Journal: The Journal of investigative dermatology

Article Title: After skin wounding, noncoding dsRNA coordinates prostaglandins and Wnts to promote regeneration

doi: 10.1016/j.jid.2017.03.023

Figure Lengend Snippet: (A) PTGS2 knockdown efficiency in NHEKs transfected with mock or PTGS2-specific siRNA. (B) qRT-PCR analysis of WNT7B mRNA expression in NHEKs (n=3) transfected with mock or PTGS2-specific siRNA, followed by treatment with or without poly(I:C), with the addition of prostaglandin E2 (PGE2), prostaglandin F2α (PGF2α), or 11β-prostaglandin F2α (11b-PGF2α). ‡Prostaglandin D2 induced significant cytotoxicity. (C) Representative western blot of Wnt7b protein expression in NHEKs treated as in Fig. 4B (n=3). (D) Western blot quantification for n=3 repeats performed as described in Fig. 4C. (E) Representative confocal scanning light microscopy (CSLM) image of regenerated hair follicles in mouse wounds injected with poly(I:C), with or without celecoxib. (F) Quantification of hair follicles in CSLM images from Fig. 4D (n=15–17). (G) Representative confocal scanning light microscopy (CSLM) image of regenerated hair follicles in mouse wounds injected with poly(I:C) and celecoxib, with or without dmPGE2. (H) Quantification of hair follicles in CSLM images from Fig. 4E (n=10–11). *denotes p<0.05.

Article Snippet: Proteins were separated under denaturing conditions on a NuPage 4–12% Bis-Tris gel (ThermoFisher), transferred onto a PVDF membrane, and probed overnight with rabbit polyclonal anti-Wnt7b antibody (1:1000, ab94915, Abcam, Cambridge, MA) followed by an HRP-conjugated secondary antibody.

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Expressing, Western Blot, Light Microscopy, Injection

Journal: The Journal of investigative dermatology

Article Title: After skin wounding, noncoding dsRNA coordinates prostaglandins and Wnts to promote regeneration

doi: 10.1016/j.jid.2017.03.023

Figure Lengend Snippet: (A) Representative confocal scanning light microscopy (CSLM) image of regenerated hair follicles in C57BL6/NJ mouse wounds injected with or without polyinosinic:polycytidylic acid (poly(I:C)). (B) Wnt ligands most strongly upregulated or downregulated in gene expression microarray comparing NHEKs treated with vehicle or poly(I:C), published under (GSE92646) (C) Representative immunofluorescent image of Wnt7b protein in C57BL6/NJ mouse wounds (scale bar = 100 μm). (D) qRT-PCR analysis of WNT7B mRNA expression in NHEKs (n=3) treated with various doses of poly(I:C). (E) Western blot analysis of Wnt7b protein expression in NHEKs treated with or without poly(I:C), with β-actin as a loading control.

Article Snippet: Proteins were separated under denaturing conditions on a NuPage 4–12% Bis-Tris gel (ThermoFisher), transferred onto a PVDF membrane, and probed overnight with rabbit polyclonal anti-Wnt7b antibody (1:1000, ab94915, Abcam, Cambridge, MA) followed by an HRP-conjugated secondary antibody.

Techniques: Light Microscopy, Injection, Gene Expression, Microarray, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: The Journal of investigative dermatology

Article Title: After skin wounding, noncoding dsRNA coordinates prostaglandins and Wnts to promote regeneration

doi: 10.1016/j.jid.2017.03.023

Figure Lengend Snippet: (A) qRT-PCR analysis of WNT7B mRNA expression in background (C57BL6/NJ) and TLR3 KO mouse wounds (n=8). (B) WNT7B mRNA expression in TLR3 KO mouse wounds injected with vehicle or recombinant IL-6 (n=8). (C) WNT7B mRNA expression in background or STAT3 KO mouse wounds (n=8). (D) WNT7B mRNA expression in NHEKs transfected with mock or TLR3-specific siRNA, followed by treatment with or without poly(I:C). *denotes p<0.05.

Article Snippet: Proteins were separated under denaturing conditions on a NuPage 4–12% Bis-Tris gel (ThermoFisher), transferred onto a PVDF membrane, and probed overnight with rabbit polyclonal anti-Wnt7b antibody (1:1000, ab94915, Abcam, Cambridge, MA) followed by an HRP-conjugated secondary antibody.

Techniques: Quantitative RT-PCR, Expressing, Injection, Recombinant, Transfection

Journal: Cell reports

Article Title: Yap/Taz inhibit goblet cell fate to maintain lung epithelial homeostasis

