EMBO/soda-nel huggingface dataset:
YAP antagonizes TEAD-mediated AR signaling and prostate cancer growth (2022)
Figure 4

Figure 4. TEAD forms a complex with AR and promotes AR signaling

(A-C) Western blot analysis of TEAD1 and TEAD4 (A), relative mRNA levels of TEAD1 and TEAD4 and the indicated AR target genes (B) and relative growth of C4-2 cells treated with two independent TEAD1/3/4 siRNA or control siRNA.

(D) Relative mRNA levels of the indicated AR target genes (left) and western blot analysis of TEAD (right) in C4-2 cells expression the control or TEAD4 constructs and treated with vehicle or XMU-MP-1.

(E-G) C4-2 cell extracts were immunoprecipitated with IgG, anti-AR (E), anti-YAP (F) or anti-TEAD4 (G) antibodies. Immunoprecipitates and cell lysates (input) were analyzed by western blot with the indicated antibodies. Asterisks in E and F indicate IgG.

(H-J) HEK-293T cells transfected with the indicated constructs. Cell lysates were immunoprecipitated with the indicate antibodies. Immunoprecipitates and cell lysates (input) were analyzed by western blot with the indicated antibodies.

(K) In vitro binding assay using GST or GST-TEAD4 purified from bacteria and Flag-AR immunopurified from HEK293 cells.

(L) Schematic diagrams of the indicated AR and TEAD constructs.

(M-R) HEK-293T cells transfected with the indicated AR, TEAD and YAP constructs. Cell lysates were immunoprecipitated with the indicate antibodies. Immunoprecipitates and cell lysates (input) were analyzed by western blot with the indicated antibodies.

Data information: Results in in B-D are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).
YAP antagonizes TEAD-mediated AR signaling and prostate cancer growth (2022)
Figure 5

Figure 5. TEAD promotes AR binding to its target promoters/enhancers

(A) Venn diagram showing the numbers of AR binding peaks overlapping with TEAD binding peaks in C4-2 cells in ChIP-Seq experiments.

(B) Heatmaps of TEAD, AR, and H3K27ac ChIP-Seq reads from C4-2 cells showing the bound loci of AR+/TEAD+, AR+/TEAD- and AR-/TEAD+.

(C) Aggregate plots showing the normalized tag density of TEAD, AR, and H3K27ac at indicated gene binding sites within 2kb frames in C4-2 cells.

(D) ChIP-seq signal tracks of AR, TEAD, and H3K27ac at promoter/enhancer regions of the indicated AR target genes in C4-2 cells.

(E) Transcription factor binding motif enrichment analyses of AR+/TEAD+, AR+/TEAD-, and AR-/TEAD+ sites.

(F) >90% (38/42) of AR target genes downregulated by XMU-MP-1 in C4-2 cells are co-bound by both TEAD and AR in their promoter/enhancer regions.

(G) ~40% (848/2136) of genes by XMU-MP-1 in C4-2 cells are co-bound by both TEAD and AR.

(H) ChIP-qPCR analysis shows that TEAD4 co-binds with AR to the promoter/enhancer regions of the indicated AR target genes in C4-2 cells.

(I) ChIP-qPCR analysis of AR binding to the promoter/enhancer regions of the indicated AR target genes in C4-2 cells treated with control (siNC) or two independent TEAD1/3/4 siRNAs.

(J) ChIP-qPCR analysis of TEAD binding to the indicated AR targets in C4-2 cells treated with control (siNC) or two independent TEAD1/3/4 siRNAs.

(K-M) ChIP-qPCR analysis of KLK2 and KLK3 promoter/enhancer occupancy of full-length AR (K), AR-V7 (L) or AR-D+L (M) exogenously expressed HEK-293T cells treated with control or TEAD siRNA.

(N) ChIP-qPCR analysis shows that exogenous expression of full-length TEAD4 (TEAD4-FL) but not truncated TEAD4 (TEAD4-N and TEAD4-C) partially rescued of AR binding to KLK2 and KLK3 promoters in HEK-293T cells with endogenous TEAD depleted by siTEAD1/3/4_2.

