1. Volume 29, Issue 10, Pages 3212-3222.e4 (December 2019)
Maintenance of Primary Hepatocyte Functions In Vitro by Inhibiting Mechanical Tension- Induced YAP Activation 
Pingxin Sun, Guanyu Zhang, Xiaohui Su, Caixia Jin, Bing Yu, Xinlu Yu, Zhuman Lv, Haoxin Ma, Mingliang Zhang, Wanguo Wei, Wenlin Li 
Cell Reports 
Volume 29, Issue 10, Pages e4 (December 2019)
DOI: /j.celrep
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2. Cell Reports 2019 29, 3212-3222.e4DOI: (10.1016/j.celrep.2019.10.128)
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3. Figure 1
Hepatocyte Dedifferentiation Occurred Concomitantly with Actomyosin Tension and Yap Activation
(A) qRT-PCR assay was performed to analyze the expression of hepatocyte functional genes and liver progenitor cell markers by FHs (freshly isolated hepatocytes without culture), and hepatocytes cultured at different time points (mean ± SD, n = 3).
(B) Immunofluorescence demonstrated that hepatocytes at the edge of the cell clusters more profoundly lost the expression of Alb and Hnf4α. Arrows indicate the cells that are negative for Alb/Hnf4α. The scale bar represents 100 μM.
(C) The stress fibers were revealed by Alexa Fluor 555 phalloidin staining at 0.5 h (showing a group of undissociated hepatocytes), 1, 3, and 7 days after hepatocyte attachment. The scale bar represents 100 μM.
(D) Cell spread was quantified after cell seeding (mean ± SEM, n = 3).
(E) The expression of the YAP downstream genes Ctgf and Cyr61 was analyzed by qRT-PCR after cell plating (mean ± SD, n = 3).
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4. Figure 2
Alleviation of Actomyosin Tension by Restricting Cell Spreading-Inhibited Hepatocyte Dedifferentiation
(A) On 700-μm2 disc domains of micropatterned slides, single hepatocytes survived up to 3 weeks as rounded cells. Phalloidin staining was used to reveal stress fibers. Yap distribution and expression of hepatocyte functional genes, including Alb and Hnf4α, were analyzed by immunostaining. The scale bar represents 50 μM.
(B) On unpatterned slides, phalloidin staining was used to reveal stress fibers. Yap distribution and expression of hepatocyte functional genes, including Alb and Hnf4α, were analyzed by immunostaining. The scale bar represents 50 μM.
(C and D) The quantification of nuclear Yap (nuc-Yap), Alb, and Hnf4α expression by hepatocytes cultured on a Matrigel-coated surface (C) or micropatterned slides (D). Data are shown as the means ± SEMs of three independent experiments. ∗∗p < 0.01.
(E) Hepatocyte spreading on micropatterned slides was quantified (means ± SEMs of three independent experiments).
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5. Figure 3
Yap Deletion Blocked Actomyosin Tension-Induced Hepatocyte Dedifferentiation
(A) AAV expressing Cre/GFP was used to transduce hepatocytes of Yapf/f mice through tail vein injection. The hepatocytes were harvested 1 week after being transduced. The scale bar represents 100 μM.
(B) Cre-mediated recombination was measured by genomic PCR.
(C) After a 3-week culture, the expression of Alb and Hnf4α by Yap−/− and Yapf/f hepatocytes was analyzed by qRT-PCR (mean ± SD, n = 3).
(D) After a 3-week culture, the expression of Ctgf and Vim by Yap−/− and Yapf/f hepatocytes was analyzed by qRT-PCR (mean ± SD, n = 3).
(E) After a 3-week culture, the expression of Alb, Hnf4α, Fah, E-cad, and YAP was analyzed by immunostaining. F-actin was revealed by phalloidin staining. The scale bar represents 100 μM.
See also Figure S1.
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6. Figure 4
A Small Molecular Cocktail LBDXL Was Able to Sustain Hepatocyte Functions by Targeting F-Actin and Yap
(A) The strategy of reiterative chemical screening is illustrated.