doi: 10.1016/j.celrep.2021.109347

Figure Lengend Snippet: (A) Representative YAP/TAZ and MUC5AC immunostaining of human airway sections (performed using an antibody recognizing YAP and TAZ). DAPI-stained nuclei are in blue. Nuclei within MUC5AC + cells are highlighted with a yellow dotted line and with a blue dotted line in MUC5AC − cells. A white dotted line marks the basal surface of the epithelium (scale bars, 10 μm). (B) Quantification of nuclear YAP/TAZ intensity in airway epithelial cells across multiple sections from two patient donors. Cells were scored as either MUC5AC-positive or -negative and the intensity of YAP/TAZ staining was measured within the nuclear area outlined by DAPI staining (minimum of n = 14; unpaired t test, ****p < 0.0001). (C) YAP/MUC5AC immunostaining of human ALI cultures imaged by confocal microscopy. A z stack view is shown in the top panels (scale bars,10 μm). (D) HBECs were transfected with control siRNA (siCTL) or siRNA targeting YAP/TAZ (siY/T). MUC5AC, SCGB1A1, YAP , and WWTR1/TAZ qPCR analysis of lysates collected 72 h after knockdown (n = 6; unpaired t test, **p = 0.001, ****p < 0.0001). (E) Heatmap of gene expression changes resulting from YAP/TAZ knockdown in HBECs analyzed by RNA sequencing (RNA-seq). 2 distinct patient isolates were treated with three independent siCTLs or siRNA targeting YAP/TAZ (siY/T), and global gene expression changes were examined by RNA-seq after 48 h of culture (n = 3 per condition, 2-fold change cutoff, FDR = 0.05). (F) Pathway enrichment of significantly upregulated and downregulated genes following YAP/TAZ depletion in human airway epithelial cells identified by GSEA. Both the −log 10 p value and the percentage representation within each gene set are displayed. In all bar plots data are represented as mean ± SEM. See also and and .

Article Snippet: Mouse Wwtr1 Taqman probe Mm01289583_m1 , ThermoFisher , 4331182.

Techniques: Immunostaining, Staining, Confocal Microscopy, Transfection, Control, Knockdown, Gene Expression, RNA Sequencing

Journal: Cell reports

Article Title: Yap/Taz inhibit goblet cell fate to maintain lung epithelial homeostasis

doi: 10.1016/j.celrep.2021.109347

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse Wwtr1 Taqman probe Mm01289583_m1 , ThermoFisher , 4331182.

Techniques: Recombinant, Lysis, TUNEL Assay, RNAscope, Positive Control, Negative Control, Staining, Transfection, Purification, cDNA Synthesis, SYBR Green Assay, Knock-Out, Microarray, Knockdown, Control, Software

Journal: British Journal of Cancer

Article Title: Epigenetic silencing of miRNA-9 is associated with HES1 oncogenic activity and poor prognosis of medulloblastoma

doi: 10.1038/bjc.2013.764

Figure Lengend Snippet: miR-9 regulates HES1 expression in MB cells. ( A ) Sequence alignment of predicted miR-9 binding site in the 3′UTR of HES1 promoter (seed-complementary region). ( B – D ) Correlation study of miR-9 and HES1 expression in three independent sets of MB patients' samples as determined by qRT–PCR and by microarray-based expression profiles. ( r =Pearson's correlation coefficient). ( E ) Correlation study of HES1 and miR-9 levels according to MB subgroups (WNT, SHH, Group 3, Group 4) ( n =88). ( F ) Luciferase reporter activity in 293T cells transfected with pre- miR-9 or negative control and co-transfected with the pisCheck2 vector containing the 3′UTR of HES1 gene downstream of the firefly luciferase reporter gene. ( G ) Relative miR-9 expression as determined by qRT–PCR 48 h following transfection with either pre- miR-9 or control in the indicated MB cell lines. Values represent fold increase of miR-9 mRNA relative to control. (*P <0.05, **P <0.01, ***P <0.001 according to Student's t-test.) ( H ) Pre- miR-9 mediated downregulation of HES1 protein expression in a representative immunoblotting experiment.

Article Snippet: Probe-primer solutions specific for the following genes were used: HES1 (Hs00172878_m1), p21 (Hs00355782_m1), MASH1/ASCL1 ( Hs00269932_m1), NEUROD1 (Hs00159598_m1), MAP2 (Hs00258900_m1), NEFH (Hs00606024_m1), GFAP (Hs00909236_m1), NES (Hs00707120_s1), and TUBB3 (Hs00964962_g1).

Techniques: Expressing, Sequencing, Binding Assay, Quantitative RT-PCR, Microarray, Luciferase, Activity Assay, Transfection, Negative Control, Plasmid Preparation, Western Blot

Journal: British Journal of Cancer

Article Title: Epigenetic silencing of miRNA-9 is associated with HES1 oncogenic activity and poor prognosis of medulloblastoma

doi: 10.1038/bjc.2013.764

Figure Lengend Snippet: Restoration of miR-9 upregulates HES1 responsive gene P21 Cip1 and impairs clonal growth of MB cells. ( A ) Cartoon depicting an overview of cell cycle-related miR-9 downstream responsive genes and their functions. ( B , C ) Flow cytometric analysis of cell cycle distribution for the indicated MB cell lines 72 h after transfection with either pre- miR-9 or control and with HES1 siRNA or siRNA scrambled control. ( D ) Pre-miR9 mediated upregulation of p21 mRNA (upper panel) and protein (lower panels) expression in the indicated MB cell lines. ( E ) Colony formation and ( F ) sphere formation analysis of human MB DAOY cells treated with pre- miR-9 or control for 6 days. Lower panels: representative pictures. (*P<0.05, **P<0.01 according to Student‘s t-test.)

Article Snippet: Probe-primer solutions specific for the following genes were used: HES1 (Hs00172878_m1), p21 (Hs00355782_m1), MASH1/ASCL1 ( Hs00269932_m1), NEUROD1 (Hs00159598_m1), MAP2 (Hs00258900_m1), NEFH (Hs00606024_m1), GFAP (Hs00909236_m1), NES (Hs00707120_s1), and TUBB3 (Hs00964962_g1).

