Journal: Theranostics

Article Title: Remodeling cancer stemness by collagen/fibronectin via the AKT and CDC42 signaling pathway crosstalk in glioma

doi: 10.7150/thno.50613

Figure Lengend Snippet: The integrin αvβ3/CDC42/F-actin/YAP/NUPR1/Nestin signaling pathway is activated in collagen/FN-cultured glioma cell (A) Expression of CDC42, F-actin, and β-actin in LN229 cells and T98G cells cultured in a flask or 3D Collagen/FN gel. (B) Expression of CDC42, F-actin, and β-actin in LN229 cells and T98G cells cultured in 3D Collagen/FN gel treated with PBS or SB273005 (5 nM, 24). (C) Cell fraction of cytosol (C) and nucleus (N) was analyzed by Western blotting. Expression of YAP1 in LN229 cells or LN229 CDC42 OE cells cultured in a flask or 3D Collagen/FN gel and treated with PBS or SB273005 (5 nM, 24 h). (D) Western blotting analysis of proteins immunoprecipitated (IP) with anti-YAP1 from LN229 cells cultured in a flask or 3D Collagen/FN gel for 48 h. (E) ChIP analysis of YAP1 or TEAD4 binding to the NUPR1 promoter in LN229 cells. YAP1 and TEAD4 increased the luciferase activity of the NUPR1 promoter in LN229 cells. (F) Expression of NUPR1 and Nestin in LN229 cells and T98G cells cultured in a flask or 3D Collagen/FN gel. (G) Expression of NUPR1in LN229 and T98G cells cultured in 3D Collagen/FN gel, treated with PBS or SB273005 (5 nM, 24 h) and transfected with CDC42 OE lentivirus. (H) Representative photographs of LN229-NC cells and LN229-CDC42 OE cells cultured in 3D Collagen/FN gel. Scale bar represents 20 µm (I) Representative photographs of LN229-NC cells and LN229-Nestin shRNA cultured in 3D Collagen/FN gel. Scale bar represents 20 µm (J) Quantification of colony sizes in (H) and (I) (K) Proliferation of 3D collagen/FN pre-cultured LN229-vec cells, LN229-CDC42 OE cells, LN229-NC cells, and LN229-Nestin shRNA cells (L) Colony formation of 3D collagen/FN pre-cultured LN229-vec cells, LN229-CDC42 OE cells, LN229-NC cells, and LN229-Nestin shRNA cells. Mean ± SEM, n.s, no significant difference, *p < 0.05, **p < 0.01.

Article Snippet: LN229 cells were harvested, lysed, and immunoprecipitated with anti-YAP1 (MAB8094, RD, USA) or IgG conjugated to protein G agarose (11243233001, Roche, USA) at 4 ºC overnight.

Techniques: Cell Culture, Expressing, Western Blot, Immunoprecipitation, Binding Assay, Luciferase, Activity Assay, Transfection, shRNA

Journal: Developmental cell

Article Title: Yorkie growth promoting activity is limited by Atg1-mediated phosphorylation

doi: 10.1016/j.devcel.2020.01.011

Figure Lengend Snippet: Confocal images of third instar wing discs stained for the ban-LacZ (A-D) reporter of Yorkie transcriptional activity, or the endogenous Diap1 (E-H) and Ex (I-L) proteins. All larvae contained the en-Gal4 driver and only UAS-mCherry (A,E,I), or, in addition, the indicated UAS-RNAi transgenes knocking down Hippo (B,F,J), Atg1 (C,G,K) or Acinus (D,H,L). The mCherry-marked posterior en-Gal4 expression domains are shown in (A’-L’). Images are representative examples from at least three independent crosses with 5 or more examples each. Scale bar = 100 μm. Detailed genotypes are listed in Table S2.

Article Snippet: Proteins used were human ULK1 (Sigma product number: SRP5096 Lot: G1163–1), human TAZ (OriGene Technologies product number: TP304082 Lot: 1160CA), and human Yap1 (OriGene Technologies product number: TP325864 Lot: 10a73e).

Techniques: Staining, Activity Assay, Expressing

Journal: Developmental cell

Article Title: Yorkie growth promoting activity is limited by Atg1-mediated phosphorylation

doi: 10.1016/j.devcel.2020.01.011

Figure Lengend Snippet: (A) Diagram of Atg1 target consensus sequence, compared to potential phosphorylation sites at serines 74, and 97 in Yorkie and corresponding sites in human and mouse YAP and TAZ proteins.

Article Snippet: Proteins used were human ULK1 (Sigma product number: SRP5096 Lot: G1163–1), human TAZ (OriGene Technologies product number: TP304082 Lot: 1160CA), and human Yap1 (OriGene Technologies product number: TP325864 Lot: 10a73e).

Techniques: Sequencing

Journal: Developmental cell

Article Title: Yorkie growth promoting activity is limited by Atg1-mediated phosphorylation

doi: 10.1016/j.devcel.2020.01.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Proteins used were human ULK1 (Sigma product number: SRP5096 Lot: G1163–1), human TAZ (OriGene Technologies product number: TP304082 Lot: 1160CA), and human Yap1 (OriGene Technologies product number: TP325864 Lot: 10a73e).

Techniques: Recombinant, SYBR Green Assay, Plasmid Preparation, Protease Inhibitor, Transfection, Chromatography, Electron Microscopy, Software

Journal: Molecular cancer therapeutics

Article Title: Concurrent Targeting of Potential Cancer Stem Cells Regulating Pathways Sensitizes Lung Adenocarcinoma to Standard Chemotherapy

doi: 10.1158/1535-7163.MCT-20-0024

Figure Lengend Snippet: Attenuation of YAP1 expression impairs cell proliferation and increases sensitivity to anti-cancer agents in LUAD. A, Western blotting confirmed YAP1-knockdown in H1299 and H1437 cells and YAP1-overexpression in A549 cells. B, Proliferation assay: YAP1-deficient cells (H1299 YAP1-sh and H1437 YAP1-sh) showed lower proliferative ratios than did control cells, whereas YAP1-overexpressing cells (A549 YAP1-LV) proliferated more rapidly. C, Cells were treated with CDDP, and cell viability was recorded at 72 hours after treatment. H1299 and H1437 YAP1-sh cells showed greater sensitivity to CDDP, and YAP1-LV cells showed higher resistance, than did their control counterparts. D, YAP1-deficient cells treated with CDDP (2 μM or 5 μM for 72 hours) showed greater percentages of cells undergoing apoptosis than did control cells; whereas YAP1-LV cells showed substantially less apoptosis. E, Both YAP1-sh cell lines showed significantly suppressed tumor growth in athymic mice, whereas YAP1-LV cells formed tumors more aggressively than did control cells (3 mice with tumors in both flanks per each group). Error bars: mean ± SEM. *P<0.05, **P<0.001.

Article Snippet: Gene silencing of YAP1 was performed using YAP1 shRNA pGFP-C-shLenti Vector (YAP1-sh) purchased from Origene (Rockville, MD, USA; #TL308332).

