1. Volume 27, Issue 4, Pages 1176-1189.e5 (April 2019)
YAP Aggravates Inflammatory Bowel Disease by Regulating M1/M2 Macrophage Polarization and Gut Microbial Homeostasis 
Xin Zhou, Weiyun Li, Shuang Wang, Panli Zhang, Qiong Wang, Jun Xiao, Chi Zhang, Xin Zheng, Xiaoyan Xu, Shengjie Xue, Lijian Hui, Hongbin Ji, Bin Wei, Hongyan Wang 
Cell Reports 
Volume 27, Issue 4, Pages e5 (April 2019)
DOI: /j.celrep
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2. Cell Reports 2019 27, 1176-1189.e5DOI: (10.1016/j.celrep.2019.03.028)
Copyright © 2019 The Author(s) Terms and Conditions

3. Figure 1 YAP Deficiency in Macrophages Protects Mice from IBD
(A) Body weight curves of YAP+/+(n = 8) and YAPΔM/ΔM (n = 9) mice in an acute model of DSS-induced colitis for 7 days.
(B) Stool consistency, fecal bleeding, and weight loss were observed on a daily basis, and the DAI (disease activity index) was scored for each YAP+/+ and YAPΔM/ΔM mouse. The DAI score was graded on a scale of 0 to 12 as described in the STAR Methods.
(C) Representative image of the DSS-induced colitis in YAP+/+ and YAPΔM/ΔM mice. The colon lengths of the DSS-induced mice were measured on day 7 (n = 17).
(D) Representative histopathological images and scores from colon tissue sections in DSS-induced WT and YAPΔM/ΔM mice (n = 3). Scale bars: 2 mm (left), 100 μm (right).
(E) RNA-seq analysis of differentially expressed genes in YAP+/+ and YAPΔM/ΔM BMMs stimulated with IL-4/IL-13 for 24 h (n = 3). Differentially expressed genes (n = 451) were identified in all pairwise comparisons and exhibited a 2-fold change in expression with an adjusted p value of 0.05.
(F) Pathway analysis of the RNA-seq data between YAP+/+ and YAPΔM/ΔM macrophages using DAVID 6.8.
See also Figure S1.
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4. Figure 2
YAP Deficiency Enhances M2 Macrophage Polarization In Vitro and in the Colon of IBD Mice
(A and B) Immunostaining (A; scale bars: 100 μm) or FACS analysis (B) of CD206+F4/80+ macrophages in colonic tissues from DSS-induced colitis mice.
(C) Immunohistochemical analysis of the M2 marker CD206 in paraffin-embedded colon sections from YAP+/+ and YAPΔM/ΔM mice (scale bars: 200 μm).
(D) The mRNA levels of Arg1, Ym-1, Fizz1, and Il-10 in immune cells from colonic lamina propria were detected by qRT-PCR.
(E and F) YAP+/+ and YAPΔM/ΔM BMMs were stimulated with IL-4/IL-13 for 24 h, and the mRNA levels of Arg1, Fizz, and Ym-1 were detected by qRT-PCR (E), or ARG1 expression at the protein level was measured by immunoblotting analysis (F). The representative data from at least three independent experiments are shown.
See also Figure S2.
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5. Figure 3
YAP Inhibits M2 Polarization via Promotion of p53 Transcription
(A) Diagram of WT or YAP mutations. The expression levels of YAP, YAP5SA, and YAP5SA-ΔC in THP1 cells were assessed by immunoblotting.
(B) THP1 cells overexpressing YAP, YAP5SA, and YAP5SA-ΔC were stimulated with IL-4/IL-13 for 24 h to assess the levels of Cd163 and Dc-sign mRNA by qRT-PCR (n = 3). ∗p < 0.05 was calculated by one-way ANOVA with Holm-Sidak multiple comparisons test.
(C) YAP+/+ and YAPΔM/ΔM PEMs were stimulated with IL-4/IL-13, followed by immunoblotting with anti-pStat6 or anti-Stat6 antibodies.
(D and E) YAP+/+ and YAPΔM/ΔM BMMs were stimulated with IL-4/IL-13 for 24 h to assess the mRNA levels of Stat6, Irf4, Cebp-β, Socs3, and Socs2 (D) and p53 expression at both the mRNA and protein levels (E).
(F) YAP+/+ and YAPΔM/ΔM BMMs were incubated with the p53 activator Nutlin-3a, followed by IL-4/IL-13 treatment for 24 h to detect Arg1 mRNA levels.
(G) YAP+/+ and YAPΔM/ΔM BMMs were transfected with p53 siRNA, followed by IL-4/IL-13 treatment to detect Arg1 mRNA levels.
(H) ChIP assays were performed with anti-HA or the IgG control antibodies in THP1 cells overexpressing HA-YAP, followed by qPCR to detect the p53 promoter. The representative data from three independent experiments are shown.
See also Figure S3.
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6. Figure 4 YAP Promotes M1 Polarization and Enhances IL-6 Production
(A and B) Immunostaining or FACS analysis of iNOS+F4/80+ (A, scale bars: 100 μm) or CD80+CD86+ (B) macrophages in colonic tissues from DSS-induced IBD mice.