(O-P) ChIP-qPCR (O) and RT-PCR analyses show that exogenous expression of wild type (TEAD4-WT) but not the DNA binding deficient form (TEAD4m) of TEAD4 rescued AR binding to the promoter/enhancer regions of KLK2 and KLK3 (O) as well as the expression of these genes (P) in C4-2 cells treated with TEAD1/3/4 siRNA.

Data information: Results in H-P are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).
YAP antagonizes TEAD-mediated AR signaling and prostate cancer growth (2022)
Figure 6

Figure 6. YAP binding to TEAD impedes AR/TEAD cooperativity

(A)­ CoIP experiments with the indicated antibodies followed by western blot using cell extracts from HEK-293T cells transfected with the indicated constructs show that increasing amounts of YAP displaced AR from TEAD4.

(B) XMU-MP-1 increased YAP but decreased AR binding to TEAD4. C4-2 cell were treated with vehicle or 2 μM XMU-MP-1 for 4 hours. Cell extracts were immunoprecipitated with IgG or anti-TEAD4 antibody. Immunoprecipitates and cell lysates (input) were analyzed by western blot with the indicated antibodies.

(C) TRULI increased YAP but decreased AR binding to TEAD4. C4-2 cell were treated with vehicle or 5 μM TRULI for 12 hours. Cell extracts were immunoprecipitated with IgG or anti-TEAD4 antibody. Immunoprecipitates and cell lysates (input) were analyzed by western blot with the indicated antibodies.

(D) YAP-5SA but not YAP-5SAS94A inhibited AR/TEAD interaction. C4-2 cells stably expressing Tet-O-GFP, Tet-O-YAP-5SA or Tet-O-YAP-5SAS94A were treated with 0.2 μg/ml doxycycline (Dox) for 10 hours, followed by CoIP and western blot analyses with the indicated antibodies.

(E) YAP-5SA inhibited AR/TEAD interaction more effectively than YAP-WT. HEK-293T cells were transfected with the indicated constructs. Nuclear extracts (NE) were immunoprecipitated with IgG or anti-TEAD4 antibody. Immunoprecipitates, nuclear extracts (input of NE), and whole cell extracts (WCE) were analyzed by western blot with the indicated antibodies.

(F) In vitro binding assays using GST-TEAD4, Flag-AR and Flag-YAP immunopurified from HEK293 cells.

(G-H) ChIP-qPCR analysis of AR (G) and TEAD (H) binding to the promoter/enhancer regions of KLK2, KLK3 and CTGF in Tet-O-YAP-5SA expressing C4-2 cells treated with 0.2 μg/ml doxycycline (Dox) for the indicated time.

(I-K) ChIP-qPCR analysis of AR binding to the promoter/enhancer regions of KLK2 and KLK3 in HEK293 cells transfected with YAP-5SA and the indicated AR constructs.

(L) ChIP-qPCR analysis to show that coexpression of TEAD4 attenuated the YAP-5SA-mediated inhibition of AR binding to the promoter/enhancer regions of KLK2 and KLK3 in HEK293 cells.

Data information: Results are representatives of three independent experiments. Data ± SD. ns: not significant, **P<0.01, ***P<0.001 (Student's t-test).
YAP antagonizes TEAD-mediated AR signaling and prostate cancer growth (2022)
Figure 7

Figure 7. YAP/TAZ depletion inhibits AR signaling due reduced TEAD expression

(A-C) Western blot analysis of YAP and TEAD (left) and mRNA levels of the indicated genes (right) in C4-2 cells treated with siYAP/TAZ for 18 (A), 36 (B), or 72 (C) hours.

(D) Western blot analysis of YAP and TEAD (left) and mRNA levels of the indicated genes (right) in C4-2 cells overexpressing TEAD4 transgenes and treated with siYAP/TAZ for 72 hours.