(B and C) The expression of Alb, Hnf4α (B), Ctgf, and Vim (C) was analyzed by qRT-PCR after treatment with the compounds and their combinations for 1 week (mean ± SD, n = 3).
(D) After a 3-week culture, the expression of Alb, Hnf4α, and Fah by LBDXL and BM hepatocytes was analyzed by immunocytochemistry. Periodic acid-Schiff staining was performed to reveal glycogen storage. F-actin was revealed by phalloidin staining. E-cad and β-cat staining was performed to demonstrate the adherens junction. The capacity of hepatocytes to take up low-density lipoprotein (LDL) was visualized by LDL-Alexa Fluor 488. The scale bar represents 100 μM.
(E and F) Alb secretion (E) and urea synthesis (F) were analyzed. Data are shown as the means ± SEMs of three independent experiments. ∗∗p < 0.01.
L, latrunculin B; B, blebbistatin; D, dasatinib; X, XAV939; Ly, LY See also Figures S2, S3, S4, and S5, and Table S1.
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7. Figure 5
LBDXL Hepatocytes Internalized and Effluxed Cholyl-l-Lysyl-Fluorescein (CLF) into Bile Canaliculi
(A–H) Hepatocytes from Rosa26loxP-mTom-stop-loxP-mGFP (mTmG) reporter mice were cultured for 3 weeks. Biliary CLF efflux from LBDXL (A) and BM hepatocytes (E) was analyzed. The cell membrane was revealed by mTomato for LBDXL (B) and BM hepatocytes (F). (C) is merged image of (A) and (B). (G) is a merged image of (E) and (F). (D) and (H) are the boxed areas in (C) and (G), respectively. The scale bar represents 100 μM.
(I and J) Bile canaliculi of LBDXL (I) and BM hepatocytes (J) were revealed by DPP4 staining after a 3-week culture. The scale bar represents 100 μM.
(K–P) Cyclosporin A (1 μM, K), troglitazone (5 μM, M), and chlorpromazine (0.5 μM, O) were tested in inhibiting CLF efflux in LBDXL 3W hepatocytes. (L), (N), and (P) are the corresponding phase contrast images of (K), (M), and (O). Inset in (O) is a higher magnification view of the area in the dotted white box. The scale bar represents 100 μM.
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8. Figure 6 LBDXL Hepatocytes Were Metabolically Functioning
(A) Expression of Cyp1a2 and Cyp2e1 by LBDXL and BM hepatocytes (with or without ethanol induction) was measured by qRT-PCR. Data are expressed as means ± SDs (n = 3).
(B) LDH leakage assay was performed to evaluate cell injury after acetaminophen (APAP) treatment under various conditions. NAC, N-acetylcysteine. Data are expressed as means ± SD (n = 3).
(C) PI staining to show compromised cell membranes of LBDXL hepatocytes after various treatments.
(D) In the assay for Cyp1a2, Cyp3a11, and Cyp2d9 activities, the metabolites of phenacetin (APAP), midazolam (1′-hydroxymidazolam), and bufuralol (1′-hydroxybufuralol) were determined by high-performance liquid chromatography/tandem mass spectrometry. FHs were used as the positive control at 8 h after plating. Data are expressed as means ± SEMs of 3 independent experiments. ∗∗p < 0.01.
See also Figure S6.
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9. Figure 7 LBDXL Hepatocytes Could Repopulate Fah−/− Mouse Livers
(A) Kaplan-Meier curve showed the survival rate of cell-transplanted Fah−/− mice after removing the supply of NTBC.
(B and C) After immunohistochemical staining of Fah, liver repopulation by LBDXL hepatocytes and FH was quantified using ImageJ software (B). Immunohistochemical staining of Fah was shown in (C). Data are expressed as means ± SEMs.
(D) The transplanted cells demonstrated mature hepatocyte phenotypes with the expression of Fah, Alb, and the bile canaliculus marker Dpp4. The scale bar represents 200 μM.
See also Figure S7.
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