Techniques: Expressing, Western Blot, Over Expression, Proliferation Assay

Journal: Molecular cancer therapeutics

Article Title: Concurrent Targeting of Potential Cancer Stem Cells Regulating Pathways Sensitizes Lung Adenocarcinoma to Standard Chemotherapy

doi: 10.1158/1535-7163.MCT-20-0024

Figure Lengend Snippet: YAP1 promotes STAT3 phosphorylation via IL-6 upregulation. A, Immunoblotting shows YAP1, STAT3 and pSTAT3 expressions in YAP1-deficient (H1299 and H1437) and YAP1-overexpressing (A549) LUAD cells. YAP1 expression was positively associated with pSTAT3 expression. B, qRT-PCR data show IL-6 mRNA expression to be positively associated with YAP1 expression in H1299, H1437 and A549 cell lines. C, Secretion of IL-6 into media was significantly reduced in YAP1-sh cells, whereas higher levels were observed in YAP1-LV cells, as measured by ELISA in cell culture supernatant. Immunoblotting of pSTAT3 and β-actin in the respective cell lines are shown below the graph. D, Addition of recombinant human IL-6 (0.1, 1 or 10 ng/ml) to H1299 and H1437 YAP1-sh cells increased pSTAT3 levels dose-dependently. E, Blocking IL-6 activity with IL-6 neutralizing antibody (1 μg/ml) inhibited pSTAT3 in a time-dependent manner without influencing total STAT3 expression in A549 YAP1-LV cells. F, ChIP-PCR assay with YAP1 antibody was conducted using H1299, H1437 and A549 cells. DNA isolation followed by PCR showed that YAP1 protein directly binds to the IL-6 gene promoter region in three LUAD cells analyzed (white arrows). “Input” indicates DNA lysate before ChIP, which was diluted to 2% for PCR reactions. Histone H3 antibody and normal rabbit IgG served as positive and negative control, respectively. G, Schematic shows YAP1 binding to IL-6 promoter and upregulating its transcription to induce STAT3 phosphorylation. Error bars: mean ± SEM. *P<0.05, **P<0.001.

Article Snippet: Gene silencing of YAP1 was performed using YAP1 shRNA pGFP-C-shLenti Vector (YAP1-sh) purchased from Origene (Rockville, MD, USA; #TL308332).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Recombinant, Blocking Assay, Activity Assay, DNA Extraction, Negative Control, Binding Assay

Journal: Molecular cancer therapeutics

Article Title: Concurrent Targeting of Potential Cancer Stem Cells Regulating Pathways Sensitizes Lung Adenocarcinoma to Standard Chemotherapy

doi: 10.1158/1535-7163.MCT-20-0024

Figure Lengend Snippet: Positive correlation between YAP1 and pSTAT3 expressions in human LUAD tissues. A, Representative images of YAP1 and pSTAT3 immunostaining in the TMA (scale bar: 100 μm). B, IHC results indicate a statistically significant correlation between YAP1 and pSTAT3 expressions (P<0.0001, χ2 test). C, YAP1 and pSTAT3 expressions were evaluated by immunoblotting in 13 LUAD PDXs. All YAP1-expressing PDXs (CTG0162, 0178, 0502, 0848, 1309, 1762, 2017 and 2708) also expressed pSTAT3.

Article Snippet: Gene silencing of YAP1 was performed using YAP1 shRNA pGFP-C-shLenti Vector (YAP1-sh) purchased from Origene (Rockville, MD, USA; #TL308332).

Techniques: Immunostaining, Western Blot, Expressing

Journal: Molecular cancer therapeutics

Article Title: Concurrent Targeting of Potential Cancer Stem Cells Regulating Pathways Sensitizes Lung Adenocarcinoma to Standard Chemotherapy

doi: 10.1158/1535-7163.MCT-20-0024

Figure Lengend Snippet: Combined genetic inhibition of YAP1 and STAT3 by inhibiting transcripts enhances cisplatin’s cytotoxicity and attenuates malignant CSC-like features more than inhibition of YAP1 or STAT3 alone. A, Functional rescue of STAT3 in YAP1-sh cells by forced expression of STAT3 (STAT3-LV). B, YAP1-sh/STAT3-LV cells (H1299 and H1437) showed less sensitivity to CDDP than YAP1-sh/STAT3-Ctrl cells but more sensitivity than YAP1-Ctrl/STAT3-Ctrl cells. Cell viability was measured at 72 hours after 5 μM CDDP treatment. C, STAT3 inhibition by RNAi in YAP1-LV cells (A549). Immunoblotting shows lower expressions of pSTAT3 and STAT3 in YAP1-LV/STAT3-si cells. D, STAT3 inhibition restored CDDP sensitivity as compared with YAP1-LV/STAT3-Ctrl cells. The YAP1-LV group was more resistant to CDDP than were the YAP1-Ctrl/STAT3-Ctrl cells. E, Effects of dual genetic inhibition of YAP1 and STAT3 in H1299 and H1437 cell lines. Immunoblotting indicated that pSTAT3 and STAT3 expressions were inhibited by STAT3 siRNA transduction. F, YAP1-sh/STAT3-si cells showed the highest sensitivity to CDDP compared with either target inhibition or control cells. G, Sphere formation assay: YAP1-sh/STAT3-si cells formed the fewest spheroids compared with the single-molecule inhibition and controls. Error bars: mean ± SEM. *P<0.05, **P<0.001.

Article Snippet: Gene silencing of YAP1 was performed using YAP1 shRNA pGFP-C-shLenti Vector (YAP1-sh) purchased from Origene (Rockville, MD, USA; #TL308332).

Techniques: Inhibition, Functional Assay, Expressing, Western Blot, Transduction, Tube Formation Assay

Journal: Molecular cancer therapeutics

Article Title: Concurrent Targeting of Potential Cancer Stem Cells Regulating Pathways Sensitizes Lung Adenocarcinoma to Standard Chemotherapy

doi: 10.1158/1535-7163.MCT-20-0024

Figure Lengend Snippet: Evaluation of verteporfin and S3I-201 as inhibitors of YAP1 and STAT3, respectively, in LUAD cells. A, Verteporfin suppressed YAP1 and STAT3 expressions in H1299 and H1437 cells in a concentration-dependent manner. STAT3 monomer (black arrows) decreased as verteporfin concentration was increased, whereas high molecular weight complexes (regions surrounded by circles) increased, indicating oligomerization of STAT3. B, S3I-201 suppressed pSTAT3 in a dose-dependent manner.

Article Snippet: Gene silencing of YAP1 was performed using YAP1 shRNA pGFP-C-shLenti Vector (YAP1-sh) purchased from Origene (Rockville, MD, USA; #TL308332).