Data information: Results are representatives of three independent experiments. Data ± SD. ns: not significant, *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).
YAP antagonizes TEAD-mediated AR signaling and prostate cancer growth (2022)
Figure 8

Figure 8. MST1/2 inhibition/YAP activation inhibits therapy resistant AR in vivo

(A-F) YAP activation inhibited AR positive prostate cancer growth in xenografts bearing C4-2 or 22RV1 tumors that stably express Tet-O-YAP-5SA. Male NOD scid gamma (NSG) mice bearing C4-2 tumor cells stably expressing a Tet-O-EGFP construct were used as control. Male NOD scid gamma (NSG) mice bearing the indicated tumors were injected i.p. with PBS containing Dox (20mg/kg) or PBS daily for the indicated time. Tumor growth curve (A, D), photograph of tumor samples (B, E), and quantification of tumor weight (C, F) at the end of treatment were shown.

(G-O) XMU-MP-1 inhibited AR positive prostate cancer growth in xenografts bearing C4-2 (G-I), 22RV1 (J-L) or R1-D567 (M-O) tumors. Male NOD scid gamma (NSG) mice bearing the indicated tumors were treated daily with XUM-MP-1 at the indicated concentrations or with vehicle for the indicated periods of time. Tumor growth curve (G, J, M), images of tumor samples (H, K, N), and quantification of tumor weight (I, L, O) at the end of treatment were shown.

Data information: Data are ± SD. **P<0.01, ***P<0.001 (Student's t-test). n=7 mice for each group.
A positive feedback loop between mTORC1 and cathelicidin promotes skin inflammation in rosacea (2021)
Figure 1

Figure 1. mTORC1 signaling is hyperactivated in skin lesions of rosacea.

(A) Heatmap of differentially regulated transcripts between HS and Rosacea as determined by RNA-sequencing of whole skin lesions (P<0.05). HS, skin biopsies from healthy individuals; Rosacea, skin biopsies from patients with rosacea. Blue color denotes low FPKM expression; red, high FPKM expression.

(B) Volcano plot of differentially regulated genes between HS and Rosacea. The red dots show the significantly upregulated genes; blue dots, significantly downregulated genes (P<0.05).

(C) Top-ranked upregulated KEGG terms related to immune responses in genes that were differentially regulated between Rosacea and HS samples revealed through Gene set enrichment analysis (GSEA).

(D) GSEA analysis on RNA-sequencing data from Rosacea versus HS skin samples shows enrichment for mTOR signaling pathway in rosacea. NES, normal enrichment score; FDR, false discovery rate; FDR value < 0.05 was considered significant. Significance was calculated by permutation test.

(E) Immunohistochemistry (IHC) of pS6 (Ser235/236) on skin sections from HS and Rosacea. Higher magnified images of boxed areas are shown at the bottom right of lower magnified images for each group. Epi, epidermis; Der, dermis. Scale bar: 50 μm.

(F) Correlation of pS6 (Ser235/236) expression (from IHC) in the epidermis of human rosacea lesions (n = 17) with the Clinician's Global Severity (CGS) scores. Sperman's correlation coefﬁcient was used for the correlation analysis (two-tailed).

(G) Immunostaining of pS6 (Ser240/244) in skin lesions from control and LL37-induced mice. Bottom right panels, magnified images of boxed areas. DAPI staining (blue) indicates nuclear localization. Scale bar: 50 μm. Right panel, the quantification of relative fluorescence intensity for pS6 (Ser240/244) in control and LL37 group (n=5 for each group). Data represent the mean ± SEM. **P < 0.01. two-tailed unpaired Student's t-test was used.

(H) Immunoblotting of pS6 (Ser235/236 and Ser240/244), total S6, pAkt (Ser473) and total Akt in cell lysates from primary human keratinocytes treated with different doses of LL37 (0 - 8 μM) and scramble LL37 (8 μM) for 2 h. Scr, scramble LL37. pS6 and pAkt protein levels were analyzed relative to total S6 and Akt respectively. Tubulin is the loading control. Data (H) are representative of at least 3 independent experiments.
A positive feedback loop between mTORC1 and cathelicidin promotes skin inflammation in rosacea (2021)
Figure 2

Figure 2. Both conditional deletion and pharmacological inhibition of mTORC1 block rosacea development.