(C and D) IL-6 levels in plasma (C) or in colon tissue (D) were evaluated from DSS-induced YAP+/+ (n = 8) and YAPΔM/ΔM (n = 10) mice.
(E) YAP+/+ and YAPΔM/ΔM PEMs were stimulated with LPS/IFN-γ to detect IL-6 and TNF-α expression by qRT-PCR or ELISA.
(F) YAP+/+ and YAPΔM/ΔM BMMs were stimulated with LPS/IFN-γ to detect Il-1β, Il-12, Inos, and Ccl2 expression by qRT-PCR.
(G) THP1 cells overexpressing the control or YAP were stimulated with LPS/IFN-γ to measure Il-6 mRNA levels.
(H) YAP+/+ and YAPΔM/ΔM BMMs were infected with Escherichia coli (E. coli), Pseudomonas aeruginosa (PAO), Salmonella typhimurium (SL1344), or Staphylococcus aureus (S. aureus) for 4 h to detect Il-6 expression. The representative data from at least three independent experiments are shown.
See also Figure S4.
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7. Figure 5 YAP Binds to the IL-6 Promoter
(A) THP1 cells overexpressing vector, YAP, YAP5SA, or YAP5SA-ΔC were stimulated with LPS/IFN-γ for 4 h to assess Il-6 expression levels via qRT-PCR.
(B) MYD88, IRAK1, TRAF6, and p65 were transfected into HEK293T cells with the vector, YAP, YAP5SA, YAP5SA-ΔC, and IL-6 luciferase reporter plasmids to measure luciferase readings.
(C) The levels of phosphorylated IKKα/β, JNK, p38, and ERK in LPS/IFN-γ-stimulated YAP+/+ and YAPΔM/ΔM BMMs were detected by immunoblotting analysis, and the relative expression was analyzed.
(D) The human IL-6 promoter region contains two putative TEAD-binding sites (T1, T2) and one AP-1 binding site. HEK293T cells were cotransfected with IRAK1 in combination with vector or YAP and the WT or mutant IL-6-luciferase reporter plasmid to measure luciferase readings.
(E) ChIP assays were performed with anti-HA antibodies followed by qPCR to measure the IL-6 and CTGF promoters in LPS/IFN-γ-treated THP1 cells overexpressing HA-YAP. The representative data from at least three independent experiments are shown.
See also Figure S5.
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8. Figure 6
YAP Expression Is Differentially Regulated during M1 and M2 Macrophage Polarization
(A) Yap expression at the mRNA level was assessed in PEMs that were stimulated with IL-4 and IL-13 for 12 or 24 h.
(B) The levels of phosphorylated p85 and pAKT in IL-4/IL-13-stimulated PEMs were detected by immunoblotting analysis.
(C) PEMs were pretreated with LY for 1 h, followed by IL-4/IL-13 treatment for 24 h to measure Yap mRNA levels.
(D–F) Expression of YAP and phosphorylated YAP S127 was assessed in PEMs (D and E) or THP1 (F) that were stimulated with IL-4 and IL-13 for the indicated times.
(G and H) Expression in YAP (G) or phosphorylated YAP S357 (H) was assessed in LPS- and IFN-γ-treated PEMs.
(I) PEMs were stimulated with LPS/IFN-γ, followed by immunostaining with Hoechst and anti-YAP (scale bars: 20 μm). A total of 10–15 viewing fields were analyzed in each experiment. The representative data from at least three independent experiments are shown.
See also Figure S6.
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9. Figure 7 YAP Regulates Colon Microbial Homeostasis
(A) Microarray data from colitis patients (n = 71) were analyzed to assess the correlation between IL-6 and YAP expression using Pearson’s test.
(B) The mRNA levels of IL-6 and YAP were analyzed at different time points during DSS-induced colitis in mice (n = 6).
(C) Microbial composition of WT and YAP cKO mice on day 7 after DSS induction (n = 3).
(D) Gut microbiota DNA was exacted from mouse stool, and the relative abundances of the interested bacteria were determined by qRT-PCR on day 7 after DSS induction (n = 3).
(E) The mRNA levels of Retn1β, Ang4, and RegIIIγ were assessed in colon tissues from DSS-induced YAP+/+ and YAPΔM/ΔM mice (n = 5).
(F) Representative images of REG3γ immunostaining in paraffin-embedded colon sections from YAP+/+ and YAPΔM/ΔM mice (scale bars: 50 μm). Quantitation was performed using Image-Pro Plus, and the average density was calculated from at least five areas of interest (AOIs) in each image.
(G) The expression of Il-22, Il-23, Il-10, Il-33, and Il-18 in immune cells from colonic lamina propria was detected by qRT-PCR (n = 5).
(H) Model: LPS/IFN-γ treatment increases YAP expression, and YAP promotes IL-6 production via activating JNK or by directly binding to the IL-6 promoter. IL-4/IL-13 treatment decreases YAP expression, and YAP inhibits M2 polarization. In addition, YAP cKO mice regulate antimicrobial peptides production and change gut microbial homeostasis. Together, YAP expression in macrophages promotes the development of IBD.
See also Figure S7.
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