Techniques: Concentration Assay, Molecular Weight

Journal: Molecular cancer therapeutics

Article Title: Concurrent Targeting of Potential Cancer Stem Cells Regulating Pathways Sensitizes Lung Adenocarcinoma to Standard Chemotherapy

doi: 10.1158/1535-7163.MCT-20-0024

Figure Lengend Snippet: Therapeutic efficacy of verteporfin and S3I-201 in cell lines and patient-derived preclinical xenografts mouse models of human LUAD. NSG mice were subcutaneously inoculated LUAD cell lines (H1299 and H1437 cells) and PDXs that endogenously expressed YAP1 and pSTAT3. Animals were randomly divided into different treatment groups (n=5) (A-D). Treatments were started when tumors reached 200 ± 50 mm3. A, Combined verteporfin + S3I-201 significantly inhibited tumor growth in a H1299 xenograft model. Verteporfin or S3I-201 treatment alone led to inhibited growth, but combined treatment had a greater inhibitory effect. B, In H1437 xenografts, only the combination of verteporfin + S3I-201 inhibited growth significantly more than controls, whereas either agent individually did not significantly affect tumor growth. C, D, Two EGFR wild-type PDXs (CTG0162 and CTG0178) that expressed both YAP1 and pSTAT3 (Fig. 3C) were implanted. The “All” combination (CDDP + GEM + verteporfin + S3I-201) dramatically impaired growth of both PDXs. Chemo + verteporfin also significantly retarded tumor growth until Day 28 in CTG0162 (C) and Day 34 in CTG0178 (D). However, after 4–5 weeks, tumors treated with chemo + verteporfin showed tumor regrowth. E, F, Pharmacodynamic analysis of PDX tumors treated with verteporfin, S3I-201 and chemotherapy drugs. Left panel: CTG0162 (E) and CTG0178 (F) tumors resected at 18 days after treatment (one sample per treatment). Both YAP1 and STAT3 were suppressed in the chemo + verteporfin and “All” treatment groups. Adding verteporfin to chemotherapy decreased NANOG and SOX2 expressions. Right panel: tumors resected at 43 days in CTG0162 (E) and 46 days in CTG0178 (F) were assessed (two samples per treatment). Expressions of YAP1 and STAT3 were higher in tumors treated with chemo + verteporfin than with the “All” group. Tumors from the “All” group had lower expression of NANOG in CTG0162, and both NANOG and SOX2 in CTG0178, than did the chemo + verteporfin group. Error bars: mean ± SEM. *P<0.05, **P<0.001. N.S., not significant.

Article Snippet: Gene silencing of YAP1 was performed using YAP1 shRNA pGFP-C-shLenti Vector (YAP1-sh) purchased from Origene (Rockville, MD, USA; #TL308332).

Techniques: Derivative Assay, Expressing

Journal: Cancer Communications

Article Title: Cytoplasmic YAP1‐mediated ESCRT‐III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

doi: 10.1002/cac2.12417

Figure Lengend Snippet: Cytoplasmic YAP1 inhibited breast cancer cell proliferation. (A) Representative IHC staining images of YAP1 in normal tissues ( n = 16) and in breast cancer tissues ( n = 119). (B) Comparisons of total YAP1 IHC staining scores between normal tissues ( n = 16) and breast cancer tissues ( n = 119) (left panel) and between the cytoplasm and nucleus of breast cancer tissues ( n = 119) (right panel). Each circle represents one patient sample. (C) Data from the TCGA database showed that YAP1 expression was lower in breast cancer tissues ( n = 1085) than in normal breast tissues ( n = 112). (D) YAP1 protein expression was measured in six pairs of primary breast cancer tissues (T) and the matched adjacent normal tissues (N) by WB analysis. β‐actin was used as a loading control. (E) YAP1 expression was determined in a normal breast epithelial cell line (MCF‐10A) and eight breast cancer cell lines with qRT‐PCR. (F) YAP1 protein expression levels were determined in a normal breast epithelial cell line (MCF‐10A) and eight breast cancer cell lines by WB. (G) CCK‐8 assays were used to determine the effect of stable YAP1 overexpression (LV‐YAP1) by lentiviral transduction on the viability of MCF7 and SKBR3 cells. (H) DNA synthesis was detected in MCF7 and SKBR3 cells after stable YAP1 overexpression with an EdU incorporation assay. Hoechst labelled with blue fluorescent signal was used to mark the nucleus, while red fluorescent signal was used to label EdU. (I) WB analysis of the expression of apoptosis markers (PARP and Caspase‐3) in MCF7 and SKBR3 cells with stable overexpression of YAP1 . (J) CCK‐8 assays were used to determine the effect of different YAP1 mutants ( NLS‐YAP1 5SA , YAP1 S94A , YAP1 S127D ) on the viability of breast cancer MCF7 and SKBR3 cells. (K) DNA synthesis was detected in MCF7 and SKBR3 cells after YAP1 WT and YAP1 S127D transient overexpression with EdU incorporation assays. Hoechst labelled with blue fluorescent signal was used to mark the nucleus, while red fluorescent signal was used to label EdU. (L) WB analysis of the expression of apoptosis markers (PARP and Caspase‐3) after YAP1 WT and YAP1 S127D transient overexpression in MCF7 and SKBR3 cells. Data were collected from three independent experiments and are expressed as the mean ± standard deviation. ** P < 0.01, *** P < 0.001 (Student's t ‐test and one‐way ANOVA). Abbreviations: YAP1, yes1‐associated transcriptional regulator; IHC, immunohistochemistry; TCGA, the Cancer Genome Atlas; WB, Western blotting; qRT‐PCR, quantitative real‐time PCR; CCK‐8, Cell Counting Kit‐8; OD450, optical density at 450nm; EdU, 5‐Ethynyl‐2’‐ deoxyuridine; PARP, poly (ADP‐ribose) polymerase 1; LV‐Con: lentivirus‐control; LV‐YAP1, YAP1 overexpression lentivirus; YAP1 WT , YAP1 wild type ; NLS‐YAP1 5SA , nuclear localization sequence‐YAP1 5SA , ANOVA, analysis of variance.

Article Snippet: YAP1 (HG17690‐UT), CHMP2B (HG14596‐UT), and NEDD4L (HG17684‐UT) overexpression plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Immunohistochemistry, Expressing, Quantitative RT-PCR, CCK-8 Assay, Over Expression, Transduction, DNA Synthesis, Standard Deviation, Western Blot, Real-time Polymerase Chain Reaction, Cell Counting, Sequencing