Techniques: Transfection, Expressing

Journal: British Journal of Cancer

Article Title: Epigenetic silencing of miRNA-9 is associated with HES1 oncogenic activity and poor prognosis of medulloblastoma

doi: 10.1038/bjc.2013.764

Figure Lengend Snippet: miR-9 restoration mediates increase of the expression of a panel of pro-neural differentiation genes in MB cells. ( A ) Cartoon depicting an overview of neuronal differentiation-related miR-9 /HES1 downstream responsive genes. ( B ) Relative mRNA expression of the indicated pro-neural differentiation genes as determined by qRT–PCR 72 h following transfection with either pre- miR-9 or control. Values represent fold change relative to control (* P <0.1, ** P <0.01 according to Student's t -test). ( C ) Morphological changes of MB cells following miR-9 restoration and immunofluorescence analysis of β -tubulin class III (TUBB3) and nestin (NES) 72 h following transfection with either pre- miR-9 or control. ( D ) Correlation study of expression of miR-9/HES1 and selected pro-neural differentiation genes in 285 MB patients' samples as determined by microarray-based expression profiles ( r =Pearson's correlation coefficient).

Article Snippet: Probe-primer solutions specific for the following genes were used: HES1 (Hs00172878_m1), p21 (Hs00355782_m1), MASH1/ASCL1 ( Hs00269932_m1), NEUROD1 (Hs00159598_m1), MAP2 (Hs00258900_m1), NEFH (Hs00606024_m1), GFAP (Hs00909236_m1), NES (Hs00707120_s1), and TUBB3 (Hs00964962_g1).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Immunofluorescence, Microarray

Journal: British Journal of Cancer

Article Title: Epigenetic silencing of miRNA-9 is associated with HES1 oncogenic activity and poor prognosis of medulloblastoma

doi: 10.1038/bjc.2013.764

Figure Lengend Snippet: Expression of miR-9 and HES1 are associated with clinicopatholigical characteristics of paediatric MB. Box plots showing ( A ) miR-9-2 and ( B ) HES1 expression according to MB histological variants: classic ( n =200), (desmo) desmoplastic ( n =21) and (Lca) large cells/anaplastic ( n =30); centre line=median. (*P<0.05, ***P<0.001 according to Student's t-test.) ( C ) Kaplan–Meier survival curves for miR-9-2 expression in paediatric MBs. Patients were sub-divided into high ( n =17) and low ( n =17) miR-9 expression groups based on the median expression value in each population. ( D ) Kaplan–Meier survival curves for HES1 expression in paediatric MBs. Patients were sub-divided into high ( n =13) and low ( n =116) HES1 expression groups based on the median expression value in each population. Optimal cutoff selection was combined with Bonferroni correction because of the low number of HES1 -expressing tumours.

Article Snippet: Probe-primer solutions specific for the following genes were used: HES1 (Hs00172878_m1), p21 (Hs00355782_m1), MASH1/ASCL1 ( Hs00269932_m1), NEUROD1 (Hs00159598_m1), MAP2 (Hs00258900_m1), NEFH (Hs00606024_m1), GFAP (Hs00909236_m1), NES (Hs00707120_s1), and TUBB3 (Hs00964962_g1).

Techniques: Expressing, Selection

Journal: Genes & Cancer

Article Title: RGS16, a novel p53 and pRb cross-talk candidate inhibits migration and invasion of pancreatic cancer cells

doi:

Figure Lengend Snippet: WI38 cells were transduced with adenoviruses carrying the transgenes p53, or RB1 /p105; a MOI of 50 was used in each case. A) Western blot analysis was used to test for p53 and pRb expression prior to microarray analysis. B) Fold change of protein expressions compared to CMV control. C) A Venn diagram shows the differentially expressed transcripts and intersects identified during the microarray analysis. The numbers in red denote transcripts that were up-regulated due to p53, pRb, or p53 and pRb expression.

Article Snippet: Membranes were probed overnight at 4°C with mouse anti-p53 (SC-DO1, 1: 1000) or mouse anti-pRb (SC-IF8, 1:500) antibodies from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Transduction, Western Blot, Expressing, Microarray, Control

Journal: Genes & Cancer

Article Title: RGS16, a novel p53 and pRb cross-talk candidate inhibits migration and invasion of pancreatic cancer cells

doi:

Figure Lengend Snippet: Fold Change of p53 and pRb common gene set cross-talk candidates

Article Snippet: Membranes were probed overnight at 4°C with mouse anti-p53 (SC-DO1, 1: 1000) or mouse anti-pRb (SC-IF8, 1:500) antibodies from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Translocation Assay

Journal: Genes & Cancer

Article Title: RGS16, a novel p53 and pRb cross-talk candidate inhibits migration and invasion of pancreatic cancer cells

doi:

Figure Lengend Snippet: Five transcripts RGS16, BCL2L11, BTG2, IL-6 and STAT4 from the p53 and pRb intersect were chosen for validation by qRT-PCR in WI38 cells expressing p53, pRb, or both p53 and pRb. The vector control (Ad.CMV) was used to calculate the fold change for each transcript. One-way ANOVA with Dunnett's test for multiple comparison were used to test for statistical significance * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, and **** p-value < 0.0001.