(A) Schematic diagram of intraperitoneal administration of tamoxifen (TM) for continuous 5 days before intradermal injection of LL37 in mice (Raptor cKO and WT mice). Mice were sacrificed on day 2 to conduct subsequent experiments. The mouse experiments were repeated for three times, and 5-8 mice were included in each group for each time. The results of a representative mouse experiment were displayed.

(B) Immunostaining of Raptor in skin sections from WT and Raptor cKO mice. Epi, epidermis. Der, dermis. Scale bar: 50 μm.

(C) The back skins of WT and Raptor cKO mice were intradermally injected with LL37 or control vehicle (n=6 for each group). Images were taken 48 h after the first LL37 administration. Below panels, magnified pictures of yellow boxed areas. Scale bar: 2 mm.

(D) HE staining of lesional skin sections from WT and Raptor cKO mice injected with LL37 or control vehicle (n=6 for each group). Scale bar: 50 μm.

(E) Dermal infiltrating cells were quantified (n = 6 for each group) for each high power field (HPF).

(F) The mRNA expression levels of Tnf-α, Il6, Mmp9 and Vegf in skin lesions (n = 6 for each group).

(G) Schematic diagram of topical application of rapamycin (RAPA) after LL37 injection in mice (BALB/c). Mice were sacrificed on day 2 to conduct subsequent experiments. The mouse experiments were repeated for three times, and 4-6 mice were included in each group for each time. The results of a representative mouse experiment were showed.

(H) The back skins of LL37 injected-mice topically applied with RAPA (n = 6 for each group). Images were taken 48 h after the first LL37 injection. Bottom panels, magnified images of yellow boxed areas. Scale bar: 2 mm.

(I) HE staining of lesional skin sections from LL37 or control mice topically applied with RAPA or vehicle. Scale bar: 50 μm.

(J) Dermal infiltrating cells were quantified (n = 6 for each group). Data represent the mean ± SEM. **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test was used.
A positive feedback loop between mTORC1 and cathelicidin promotes skin inflammation in rosacea (2021)
Figure 3

Figure 3. Hyperactivation of mTORC1 accelerates rosacea development.

(A) The back skins of WT (Tsc2+/+) and Tsc2+/- mice were intradermally injected with LL37 or control vehicle (n=5 for each group). Pictures were taken on day 0.5, 2, 5, 8, 12 after the first LL37 injection. The mouse experiments were repeated for three times, and 5 mice were included in each group for each time. The results of a representative mouse experiment were displayed. Scale bar: 2 mm.

(B, C) The severity of the rosacea-like phenotypes was evaluated with the redness area (B) and score (C) (n = 5 for each group). **P <0.01, comparison between Tsc2+/- (LL37) and Tsc2+/+ (LL37) group. ##P <0.01, comparison between Tsc2+/+ (LL37) and Tsc2+/+ (Control).

(D) HE staining of lesional skin sections from Tsc2+/+ and Tsc2+/- mice injected with LL37 or control vehicle (n = 5 for each group). Scale bar: 50 μm.

(E) Dermal infiltrating cells were quantified (n = 5 for each group). Data represent the mean ± SEM. **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test was used.
A positive feedback loop between mTORC1 and cathelicidin promotes skin inflammation in rosacea (2021)
Figure 4

Figure 4. mTORC1 regulates cathelicidin LL37 via a positive feedback loop in keratinocytes.

(A) The mRNA expression level of human cathelicidin (CAMP) in skin lesions from healthy individuals (n = 10) and patients with rosacea (n = 15).

(B) Co-immunostaining of cathelicidin and pS6 on skin sections from HS and rosacea. Scale bar, 50 μm. Epi, epidermis.

(C) Immunostaining of mouse cathelicidin (CRAMP) in skin sections from WT and Raptor cKO mice injected with LL37 or control vehicle (n = 6 for each group). Bottom right panels, magnified images of dotted line areas. Epi, epidermis; Der, dermis; Scale bar: 50 μm.