Journal: Cancer Communications

Article Title: Cytoplasmic YAP1‐mediated ESCRT‐III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

doi: 10.1002/cac2.12417

Figure Lengend Snippet: YAP1 overexpression promoted the formation of autophagosomes and autophagy flow was smooth in breast cancer cells. (A) WB analysis was used to examine expression of the autophagy markers LC3 and p62 after YAP1 overexpression for 48 h in MCF7 and SKBR3 cells. (B) Representative IF staining images and quantitative analysis of LC3 fluorescence puncta representing the formation of autophagosomes after YAP1 overexpression for 48 h in MCF7 and SKBR3 cells. DAPI labelled with blue fluorescent signal was used to mark the nucleus, while green fluorescent signal was used to label LC3. (C) WB analysis of LC3 and p62 expression in MCF7 and SKBR3 cells after YAP1 transient overexpression for 48 h with or without EBSS (6 h) starvation or CQ (20 µmol/L, 6 h) treatment. (D) Representative confocal laser microscopy images and quantitative analysis of red and green fluorescent puncta in MCF7 and SKBR3 cells. Cells were transfected with mRFP‐GFP‐LC3 adenovirus for 24 h after YAP1 overexpression to visualize autophagic flux. Yellow puncta indicate the presence of GFP and mRFP. Red puncta indicate the fusion of the autophagosome with the lysosome and quenching of GFP. DAPI labelled with blue fluorescent signal was used to mark the nucleus. (E) Autophagic structures (indicated with red arrows) were visualized with TEM in MCF7 cells after YAP1 overexpression for 48 h. (F) WB of LC3 and p62 expression in breast cancer MCF7 and SKBR3 cells transiently transfected with different YAP1 mutants ( NLS‐YAP1 5SA , YAP1 S94A , YAP1 S127D ). Data were collected from three independent experiments and are shown as the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001 (Student's t ‐test). Abbreviations: YAP1, Yes1‐associated transcriptional regulator; WB, Western blotting; LC3, microtubule‐associated protein 1 light chain 3; SQSTM1/p62, sequestosome 1; IF, immunofluorescence staining; DAPI, 4',6‐diamidino‐2‐phenylindole; EBSS, Earle's Balanced Salt Solution; CQ, chloroquine; GFP, green fluorescent protein; mRFP: monomer red fluorescent protein; TEM, transmission electron microscopy; EGFP, enhanced green fluorescent protein; YAP1 WT , YAP1 wild type ; NLS‐YAP1 5SA , nuclear localization sequence‐YAP1 5SA .

Article Snippet: YAP1 (HG17690‐UT), CHMP2B (HG14596‐UT), and NEDD4L (HG17684‐UT) overexpression plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Over Expression, Expressing, Staining, Fluorescence, Microscopy, Transfection, Standard Deviation, Western Blot, Immunofluorescence, Transmission Assay, Electron Microscopy, Sequencing

Journal: Cancer Communications

Article Title: Cytoplasmic YAP1‐mediated ESCRT‐III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

doi: 10.1002/cac2.12417

Figure Lengend Snippet: Inhibition of autophagy weakened the anti‐proliferation effect of YAP1 on breast cancer cells. (A) MCF7 and SKBR3 cells were transfected with ATG7‐siRNAs ( siATG7#1 , siATG7#2 , siATG7#3 ), and the knockdown efficiency of ATG7 was determined via WB analysis. siATG7#1 was selected for additional experimentation (upper panel). WB analysis was also used to examine expression of the autophagy markers LC3 and p62 after YAP1 overexpression with and without siATG7 in MCF7 and SKBR3 cells (lower panel). (B) CCK‐8 assays were performed to determine the effect of YAP1 overexpression on cell viability with and without ATG7 knockdown in MCF7 and SKBR3 cells. (C‐D) DNA synthesis in MCF7 and SKBR3 cells overexpressing YAP1 with and without ATG7 knockdown was determined using an EdU incorporation assay (upper panel). Hoechst labelled with blue fluorescent signal was used to mark the nucleus, while red fluorescent signal was used to label EdU. Statistical analysis is shown in the lower panel. (E) CCK‐8 assays were used to determine the effect of YAP1 overexpression on viability of MCF7 and SKBR3 cells with or without the autophagy inhibitor CQ (20 µmol/L). (F‐G) DNA synthesis was examined in MCF7 and SKBR3 cells transiently overexpressing YAP1 with or without CQ (20 µmol/L) using an EdU incorporation assay (upper panel). Hoechst labelled with blue fluorescent signal was used to mark the nucleus, while red fluorescent signal was used to label EdU. Statistical analysis is shown in the lower panel. Experiments were conducted in triplicate, and data are shown as the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001 (Student's t ‐test and one‐way ANOVA). Abbreviations: YAP1, Yes1‐associated transcriptional regulator; WB, Western blotting; ATG7, autophagy related 7; LC3, microtubule‐associated protein 1 light chain 3; SQSTM1/P62, sequestosome 1; CCK‐8, Cell Counting Kit‐8; OD450, optical density at 450nm; EdU, 5‐Ethynyl‐2’‐ deoxyuridine; CQ, chloroquine; ANOVA, analysis of variance.

Article Snippet: YAP1 (HG17690‐UT), CHMP2B (HG14596‐UT), and NEDD4L (HG17684‐UT) overexpression plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Inhibition, Transfection, Expressing, Over Expression, CCK-8 Assay, DNA Synthesis, Standard Deviation, Western Blot, Cell Counting

Journal: Cancer Communications

Article Title: Cytoplasmic YAP1‐mediated ESCRT‐III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

doi: 10.1002/cac2.12417

Figure Lengend Snippet: YAP1 bound to VPS4B and CHMP2B and promoted CHMP2B‐VPS4B assembly. (A) PPI network of autophagy‐related genes that interact with YAP1. The three genes labeled in pink (VPS25, VPS4B, and CHMP3) belong to the ESCRT‐III complex. (B) SKBR3 cells were transfected with YAP1 overexpression plasmid, MDA‐MB‐231 cells were transfected with siYAP1 , and mRNA expression levels of ESCRT‐III subunits were determined by qRT‐PCR. (C) VPS4B and CHMP2B expression levels were detected via WB in MDA‐MB‐231 and SKBR3 cells after YAP1 overexpression. (D) Left panel: magnified view of CHMP2B‐YAP1‐VPS4B binding. The R87–P99 residues of YAP1 were embedded in the binding region of CHMP2B and VPS4B; YAP1 serves as an anchor protein, stabilizing CHMP2B‐VPS4B binding. Right panel: the side chain phenyl ring of YAP1 (F95) interacts through π stacking with the imidazole ring of VPS4B (H36) and the phenyl ring of VPS4B (Y40). The amino group of the YAP1 side chain (R87) forms a hydrogen bond with the hydroxyl group of CHMP2B (D129). (E) MDA‐MB‐231 and SKBRS parent cell lysates were immunoprecipitated with anti‐YAP1, anti‐VPS4B, or anti‐CHMP2B antibodies, and YAP1 binding to VPS4B and CHMP2B was examined with WB analysis. (F) Upper panel: IF staining shows the co‐localization and subcellular localization of YAP1 (red) and VPS4B (green) in MDA‐MB‐231 and SKBR3 cells. DAPI labelled with blue fluorescent signal was used to mark the nucleus, while red fluorescent signal was used to label YAP1 and green fluorescent signal was used to label VPS4B. Lower panel: IF staining shows the co‐localization and subcellular localization of YAP1 (green) and CHMP2B (red) in MDA‐MB‐231 and SKBR3 cells. DAPI labelled with blue fluorescent signal was used to mark the nucleus, while red fluorescent signal was used to label CHMP2B and green fluorescent signal was used to label YAP1. (G) MDA‐MB‐231 and SKBR3 cells transfected with vector control or YAP1 overexpression plasmid were immunoprecipitated with anti‐VPS4B or anti‐CHMP2B antibody, and WB analysis was used to detect CHMP2B and VPS4B. Overexpression of YAP1 increased the interactions of VPS4B and CHMP2B. (H) MDA‐MB‐231 cells transfected with negative control or siYAP1 were immunoprecipitated with anti‐VPS4B or anti‐CHMP2B antibody, and WB analysis was used to detect CHMP2B and VPS4B. Downregulation of YAP1 weakened the interaction of VPS4B and CHMP2B. (I) MCF7 cells transfected with vector control or 3 truncated mutants of YAP1 were immunoprecipitated with anti‐Flag antibody, YAP1(1‐159aa) binds to VPS4B and CHMP2B. Blue arrows represent Flag‐specific bands. (J) MCF7 cells transfected with vector control or mutants of YAP1 were immunoprecipitated with anti‐Flag antibody; YAP1(F95) binds to VPS4B, and YAP1 (R87) binds to CHMP2B. Each experiment was repeated three times, and data are shown as the mean ± standard deviation. Abbreviations: PPI, protein‐protein interaction; ESCRT, endosomal sorting complexes required for transport; YAP1, Yes1‐associated transcriptional regulator; qRT‐PCR, quantitative real‐time PCR; WB, Western blotting; IP, immunoprecipitation; VPS4B, vacuolar protein sorting 4 homolog B; CHMP2B, charged multivesicular body protein 2B; IgG, immunoglobulin G; IF, immunofluorescence staining; DAPI, 4',6‐diamidino‐2‐phenylindole.