Article Snippet: Membranes were probed overnight at 4°C with mouse anti-p53 (SC-DO1, 1: 1000) or mouse anti-pRb (SC-IF8, 1:500) antibodies from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Expressing, Plasmid Preparation, Control, Comparison

Journal: Genes & Cancer

Article Title: RGS16, a novel p53 and pRb cross-talk candidate inhibits migration and invasion of pancreatic cancer cells

doi:

Figure Lengend Snippet: Five transcripts RGS16, BCL2L11, BTG2, IL-6 and STAT4 from the p53 and pRb intersect were chosen for validation by qRT-PCR in SAOS-2 cells expressing p53, pRb, or both p53 and pRb. The vector control (Ad.CMV) was used to calculate the fold change for each transcript. Oneway ANOVA with Dunnett's test for multiple comparison were used to test for statistical significance * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, and **** p-value < 0.0001.

Article Snippet: Membranes were probed overnight at 4°C with mouse anti-p53 (SC-DO1, 1: 1000) or mouse anti-pRb (SC-IF8, 1:500) antibodies from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Expressing, Plasmid Preparation, Control, Comparison

Journal: Genes & Cancer

Article Title: RGS16, a novel p53 and pRb cross-talk candidate inhibits migration and invasion of pancreatic cancer cells

doi:

Figure Lengend Snippet: Characterization of pancreatic cancer cell lines [ 6 , 7 , 89 , 90 ]

Article Snippet: Membranes were probed overnight at 4°C with mouse anti-p53 (SC-DO1, 1: 1000) or mouse anti-pRb (SC-IF8, 1:500) antibodies from Santa Cruz Biotechnology (Dallas, TX).

Techniques:

Journal: PLoS ONE

Article Title: Mesenchymal stromal cell-derived extracellular vesicles reduce lung inflammation and damage in nonclinical acute lung injury: Implications for COVID-19

doi: 10.1371/journal.pone.0259732

Figure Lengend Snippet: (A) Antibody microarray particle analysis of tetraspanin proteins. (B) Electrochemiluminescent multiplexed immunoassay detection of tetraspanin proteins. (C) Western blot analysis of MSC-EV proteins flotillin-1, Annexin-2, Syntenin-1, MHC-I and contaminating proteins MHC-II and calreticulin. (D) Immunogold cryo-transmission electron microscopy imaging for CD63 and phosphatidylserine. Atomic force microscopy for MSC-EV morphology and size. (E) Nanoparticle tracking for MSC-EV size and concentration. (F) MSC-EV particle density correlated to phospholipid content. Error bars are SEM of the mean. *p ≤0.05 vs CD63. Scale bar denotes 100 nm.

Article Snippet: Lysates were volume loaded and separated on a 12% Bolt Bis-Tris Gel and probed using Abcam antibodies against flotillin-1 (ab133497 at 1:10,000 dilution), Annexin-2 (ab41803 at 1:1000 dilution), Syntenin-1 (ab19903 at 1:1000 dilution), MHC-I (ab110645 at 1:1000 dilution), MHC-II (157210 at 1:10000 dilution), and Calreticulin (ab92516 at 1:1000 dilution).

Techniques: Microarray, Western Blot, Transmission Assay, Electron Microscopy, Imaging, Microscopy, Concentration Assay

Journal: bioRxiv

Article Title: TCF4 induces enzalutamide resistance via neuroendocrine differentiation in prostate cancer

doi: 10.1101/560821

Figure Lengend Snippet: TCF4 mediates NED in human prostate cancer cell lines. A. Unsupervised comparison of transcriptome of LNCaP cultured in FBS vs charcoal-stripped FBS (cFBS) was carried out. B. Comparison of transcriptome between LNCaP-cFBS and LNCaP-Enz (resistant to enzalutamide). Markers of NED (ChgA, NSE, and PTHrP) were significantly increased in LNCaP-EnzR. C . Human prostate cancer cell lines LNCaP and 22Rv1 were treated with increasing concentrations of enzalutamide (0-10 μM) under an androgen-deprived condition (RPMI-1640/10% cFBS) for 48 hours. Immunoblot demonstrated that NED markers (ChgA, NSE, and PTHrP) were induced by enzalutamide. D. QPCR demonstrated that NED marker (ChgA, NSE, and PTHrP) mRNA expression levels increased also after treatment with enzalutamide at the indicated concentrations (0-10 μM) for 48 hours.

Article Snippet: TCF4 cDNA (Origene, Cat# 224345, Rockville, MD) was cloned into pLenti4/V5-Dest expression vector (ThermoFisher, Waltham, MA).

