor647 and anti-mouse IgG-AlexaFluor488, for 1h at room temperature. Finally, cells were again washed with PBS containing 0.05% Tween20 and then counterstained with Hoechst 3342 (ThermoFisher Scientific). Cells were imaged using the Zeiss LSM 710-Live Duo scan confocal microscope and images were processed with ImageJ software.
Kaserman J.E., Werder R.B., Wang F., Matte T., Higgins M.I., Dodge M., Lindstrom-Vautrin J., Bawa P., Hinds A., Bullitt E., Caballero I.S., Shi X., Gerszten R.E., Brunetti-Pierri N., Liesa M., Villacorta-Martin C., Hollenberg A.N., Kotton D.N, & Wilson A.A. (2022). Human iPSC-hepatocyte modeling of alpha-1 antitrypsin heterozygosity reveals metabolic dysregulation and cellular heterogeneity. Cell reports, 41(10), 111775.
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