Article Snippet: YAP1 (HG17690‐UT), CHMP2B (HG14596‐UT), and NEDD4L (HG17684‐UT) overexpression plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Labeling, Transfection, Over Expression, Plasmid Preparation, Expressing, Quantitative RT-PCR, Binding Assay, Immunoprecipitation, Staining, Negative Control, Standard Deviation, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence

Journal: Cancer Communications

Article Title: Cytoplasmic YAP1‐mediated ESCRT‐III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

doi: 10.1002/cac2.12417

Figure Lengend Snippet: EGCG promoted retention of YAP1 in the cytoplasm by activating the Hippo pathway and promoting autophagy in breast cancer cells. (A) Natural small‐molecule compounds predicted to activate the Hippo pathway in breast cancer cell lines. (B) Bioinformatics analysis of pathways related to EGCG. (C) WB analysis was performed to examine expression of Hippo pathway components and YAP1 target genes ( CTGF and CYR61 ) in MCF7 and MDA‐MB‐231 cells treated with EGCG of various concentrations for 6 h. (D) Expression levels of YAP1 and p‐YAP1 were determined in the nucleus and cytoplasm of MCF7 and MDA‐MB‐231 cells via WB analysis. LaminB1 and β‐actin were used as extraction controls for the nucleus and cytoplasm, respectively. (E‐F) Expression levels of the autophagy markers LC3 and p62 were detected by WB in MCF7 and MDA‐MB‐231 cells after treatment with EGCG of various concentrations or for different lengths of time. (G) IF staining images showing LC3 fluorescence puncta and quantitative analysis of MCF7 and MDA‐MB‐231 cells treated with EGCG (50 µg/mL, 6 h). DAPI labelled with blue fluorescent signal was used to mark the nucleus, while green fluorescent signal was used to label LC3. (H) Autophagic structures (indicated with red arrows) were detected with TEM in MCF7 and MDA‐MB‐231 cells treated with EGCG (50 µg/ml, 6 h). Data are from three independent experiments and are shown as the mean ± standard deviation. ** P < 0.01 (Student's t ‐test). Abbreviations: YAP1, Yes1‐associated transcriptional regulator; EGCG, epigallocatechin gallate; WB, Western blotting; MST1, macrophage stimulating 1; p‐MST1, phosphorylated‐macrophage stimulating 1; MOB1A: MOB kinase activator 1A; p‐MOB1A: phosphorylated‐MOB1A; CTGF, connective tissue growth factor; CYR61, cysteine rich angiogenic inducer 61; LC3, microtubule‐associated protein 1 light chain 3; SQSTM1/p62, sequestosome 1; IF, immunofluorescence staining; DAPI, 4',6‐diamidino‐2‐phenylindole; TEM, transmission electron microscopy.

Article Snippet: YAP1 (HG17690‐UT), CHMP2B (HG14596‐UT), and NEDD4L (HG17684‐UT) overexpression plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Expressing, Staining, Fluorescence, Standard Deviation, Western Blot, Immunofluorescence, Transmission Assay, Electron Microscopy

Journal: Cancer Communications

Article Title: Cytoplasmic YAP1‐mediated ESCRT‐III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

doi: 10.1002/cac2.12417

Figure Lengend Snippet: Cytoplasmic retention of YAP1 promoted CHMP2B‐VPS4B assembly and autophagic death of breast cancer cells after EGCG treatment. (A) CCK‐8 assays were used to detect the viability of MCF7 and MDA‐MB‐231 cells treated with EGCG (50 µg/mL) with or without the autophagy inhibitor CQ (20 µmol/L). (B) DNA synthesis was detected in MCF7 and MDA‐MB‐231 cells treated with EGCG (50 µg/ mL, 6 h) with or without CQ (20 µmol/L) using an EdU incorporation assay (left panel). Hoechst labelled with blue fluorescent signal was used to mark the nucleus, while red fluorescent signal was used to label EdU. Statistical analysis is shown in the right panel. (C) Upper panel: WB analysis showing the effects of EGCG (50 µg/mL, 6 h) on expression of the autophagy markers LC3 and p62 in MCF7 and MDA‐MB‐231 cells with or without the MST1 kinase inhibitor XMU‐MP‐1 (8 µmol/L, 6 h). Lower panel: WB analysis showing the effects of EGCG (50 µg/mL, 6 h) on expression of the LC3 and p62 in MCF7 and MDA‐MB‐231 cells after YAP1 knockdown. (D) CCK‐8 assays showing the effects of EGCG (50 µg/mL) on the viability of MCF7 and MDA‐MB‐231 cells after YAP1 knockdown. (E) EdU incorporation assay showing the effects of EGCG (50 µg/mL) on DNA synthesis in MCF7 and MDA‐MB‐231 cells after YAP1 knockdown (upper panel). Hoechst labelled with blue fluorescent signal was used to mark the nucleus, while red fluorescent signal was used to label EdU. Statistical analysis is shown in the lower panel. (F) MDA‐MB‐231 cells treated with EGCG (50 µg/mL, 48 h) or a control were immunoprecipitated with anti‐VPS4B or anti‐CHMP2B antibody, then WB analysis was used to detect CHMP2B and VPS4B. Treatment with EGCG increased interactions between VPS4B and CHMP2B. Data are from three independent experiments and are shown as the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001 (Student's t ‐test and one‐way ANOVA). Abbreviations: CCK‐8, Cell Counting Kit‐8; OD450, optical density at 450nm; EGCG, epigallocatechin gallate; CQ, chloroquine; EdU, 5‐Ethynyl‐2’‐ deoxyuridine; WB, Western blotting; MST1, macrophage stimulating 1; p‐MST1, phosphorylated‐macrophage stimulating 1; LC3, microtubule‐associated protein 1 light chain 3; SQSTM1/p62, sequestosome 1; YAP1, Yes1‐associated transcriptional regulator; IP, immunoprecipitation; VPS4B, vacuolar protein sorting 4 homolog B; CHMP2B: charged multivesicular body protein 2B; IgG, immunoglobulin G; ANOVA, analysis of variance.