Techniques: Cell Culture, Western Blot, Marker, Expressing

Journal: bioRxiv

Article Title: TCF4 induces enzalutamide resistance via neuroendocrine differentiation in prostate cancer

doi: 10.1101/560821

Figure Lengend Snippet: TCF4 mediated enzalutamide resistance in human prostate cancer cell lines. A. TCF4 cDNA was transiently transfected into LNCaP, 22Rv1, and VCaP using lipofectamine. Cells were analyzed 48 hours after transfection. The results demonstrated that the overexpression of TCF4 induced the mRNA expression levels of LNCaP, 22Rv1, and VCaP. As control, parental lines transfected with the plasmid backbone was used. B . In addition to increasing NED, overexpression of TCF4 increased the cellular proliferation rate of LNCaP, VCaP, and 22Rv1. The result shows cell counts at 72 hours after transfection. C. LNCaP and 22Rv1 were treated with enzalutamide for 48 hours. Where indicated, TCF4 or the control POU2F2 expression was silenced using shRNA approach. Increased protein levels of neuroendocrine markers following enzalutamide (enz) treatment was blocked by TCF4 shRNA in LNCaP and 22Rv1. POU2F2 was used as a negative control because it is a transcription factor whose consensus binding element was also found commonly in the promoter regions of the neuroendocrine markers. D. LNCaP was treated with 10 μM enzalutamide and cells were harvested at the indicated time. Kinetics of NED markers and TCF4 transcription following enzalutamide treatment was analyzed using QPCR. The results demonstrated increased mRNA levels of TCF4 in 3 hours while the expression levels of NED markers (ChgA, NSE, and PTHrP) was induced at 24 hours. This observation suggests that TCF4 signals upstream of NED markers.

Article Snippet: TCF4 cDNA (Origene, Cat# 224345, Rockville, MD) was cloned into pLenti4/V5-Dest expression vector (ThermoFisher, Waltham, MA).

Techniques: Transfection, Over Expression, Expressing, Plasmid Preparation, shRNA, Negative Control, Binding Assay

Journal: bioRxiv

Article Title: TCF4 induces enzalutamide resistance via neuroendocrine differentiation in prostate cancer

doi: 10.1101/560821

Figure Lengend Snippet: Effect of blocking TCF4/β-catenin (PKF118-310) or PTHrP (PTHrP (7-34) ) on enzalutamide resistant prostate cancer cells. A. LNCaP, 22Rv1, and VCaP were treated with enzalutamide (10 μM) and/or XAV939 (10 μM), β-catenin degradation activator for 48 hours. XAV939 treatment was carried out 5 min prior to the addition of enzalutamide. PTHrP mRNA induction after 48 hours of treatment with 10 μM enzalutamide was completely blocked by XAV939, β-catenin inhibitor in LNCaP, 22Rv1 and VCaP. B. LNCaP, 22Rv1, and VCaP were treated with 10 uM enzalutamide for 48 hours. The PTHrP receptor, PTH1R, mRNA level significantly increased after enzalutamide treatment in all three cell lines. C. Three enzalutamide-resistant human prostate cancer cell lines (LNCaP-EnzR, 22Rv1-EnzR, and VCaP-EnzR) were treated with 10 μM enzalutamide and increasing concentrations of PKF118-310 and PTHrP (7-34) as indicated. After 48 hours, viable cells were counted. In the presence of 10 μM Enz, PKF118-310 or PTHrP (7-34) increased enzalutamide sensitivity in the enzalutamide resistant prostate cancer cell lines, LNCaP-EnzR, VCaP-EnzR, and 22Rv1-EnzR. D. Enzalutamide-resistant human prostate cancer cell lines were treated with a fixed concentration of PKF118-310 (5 μM) or PTHrP (7-34) (10 μM) and varying concentrations of enzalutamide (0-10 μM). After 48 hours, viable cells were counted. In the presence of 5 μM of PKF118-310 or 10 μM of PTHrP (7-34) , enzalutamide inhibited cellular proliferation in a concentration-dependent manner.

Article Snippet: TCF4 cDNA (Origene, Cat# 224345, Rockville, MD) was cloned into pLenti4/V5-Dest expression vector (ThermoFisher, Waltham, MA).

Techniques: Blocking Assay, Concentration Assay

Journal: bioRxiv

Article Title: TCF4 induces enzalutamide resistance via neuroendocrine differentiation in prostate cancer

doi: 10.1101/560821

Figure Lengend Snippet: Human CRPC tissue microarray (TMA) analysis. TMA was obtained from the rapid autopsy program at the University of Michigan. This array contains 51 CRPC samples as well as 16 benign prostate tissues and 12 localized prostate cancer tissues for controls. A. Immunofluorescence microscopy demonstrated a consistent co-localization of TCF4 (red) and PTHrP (green) in CRPC tissues. B. There was a correlation between PTHrP and TCF4 expression. C. Protein expression levels of TCF4 and D. PTHrP in patients with localized CaP (Local CaP) and metastatic CaP (Meta CaP). TCF4 and PTHrP protein levels were higher in CaP when compared with benign and increased even further in metastatic group when compared with localized CaP group. Benign n=16, Local CaP n=12, meta CaP n=51.

Article Snippet: TCF4 cDNA (Origene, Cat# 224345, Rockville, MD) was cloned into pLenti4/V5-Dest expression vector (ThermoFisher, Waltham, MA).