(D) Quantification of relative fluorescence intensity for CRAMP in epidermis (n = 6).

(E) Immunoblotting of cathelicidin in cell lysates from primary human keratinocytes treated with different doses of LL37 (0 - 8 μM) and scramble LL37 (8 μM) for 24 h. Cathelicidin protein levels were analyzed.

(F) Immunoblot analysis of cathelicidin and pS6 in cell lysates from primary human keratinocytes treated with LL37 (4 μM) ± RAPA for 24 h. Cathelicidin protein levels were analyzed.

(G) Immunostaining of cathelicidin in HaCaT keratinocytes treated with LL37 (4 μM) ± RAPA for 24 h. DAPI staining (blue) indicates nuclear localization. Scale bar: 50 μm. All results are representative of at least 3 independent experiments. Data represent the mean ± SEM. **P < 0.01. two-tailed unpaired Student's t-test (A) or 1-way ANOVA with Bonferroni's post hoc test (D) was used.
A positive feedback loop between mTORC1 and cathelicidin promotes skin inflammation in rosacea (2021)
Figure 5

Figure 5. Cathelicidin LL37 activates mTORC1 through binding to TLR2 in keratinocytes.

(A) Representative images showing cellular localization of LL37 or sLL37 analyzed by fluorescent microscope in live primary human keratinocytes treated with FITC-labeled LL37 or sLL37 (4 μM) for 30 min followed by PBS washed. BF, bright field. Scale bar: 50 μm.

(B) Representative images showing membrane localization of LL37 or sLL37 in live primary human keratinocytes treated with FITC-labeled LL37 / sLL37 and PKH26 for 30 min. Scale bar: 50 μm.

(C) Immunoblotting analysis of TLR2 and pS6 in cell lysates from HaCaT keratinocytes carrying either a vector expressing a scramble shRNA or TLR2 shRNAs were exposed to LL37 (4 μM) for 2 h. pS6 protein levels were analyzed relative to total S6 (fold change).

(D) Immunoblot analysis of TLR2 and pS6 in cell lysates from HaCaT keratinocytes carrying either a vector expressing TLR2 or an empty vector were exposed to LL37 (4 μM) for 2 h. pS6 protein levels were analyzed relative to total S6. Tubulin or Actin was used as the loading control.

(E) Primary human keratinocytes expressing TLR2 shRNAs or scramble shRNA were treated with FITC-labeled LL37 or sLL37 ± PKH26 for 30 min followed by PBS washed, then live cells were analyzed by fluorescent microscope. Scale bar: 50 μm.

(F, G) Primary human keratinocytes were incubated with sLL37-flag or LL37-flag for 1 h. Cell lysates were immunoprecipitated with anti-flag or anti-TLR2 antibodies, showing an interaction between LL37 and TLR2. Medium, the medium of primary human keratinocytes treated with sLL37-flag or LL37-flag. All results are representative of at least 3 independent experiments.
A positive feedback loop between mTORC1 and cathelicidin promotes skin inflammation in rosacea (2021)
Figure 6

Figure 6. Cathelicidin LL37 stimulates NF-κB signaling via mTORC1 in keratinocytes.

(A) Heatmap of differentially regulated genes in skin lesions from LL37-injected mice administrated with RAPA or vehicle determined by RNA-sequencing (n = 3 independent biological samples for each group). Blue color denotes low FPKM expression; red, high FPKM expression.

(B) Venn diagram showing the overlap genes between the two comparisons. LL37 vs control, LL37-injected mice administrated with vehicle vs control mice administrated with vehicle; LL37 vs LL37+RAPA, LL37-injected mice administrated with vehicle vs LL37-injected mice administrated with RAPA.

(C) Top-ranked enriched KEGG terms in genes that were differentially regulated respectively in the two comparisons (LL37 vs control, and LL37 vs LL37+RAPA) revealed by GSEA analysis. NF-κB signaling pathway was highlighted in red box.

(D) GSEA on RNA-sequencing data from the two comparisons both shows enrichment for NF-κB signaling pathway in LL37 group. Significance was calculated by permutation test.