Article Snippet: YAP1 (HG17690‐UT), CHMP2B (HG14596‐UT), and NEDD4L (HG17684‐UT) overexpression plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: CCK-8 Assay, DNA Synthesis, Expressing, Immunoprecipitation, Standard Deviation, Cell Counting, Western Blot

Journal: Cancer Communications

Article Title: Cytoplasmic YAP1‐mediated ESCRT‐III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

doi: 10.1002/cac2.12417

Figure Lengend Snippet: Cytoplasmic YAP1 promoted autophagic cell death in vivo. (A) Tumor‐bearing nude mice from different treatment groups ( n = 6 per group). (B) Tumors taken from mice in different treatment groups. (C) The weight of tumors from mice in each treatment group. (D) The tumor volume from mice in each treatment group over time. (E) WB analysis was performed to detect the expression of YAP1, p‐YAP1, the autophagy markers LC3 and p62, and YAP1 target genes CTGF and CYR61 in tumor tissues. (F) IHC staining of LC3, p62, YAP1, CTGF, and CYR61 in tumor tissues from mice in different treatment groups. (G) Autophagic structures (indicated with red arrows) in tumor tissues from the control and EGCG treatment groups were detected with TEM. Data are shown as the mean ± standard deviation. *** P < 0.001 (Student's t ‐test and one‐way ANOVA). Abbreviations: EGCG, epigallocatechin gallate; LC3, microtubule‐associated protein 1 light chain 3; SQSTM1/p62, sequestosome 1; WB, Western blotting; YAP1, Yes1‐associated transcriptional regulator; CTGF, connective tissue growth factor; CYR61, cysteine rich angiogenic inducer 61; IHC, immunohistochemistry; TEM, transmission electron microscopy; ANOVA, analysis of variance.

Article Snippet: YAP1 (HG17690‐UT), CHMP2B (HG14596‐UT), and NEDD4L (HG17684‐UT) overexpression plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: In Vivo, Expressing, Immunohistochemistry, Standard Deviation, Western Blot, Transmission Assay, Electron Microscopy

Journal: Cancer Communications

Article Title: Cytoplasmic YAP1‐mediated ESCRT‐III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

doi: 10.1002/cac2.12417

Figure Lengend Snippet: NEDD4L mediated ubiquitylation and degradation of YAP1 through binding to YAP1. (A) MDA‐MB‐231 parent cell lysates were immunoprecipitated with anti‐YAP1 antibody. The red arrow indicates the YAP1‐interacting protein identified by mass spectrometry. (B) The E3 ubiquitin ligases acting on YAP1 were predicted with the online database UbiBrowser, an integrated bioinformatics platform. The eight dots in red refer to those verified as E3 ubiquitin ligases of YAP1 in the literatures, and the 10 dots in blue are predicted as E3 ubiquitin ligases of YAP1. The capital letters in dots indicate the initial letters of E3 ubiquitin ligases‐domains: F refers to F‐box domain, R refers to RING domain, H refers to HECT domain, U refers to UBOX domain. The predicted interactions are arranged in descending order clockwise based on the confidence score. (C) NEDD4L and YAP1 were immunoprecipitated from MCF7 and SKBR3 parent cell lysates with anti‐YAP1 and anti‐NEDD4L antibodies, then detected via WB. This confirmed the interaction between YAP1 and NEDD4L. (D) Representative IHC staining images of NEDD4L and YAP1 in breast cancer tissues ( n = 51) (left panel). Expression levels of NEDD4L and YAP1 were negatively correlated with one another (right panel). (E) WB analysis of NEDD4L expression in six matched pairs of primary breast cancer tissues (T) and adjacent normal tissues (N). (F) MCF7 and SKBR3 cells were transfected with NEDD4L ‐ siRNAs ( siNEDD4L#1 , siNEDD4L#2 , siNEDD4L#3 ), and NEDD4L expression was detected with WB analysis. siNEDD4L#3 was selected for subsequent experiments. (G) Stability analysis of YAP1. MCF7 and SKBR3 cells were transfected with NEDD4L overexpression plasmid or siNEDD4L for 48 h, then exposed to CHX (100 µg/mL) for 0, 8, 10, or 12 h. WB analysis of YAP1 expression was then performed. (H‐I) YAP1 ubiquitination degradation assay. MCF7 and SKBR3 cells were transfected with NEDD4L overexpression plasmid or siNEDD4L and treated with MG132 (20 µmol/L, 6 h). YAP1 expression was detected with WB analysis. (J) Ubiquitination assays showing the effects of NEDD4L on YAP1 ubiquitination. MCF7 and SKBR3 cells overexpressing Flag‐ub were transfected with NEDD4L overexpression plasmid or siNEDD4L then treated with MG132 (20 µmol/L, 6 h). WB analysis was used to detect the ubiquitination of YAP1. Each experiment was repeated three times. Abbreviations: NEDD4L, NEDD4 like E3 ubiquitin protein ligase; IP, immunoprecipitation; IgG, immunoglobulin G; WB, Western blotting; YAP1, Yes1‐associated transcriptional regulator; IHC, immunohistochemistry; CHX, cycloheximide; Ub, ubiquitin.

Article Snippet: YAP1 (HG17690‐UT), CHMP2B (HG14596‐UT), and NEDD4L (HG17684‐UT) overexpression plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Binding Assay, Immunoprecipitation, Mass Spectrometry, Immunohistochemistry, Expressing, Transfection, Over Expression, Plasmid Preparation, Degradation Assay, Western Blot

Journal: Cancer Communications

Article Title: Cytoplasmic YAP1‐mediated ESCRT‐III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

doi: 10.1002/cac2.12417

Figure Lengend Snippet: Cytoplasmic YAP1 was beneficial for breast cancer survival and could be used to establish survival prediction models. (A) Kaplan‐Meier survival plots of total YAP1 expression, cytoplasmic YAP1 expression, and nuclear YAP1 expression in breast cancer tissues. (B‐C) A constructed nomogram for survival prediction of a patient with breast cancer (left panel). C‐index = 0.76 for DFS; C‐index = 0.80 for OS. The ROC curves show the prediction efficiency of the nomogram prediction models (AUC = 0.78 for DFS; AUC = 0.84 for OS). Abbreviations: YAP1, Yes1‐associated transcriptional regulator; DFS, disease‐free survival; OS, overall survival; HR, hazard ratio; CI, confidence interval; ROC, receiver operating characteristic; AUC: area under the curve.