Techniques: Microarray, Immunofluorescence, Microscopy, Expressing

Journal: bioRxiv

Article Title: TCF4 induces enzalutamide resistance via neuroendocrine differentiation in prostate cancer

doi: 10.1101/560821

Figure Lengend Snippet: TCF4 induces enzalutamide resistance in the human prostate cancer cell line, LNCaP. LNCaP transfected with tetracycline-inducible TCF4 plasmid (LNCaP-TCF4) was injected into the flanks of twenty Rag2-/-, γ c -/- immunodeficient mice. When tumors reached an average size of 3 mm, the mice were divided into four groups of five each. Where indicated, 10 mg/kg enzalutamide was delivered orally daily. In the designated groups, TCF4 was delivering doxycycline via the drinking water. At the end of the indicated duration, all tumors were harvested and analyzed for protein and mRNA expression. A. When TCF4 expression was induced with doxycycline (Doxy), tumor growth rate increased when compared to the control group. In the absence of TCF4 induction, enzalutamide treatment slowed tumor growth rate. However, enzalutamide treatment had no demonstrable inhibitory effect in TCF4-induced doxycycline group. Con = LNCaP-TCF4 without doxycycline. Enz = enzalutamide. B. H&E staining. There was no difference among all groups. C. Immunofluorescence staining for PTHrP (green), TCF4 (red) with DAPI (blue) staining. Increased TCF4 and PTHrP protein levels were observed following the induction of TCF4 with doxycycline. Directly supporting the tissue culture data, enzalutamide treatment also increased protein levels of TCF4 and PTHrP. D and E . Effect of enzalutamide and TCF4 overexpression on TCF4 and PTHrP by western blot analysis ( D) and QPCR ( E ). Immunoblot and QPCR both demonstrated increased expression of TCF4 and PTHrP following TCF4 induction (Doxy). A more modest increase in TCF4 and PTHrP expression was observed with enzalutamide (enz) treatment. Error bars indicate average ± SE and * p-value<0.05.

Article Snippet: TCF4 cDNA (Origene, Cat# 224345, Rockville, MD) was cloned into pLenti4/V5-Dest expression vector (ThermoFisher, Waltham, MA).

Techniques: Transfection, Plasmid Preparation, Injection, Expressing, Staining, Immunofluorescence, Over Expression, Western Blot

Journal: bioRxiv

Article Title: TCF4 induces enzalutamide resistance via neuroendocrine differentiation in prostate cancer

doi: 10.1101/560821

Figure Lengend Snippet: Effect of TCF4/β-catenin inhibitor (PKF118-310) and PTHrP antagonist (PTHrP (7-34) ) in enzalutamide-resistant prostate cancer. After injection of LNCaP-EnzR into the flanks of forty Rag2-/-, γ c -/- immunodeficient mice, all mice were surgically castrated divided into four groups of ten each. Animals in predesignated groups were treated daily with PKF118-310 (0.85 mg/kg intraperitoneal) and/or PTHrP (7-34) (0.2 mg/kg subcutaneous). All mice were administered daily 10 mg/kg enzalutamide orally. A. Treatment of PKF118-310 and/or PTHrP (7-34) with 10 mg/kg enzalutamide decreased tumor growth compare with vehicle treatment control group (con). B. H&E staining. There was no difference among all groups. C. Immunofluorescence staining for TCF4 (green), PTHrP (red) with DAPI (blue) staining. Consistent with its mechanism of action, PFK118-310 treatment decreased PTHrP protein levels. However, there was no effect on TCF4 levels. In contrast, PTHrP (7-34) had no demonstrable effect on the protein levels of both TCF4 and PTHrP. Treatment of PKF118-310 decreased PTHrP protein ( D ) and mRNA expression ( E ). Error bars indicate average ± SE and * p-value<0.05.

Article Snippet: TCF4 cDNA (Origene, Cat# 224345, Rockville, MD) was cloned into pLenti4/V5-Dest expression vector (ThermoFisher, Waltham, MA).

Techniques: Injection, Staining, Immunofluorescence, Expressing

Journal: Cell Reports Medicine

Article Title: Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma

doi: 10.1016/j.xcrm.2023.101091

Figure Lengend Snippet: Identification of PTK7 through cell surface glycoproteomics (A) Schematic depicting in vivo modeling and cell surface capture technique with glycoproteomics used to identify NB cell surface immunotherapy targets. (B) Hierarchical clustering of glycoprotein expression in different xenograft tumor cells following vehicle or chemotherapy treatment for 5 days in vivo . (C) Protein prioritization strategy to narrow the pool of identified cell surface proteins into actionable immunotherapy targets. (D) Screening protocol used to validate cell surface localization and rule out proteins potentially expressed in normal tissues using the Human Protein Atlas. (E) Characterization of 15 potential immunotherapy targets identified and narrowed in (A)–(E), highlighting impact on patient survival and cancer cell line dependency. (F) Abundance plots from cell surface glycoproteomics demonstrating stable PTK7 over the course of chemoimmunotherapy relative vehicle control in both CDX and PDX models. Proteins that don’t change in expression following chemotherapy are demonstrated in the orange/red range of the curve, while those downregulated are marked as blue and upregulated as gray. (G) Survival analysis using the R2 database (Cangelosi dataset ) depicting the impact of PTK7 expression towards patient prognosis. INSS stage 4 or MYCN amplification categories were chosen as they most closely represent high-risk disease. Cutoff was determined using Kaplan Scan as described in . Additional categories can be found in .

Article Snippet: Membranes were blocked (5% Blotting-Grade Blocker w/v in TBS-T, BioRad) and probed overnight at 4°C using rabbit anti-PTK7 primary antibody (1:1000).