(E) Immunostaining of phospho-p65 (p-p65) in skin sections. Bottom right panels, magnified images of dotted line areas. Epi, epidermis. Der, dermis. Scale bar: 50 μm.

(F) Quantification of relative fluorescence intensity for p-p65 in epidermis (n=6).

(G) The mRNA expression levels of NF-κB family of transcription factors (Nfκb1, Nfκb1, Rela and Relb) in mice skin lesions (n=6 for each group).

(H) GSEA on RNA-sequencing data shows enrichment for NF-κB signaling pathway in rosacea patients. Significance was calculated by permutation test.

(I) IHC of p65 on skin sections from HS and rosacea. Scale bar, 50 μm.

(J) Percentage of nuclear p65 positive cells in the epidermis from HS (n = 8) and rosacea (n = 13). All results are representative of at least 3 independent experiments. Data represent the mean ± SEM. **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test (F and G) or two-tailed unpaired Student's t-test (J) was used.
A positive feedback loop between mTORC1 and cathelicidin promotes skin inflammation in rosacea (2021)
Figure 7

Figure 7. Cathelicidin LL37 stimulates rosacea-characteristic chemokines and cytokines via mTORC1. (A) GSEA on RNA-sequencing data from the two comparisons (LL37 vs control, and LL37 vs LL37+RAPA) both show enrichment for chemokine signaling pathway in LL37 group. Significance was calculated by permutation test.

(B) The mRNA expression levels of mouse chemokines (Cxcl11, Cxcl12, Ccl2 and Ccl3) in skin lesions (n=6 for each group).

(C) The mRNA expression levels of rosacea-associated chemokines in primary human keratinocytes treated with LL37 ± RAPA in vitro. Data represent the mean ± SEM (from 3 replicates).

(D) GSEA on RNA-sequencing data from rosacea skin lesions versus HS skin samples shows enrichment for chemokine signaling pathway in rosacea. Significance was calculated by permutation test.

(E) Heatmap of upregulated chemokines and cytokines in rosacea skin lesions determined by RNA-sequencing. All results are representative of at least 3 independent experiments. Data represent the mean ± SEM. *P < 0.05, **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test was used.
Commensal microbiota influence systemic autoimmune responses (2014)
Figure 1

Systemic autoimmune responses in mice lacking secondary lymphoid organs

3‐month‐old wild‐type (C57BL/6), Ltbr−/−, Lta−/−, LIGHT−/−, and Ltbr−/−Hox11−/− mice; percentage of mice with at least one ANA.
6‐month‐old Ltbr−/− mice (n = 15); titers of individual mice.
LIA of 6‐month‐old Ltbr−/− mice, wild‐type mice, and 3‐month‐old Rorγt‐Ltb−/− mice.
LTβR‐Fc or control immunoglobulin (Ig)‐treated wild‐type mice; percentage of mice with at least one ANA. E denotes gestational day. *ANA were determined with LIA at the age of 3 months. **ANA were determined with LIA 3 months after the start of treatment.
3‐month‐old cell‐specific LT knockout mice; percentage of mice with at least one ANA.
Bone marrow cells from wild‐type or Ltbr−/− mice were transferred at neonatal age into lethally irradiated wild‐type or Ltbr−/− mice; percentage of mice with at least one ANA. ANA were determined with LIA at the age of 3 months.
Commensal microbiota influence systemic autoimmune responses (2014)
Figure 2

Central tolerance against nuclear antigens is not impaired in lymphotoxin‐deficient mice

Thymic reconstitution was analyzed by counting total cell number and flow cytometry for cell percentages. Numbers represent the percentages within the indicated regions (left panel). No significant differences were found. Data represent means ± s.e.m.
T‐cell reconstitution of spleen was assessed by flow cytometry. Numbers represent the percentages within the indicated regions.
Total IgG from nude mice engrafted with Ltbr−/− or wild‐type thymi was determined by ELISA. No significant differences were found. Data represent means ± s.e.m.
Sera were collected 3 months after transplantation and tested for ANA; percentage of mice with at least one ANA reactivity.