Article Snippet: YAP1 (HG17690‐UT), CHMP2B (HG14596‐UT), and NEDD4L (HG17684‐UT) overexpression plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Expressing, Construct

Journal: Cancer Communications

Article Title: Cytoplasmic YAP1‐mediated ESCRT‐III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

doi: 10.1002/cac2.12417

Figure Lengend Snippet: Results of univariate and multivariate cox analysis for disease‐free survival (DFS) and overall survival (OS) in breast cancer patients

Article Snippet: YAP1 (HG17690‐UT), CHMP2B (HG14596‐UT), and NEDD4L (HG17684‐UT) overexpression plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Mutagenesis

Journal: Cancer Communications

Article Title: Cytoplasmic YAP1‐mediated ESCRT‐III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

doi: 10.1002/cac2.12417

Figure Lengend Snippet: The mechanism of cytoplasmic YAP1‐mediated ESCRT‐III assembly promoting autophagic cell death. Abbreviations: YAP1, Yes1‐associated transcriptional regulator; ESCRT, endosomal sorting complexes required for transport; EGCG, epigallocatechin gallate; NEDD4L, NEDD4 like E3 ubiquitin protein ligase; CHMP2B, charged multivesicular body protein 2B; VPS4B, vacuolar protein sorting 4 homolog B; LC3, microtubule associated protein 1 light chain 3; Ub, ubiquitin.

Article Snippet: YAP1 (HG17690‐UT), CHMP2B (HG14596‐UT), and NEDD4L (HG17684‐UT) overexpression plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques:

Journal: Experimental Hematology & Oncology

Article Title: Polyunsaturated fatty acids promote M2-like TAM deposition via dampening RhoA-YAP1 signaling in the ovarian cancer microenvironment

doi: 10.1186/s40164-024-00558-8

Figure Lengend Snippet: TAMs polarized in OCM/AS via inactivating RhoA-YAP1 signaling cascade. ( A ) M0 MФs (THP-1) were stimulated with LPS/LFN-γ to make M1 MФs. After that, M1 MФs were treated with OCM or a control medium. RNA-seq analysis compared the OCM-treated M1 MФs versus non-OCM-treated M1 MФs. Functional enrichment and pathway analysis of significantly differentially expressed mRNAs. The results show the enriched related pathways. ( B and C ) Pull-down assays for the active RhoA in OCM-MФs, AS-MФs (PBMC MΦs (upper) or THP-1 MΦs (lower)). ( D ) Linoleic acid (LA, 200 µM) and methyl arachidonate (MAA, 200 µM), but not oleic acid (OA, 200 µM), led to a reduction in RhoA activity in M0 MФs (PBMC) after 24 h treatment. ( E ) Knockdown of RhoA is confirmed by Western blotting. After transfecting M0 MΦs (THP-1 MФs) by RhoA siRNAs (#1 - #3) or scrambled siRNA (SC) for 48 h, cellular proteins were collected, and RhoA, YAP1, TAZ, MST1, and β-actin were detected by immunoblotting. ( F ) The localization of YAP and TAZ in the cytoplasm and nuclear was measured in MФs cells after 48 h post-transfection of RhoA siRNAs (#1 - #3) or scrambled siRNA (SC). ( G ) The protein expression of YAP1 and MST1 were measured in M0 MФs (PBMC MФs) treated with the control medium, OCM, or OCM-C. The expression of β-actin was used as the internal control. ( H ) Immunofluorescence analyzed the expression of F-actin (pink) and YAP1 (green) in M0 MΦs treated with the control medium. Nuclei were counterstained with Hoechst (blue). ( I ) The results of relative F-actin and YAP1 expression in macrophages are shown in the figures. ( n = 3). Scale bar: 5 μm. ( J ) The localization of YAP1 in the cytoplasm and nuclear was measured in M0 MΦs via western blot after OCM stimulation for 24 h. ( K ) Linoleic acid (LA) reduced nuclear YAP1 activity in a dose-dependent in MΦs after 24 h treatment. XMU MP1 (5µM) restores nuclear YAP1 in LA-treated MΦs

Article Snippet: This was accomplished using YAP1 Human siRNA Oligo Duplex (Locus ID 10413, OriGene) and RHOA Human siRNA Oligo Duplex (Locus ID 387, OriGene) to specifically target YAP1 and RhoA, respectively.

Techniques: Control, RNA Sequencing Assay, Functional Assay, Activity Assay, Knockdown, Western Blot, Transfection, Expressing, Immunofluorescence

Journal: Experimental Hematology & Oncology

Article Title: Polyunsaturated fatty acids promote M2-like TAM deposition via dampening RhoA-YAP1 signaling in the ovarian cancer microenvironment

doi: 10.1186/s40164-024-00558-8

Figure Lengend Snippet: Inhibition of MST1/2 Activates YAP1 while impeding M2-like macrophage polarization. ( A ) WT and Yap1 −/− BMDMs were stimulated with murine IL-4/IL-13 (10 ng/mL, 10 ng/mL) or Ctrl for 24 h, and the mRNA levels of Arg1 , Fizz1 , and Ym1 were detected by qPCR. Gene expression data were normalized to the reference gene Gapdh , and the results are presented as the fold change relative to the control. ( B ) The localization of YAP1 and TAZ in the cytoplasm and nuclear was measured in OCM-MФs or OCM-MФs treated with XMU MP1 (5 µM) for 24 h. ( C ) Comparison of the population of CD206 or CD163 in OCM-MФs, AS-MФs, and XMU MP1(5 µM) treated OCM/AS-MФs. ( D ) The mRNA levels of IL10 , TGFβ , NOS2 , and TNF in M0 MФs, OCM-MФs, or OCM-MФs treated with XMU MP1 (5 µM) were detected by qRT–PCR. Gene expression data were normalized to the reference gene 18 S , and the results were presented as the fold change relative to the control treatment. ( E ) Immunofluorescent staining of YAP1 + CD163 + and YAP1 − CD86 + cells in ascites spheroids derived from EOC patients ( n = 3). Hoechst was used to stain nuclei. Scale bar: 50 μm. The data were shown as the mean ± SEM and were analyzed by unpaired Student’s t-tests. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001

Article Snippet: This was accomplished using YAP1 Human siRNA Oligo Duplex (Locus ID 10413, OriGene) and RHOA Human siRNA Oligo Duplex (Locus ID 387, OriGene) to specifically target YAP1 and RhoA, respectively.