Techniques: In Vivo, Expressing, Control, Amplification

Journal: Cell Reports Medicine

Article Title: Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma

doi: 10.1016/j.xcrm.2023.101091

Figure Lengend Snippet: PTK7 expression and functional relevance in NB (A) Western blot analysis of PTK7 expression in a panel of NB cell lines along with their MYCN and ALK genetic signatures. MCYN-A, amplified; MYCN-NA, non-amplified; ALK, ALK mutation present. (B) Histograms depicting PTK7 expression in an array of NB cell lines (left) and patient-derived xenografts (right). Isotype control staining is provided directly underneath as solid gray histogram for each model. (C) Histograms depicting PTK7 expression in primary patient tumors (solid histogram, hNB-4r and hNB-26) from 2 biological donors and their corresponding patient-derived xenografts (dotted histogram, NBx-4r and NBx-26) isolated after growth in NSG mice. (D) PTK7 expression, represented as median fluorescence intensity (MFI), in IMR5 cells treated with 50 μM topotecan for 48 h relative vehicle control, n = 3 biological replicates (as defined in methods), and corresponding table summarizing PTK7 expression (as MFI) of IMR5 cells treated with increasing doses of topotecan for 48 h. (E) PTK7 expression represented as MFI in IMR5 cells made resistant to melphalan (melphalan-R) through continuous culture compared with wild-type control IMR5 cells. n = 3 biological replicates. (F) Histograms depicting PTK7 expression in primary patient tumor tissue (NB-26) procured at diagnosis and following induction chemotherapy from the same patient. (G) Cell surface (left) and total protein (right) expression of PTK7 in CRISPR-cas9 PTK7 knockout models. (H) Normalized proliferation (relative to day 0) determined using Cell Titer Glo (CTG) of PTK7 KO cells for NB cell models. n = 3 biological replicates. (I) Cell viability (relative vehicle control) determined by CTG of PTK7 KO cells following treatment with doxorubicin (0.1 nM to 10 μM) for 48 h. IC 50 values calculated using non-linear regression. n.s., no significant difference in value in KOs vs. NT gRNA control. n = 3 biological replicates.

Article Snippet: Membranes were blocked (5% Blotting-Grade Blocker w/v in TBS-T, BioRad) and probed overnight at 4°C using rabbit anti-PTK7 primary antibody (1:1000).

Techniques: Expressing, Functional Assay, Western Blot, Amplification, Mutagenesis, Derivative Assay, Control, Staining, Isolation, Fluorescence, CRISPR, Knock-Out

Journal: Cell Reports Medicine

Article Title: Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma

doi: 10.1016/j.xcrm.2023.101091

Figure Lengend Snippet: PTK7 expression in pediatric normal tissues and blood (A) Schematic depicting pediatric microarray of normal tissues designed by the pathology team at Children’s Healthcare of Atlanta (CHOA). IHC of PTK7 expression in primary colorectal cancer tissue as a control for positive signal. IHC score = 3+. Scale bar provided applies to all images in A-B. (B) PTK7 expression in pediatric normal tissues representative of all major organs. Where possible, duplicate tissues were analyzed and scored representative of both male and female. Left: tissues stained IHC score = 0; right: tissues stained IHC score = 1. (C) Representative flow plots depicting PTK7 expression in normal pediatric kidney tissue and various normal adult immune cells. A total of n = 2 and n = 3 biological replicates were performed respectively, on the basis of tissue availability. (D) Histograms depicting PTK7 expression in primary patient tumors (black line, hNB-4r and hNB-26) and their corresponding immune cell infiltrate (red line, CD45 + ). Each plot represents n = 1 replicate of the two different biological donors.

Article Snippet: Membranes were blocked (5% Blotting-Grade Blocker w/v in TBS-T, BioRad) and probed overnight at 4°C using rabbit anti-PTK7 primary antibody (1:1000).

Techniques: Expressing, Microarray, Control, Staining

Journal: Cell Reports Medicine

Article Title: Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma

doi: 10.1016/j.xcrm.2023.101091

Figure Lengend Snippet: Design, expression, and specificity of UBC/EF1a-based PTK7 CARs (A) Lentiviral vector-based UBC PTK7 CAR DNA constructs. (B) Percentage GFP + Jurkat T cells transduced with PTK7 VL-L-VH CAR or PTK7 VH-L-VL CAR, gated on live cells, n = 3 biological replicates. (C) Percentage CAR+ (PTK7-FC chimera+) Jurkat T cells transduced with PTK7 VL-L-VH CAR or PTK7 VH-L-VL CAR. Gated on live GFP + cells, n = 3 biological replicates. (D) Representative flow plot showing GFP vs. CAR expression in Jurkat T cells, gated on live cells. (E) CAR western blot from whole-cell lysate of Jurkat T cells transduced with PTK7 VL-L-VH CAR or PTK7 VH-L-VL CAR, 3 days post-transduction. Western blot antibody against human CD3ζ. (F) Percentage CD69 + Jurkat T cells co-cultured with (+) or without (-) target cells, gated on live GFP + cells, n = 3 biological replicates. (G) Top: PTK7 and GAPDH western blot of whole-cell lysate of Jurkat T cells and primary T cells. Bottom: histogram depicting cell surface PTK7 expression on T cells pre- and post-stimulation with CD3/CD28 beads relative isotype control. (H) Left: percentage CD69 + Jurkat T cells transduced with PTK7 VH-L-VL CAR or CD19 CAR co-cultured with target cells (SK-N-AS non-targeting gRNA control or SK-N-AS PTK7 KO), gated on live GFP + cells, n = 3 biological replicates. Right: representative flow plots showing CD69 expression within GFP + Jurkat T cells. (I) Lentiviral vector-based EF1α PTK7 CAR DNA construct. (J) Left: representative flow plot showing CAR expression (PTK7-FC chimera) in primary T cells transduced with indicated CAR AT MOI 20. Right: MFI of PTK7 CAR expression in primary T cells transduced with indicated CAR, n = 2 or 3 biological replicates. (K) Western blot from whole-cell lysate of primary T cells transduced with indicated CAR at MOI 20. Western blot antibody against human CD3ζ.