Techniques: Inhibition, Expressing, Control, Comparison, Quantitative RT-PCR, Staining, Derivative Assay

Journal: Experimental Hematology & Oncology

Article Title: Polyunsaturated fatty acids promote M2-like TAM deposition via dampening RhoA-YAP1 signaling in the ovarian cancer microenvironment

doi: 10.1186/s40164-024-00558-8

Figure Lengend Snippet: Abolishing Yap1 in MΦs results in an immunosuppressive microenvironment that facilitates EOC peritoneal metastases. ( A ) Quantification of the CD86 + and CD206 + mean of fluorescence intensity (MFI) in the LPMs and SPMs of WT and Yap1 −/− mice ( n = 4) after 28 days’ intraperitoneal ( i.p. ) injection of ID8 cells. ( B ) Distinct distributions of iNOS + F4/80 + , F4/80 + CD206 + Arg1 + population densities and infiltrating CD8 + T cells in WT and Yap1 −/− mice after 28 days i.p. injection of ID8 cells. ( C ) Ki67 expression was evaluated in omental isolated from WT and Yap1 −/− mice after 28 days i.p. , injection of ID8 cells. ( D ) Metastasis in WT and Yap1 −/− mice following i.p. injection was evaluated every 7 days by bioluminescence imaging. ( E ) Quantitative fluorescent analysis is calculated as the total fluorescence intensity per organ within the peritoneum post-49 days (left). The results of fluorescence quantification are presented on the right. ( F ) For the immunofluorescence staining, an equal volume of ascites (500 µL) from each mouse was collected and then centrifuged to harvest cell spheroids. Then, a 40 μm mesh filter was employed to recover cell clusters in reverse filtration. These cells were subsequently utilized for immunofluorescent staining of Ki67 + F4/80 +, CD206 + CD163 + , CD8 + CD163 + , and iNOS + CD163 + cells in ascites spheroids from WT and Yap1 −/− mice ( n = 4) after 49 days. Scale bar: 50 μm. ( G ) Quantification of the CD86 + and CD206 + MFI in peritoneal MФs (LPMs and SPMs) isolated from tumor-bearing mice ( i.p. injection of ID8 cells) that were treated with vehicle or XMU MP1 ( n = 4) for 28 days. ( H ) Distinct distributions of iNOS + F4/80 + , F4/80 + CD206 + Arg1 + population densities and infiltrating CD8 + T cells in omental were evaluated by m-IHC after vehicle or XMU MP1 for 28-day treatment. ( I ) Quantification of omental metastases number ( n = 4). The data were shown as the mean ± SEM and were analyzed by unpaired Student’s t-tests. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001

Article Snippet: This was accomplished using YAP1 Human siRNA Oligo Duplex (Locus ID 10413, OriGene) and RHOA Human siRNA Oligo Duplex (Locus ID 387, OriGene) to specifically target YAP1 and RhoA, respectively.

Techniques: Fluorescence, Injection, Expressing, Isolation, Imaging, Immunofluorescence, Staining, Filtration

Journal: International Journal of Biological Sciences

Article Title: Crosstalk between hypoxia-sensing ULK1/2 and YAP-driven glycolysis fuels pancreatic ductal adenocarcinoma development

doi: 10.7150/ijbs.60018

Figure Lengend Snippet: Correlation between ULK1/2, YAP and PKM2 in PDAC. (A) Representative images of immunohistochemistry comparing expression of ULK2, YAP and PKM2 in pancreas tissues from the wild-type mice and PDAC tissues from the KPC ( Pdx1 -Cre; LSL-Kras G12D/+ ; Trp53 fl/+ ) mice. Scale bar = 100 μm. (B) Comparison of ULK2, YAP1 and PKM2 mRNA levels between normal pancreatic tissues and PDAC tumor samples from three independent cohorts in the Oncomine database. (C and D) Representative cases stained by immunohistochemistry (C) and pie charts (D) showing expression of ULK2, YAP and PKM2 in 95 primary human PDAC specimens are positively correlated with each other. Scale bar = 50 μm. (E and F) Western blotting analyses of ULK2, YAP and PKM2 protein correlation in 6 freshly collected human PDAC specimens. The P value shown was calculated by Pearson's correlations. (G) Gene set enrichment analysis (GSEA) plot showing the correlation of YAP1 and PKM signature with ULK2 in GSE55643, respectively. (H) Schematic model proposing the nuclear localization of ULK1/2 within PDAC cells under hypoxic microenvironment and its role in YAP Ser227 phosphorylation, stabilization and subsequent transcriptional coactivation of PKM2 together with HIF-1α.

Article Snippet: Commercial human YAP1 sgRNAs (HCP255077-SG01-3) purchased from GeneCopoeia (Rockville, USA) were used to generate YAP knockout (KO) cells.

Techniques: Immunohistochemistry, Expressing, Comparison, Staining, Western Blot, Phospho-proteomics

Journal: International Journal of Biological Sciences

Article Title: Crosstalk between hypoxia-sensing ULK1/2 and YAP-driven glycolysis fuels pancreatic ductal adenocarcinoma development

doi: 10.7150/ijbs.60018

Figure Lengend Snippet: YAP transactivates PKM2 in an ULK1/2-dependent fashion under hypoxia. (A) Western-blotting determining the amount of PKM2 expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Scr, scrambled shRNA. (B) RT-qPCR analyses of PKM2 gene expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. * P <0.05 and ** P <0.01 versus Scr. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (C) Representative IHC staining images (c1) and quantification (c2) for PKM2 staining in the xenografts from mice injected with SW-1990 cells expressing ULK1, ULK2 or ULK1/2 shRNA ( n =6). Scale bar = 50 μm. (D) Luciferase-reporter PKM2 promoter activity analysis of SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Data are expressed as mean ± s.d. of three independent experiments. * P <0.05 and ** P <0.01 versus Scr. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (E) ChIP analysis for YAP and HIF-1α binding to PKM2 gene promoter in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA in the presence of hypoxia, respectively. (F) Coimmunoprecipitation assay testing the interaction between nuclear YAP and HIF-1α in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. (G) RT-qPCR analyses of YAP1 gene expression in SW-1990 cells transfected with ULK1, ULK2 and ULK1/2 shRNA under hypoxia, respectively. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. ** P <0.01 and *** P <0.001 versus Scr. Two-sided ANOVA with Bonferroni post hoc t test correction was used to calculate the P value. (H) Western-blotting comparing the abundance of PKM2 expression in SW-1990 cells with or without ULK1/2 shRNA transfection in the presence or absence of YAP knockout (KO) under hypoxia. (I) RT-qPCR analyses of PKM2 gene expression in SW-1990 cells with or without ULK1/2 shRNA transfection in the presence or absence of YAP knockout (KO) under hypoxia, respectively. Experiments were performed three times, each with quantitative RT-PCR in technical duplicate and real-time values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are expressed as mean ± s.d. ** P <0.01 versus -. Two-sided Student's t test was used to calculate the P value. (J) Luciferase-reporter PKM2 promoter activity analysis of SW-1990 cells with or without ULK1/2 shRNA transfection in the presence or absence of YAP knockout (KO) under hypoxia, respectively. Data are expressed as mean ± s.d. of three independent experiments. ** P <0.01 versus -. Two-sided Student's t test was used to calculate the P value.

Article Snippet: Commercial human YAP1 sgRNAs (HCP255077-SG01-3) purchased from GeneCopoeia (Rockville, USA) were used to generate YAP knockout (KO) cells.

Techniques: Western Blot, Expressing, Transfection, shRNA, Quantitative RT-PCR, Gene Expression, Immunohistochemistry, Staining, Injection, Luciferase, Activity Assay, Binding Assay, Co-Immunoprecipitation Assay, Knock-Out