Article Snippet: Membranes were blocked (5% Blotting-Grade Blocker w/v in TBS-T, BioRad) and probed overnight at 4°C using rabbit anti-PTK7 primary antibody (1:1000).

Techniques: Expressing, Plasmid Preparation, Construct, Transduction, Western Blot, Cell Culture, Control

Journal: Cell Reports Medicine

Article Title: Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma

doi: 10.1016/j.xcrm.2023.101091

Figure Lengend Snippet: PTK7 CAR T cells have specific in vitro cytotoxicity (A) Twelve-hour flow cytometry cytotoxicity assay. Percentage cytotoxicity = the sum of 7AAD + , annexin V + , and 7AAD + annexin V+ cells, gated on target cells only. n = 3 donors with n = 2 biological replicates. Four-hour cytotoxicity shown in Figure S5 A. (B) Target cells and CAR T cells or mock T cell control were incubated with caspase-3/7 dye and live cell analysis was performed at the indicated time points. Processing definitions of caspase-3/7 activity (green object count) on target cells were determined. n = 1 donor. Error bars represent SD of 4 technical replicates. (C) Percentage CD69 expression in mock T cells or CAR T cells co-cultured with indicated target cells from 4-h cytotoxicity assay, gated on live primary T cells. n = 3 donors, statistical analysis represents Student’s t test (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns, p > 0.05). (D) PTK7 MFI expression in MR5, NLF, and CMK target cells, n = 6 biological replicates. (E) Representative image of mock or CAR T cells with target cell co-culture at 4 h. Scale bar provided is applicable to each image in E.

Article Snippet: Membranes were blocked (5% Blotting-Grade Blocker w/v in TBS-T, BioRad) and probed overnight at 4°C using rabbit anti-PTK7 primary antibody (1:1000).

Techniques: In Vitro, Flow Cytometry, Cytotoxicity Assay, Control, Incubation, Cell Analysis, Activity Assay, Expressing, Cell Culture, Co-Culture Assay

Journal: Cell Reports Medicine

Article Title: Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma

doi: 10.1016/j.xcrm.2023.101091

Figure Lengend Snippet: PTK7 CAR T cells improve survival in vivo (A and B) NSG mice were injected with 3 × 10 5 IMR5 cells on day −1 IV, then treated with 5 × 10 6 mock T cells or PTK7 CAR T cells on day 0 IV. (A) Bioluminescence images and (B) Kaplan-Meier survival analysis of arms. n = 5 untreated, n = 7 mock T cell, n = 7 PTK7 CAR T cell. Statistical analysis represents log rank (Mantel-Cox) test (∗p < 0.05 and ∗∗p < 0.01; ns, p > 0.05). (C) Top: representative flow plot showing percentage PTK7 CAR binding/expression to human and mouse PTK7-FC chimera. Bottom: histogram and MFI of CAR binding/expression. (D) Weight change (%) of mice in (A) and (B) Top: females. Bottom: males. (E) Histogram of PTK7 expression in IMR5 cells in the liver of mice on day 16.

Article Snippet: Membranes were blocked (5% Blotting-Grade Blocker w/v in TBS-T, BioRad) and probed overnight at 4°C using rabbit anti-PTK7 primary antibody (1:1000).

Techniques: In Vivo, Injection, Binding Assay, Expressing

Journal: Cell Reports Medicine

Article Title: Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma

doi: 10.1016/j.xcrm.2023.101091

Figure Lengend Snippet: PTK7 expression in pediatric solid tumors beyond NB (A) Box-and-whisker plots show PTK7 expression for bone cancer, sarcoma, leukemia, and brain cancer from Cancer Cell Line Encyclopedia (CCLE; sites.broadinstitute.org/ccle/). Dotted line represents the mean. Solid line represents the median. Dots represent outliers. Error bars represent SD. (B) PTK7 mRNA expression across a panel of pediatric tumor tissues using the R2:Genomics Analysis and Visualization Platform ( http://r2.amc.nl ). ∗Extracranial solid tumor types. (C) IHC demonstrating PTK7 expression in osteosarcoma PDX tissue and rhabdomyosarcoma primary and paired PDX tissue. Scores of IHC staining for each tissue are provided in top left. (D) Western blot analysis of PTK7 expression in a panel of osteosarcoma (n = 2) and rhabdomyosarcoma (n = 1) cell lines. GAPDH serves as the loading control. (E) Four-hour flow cytometry cytotoxicity assay of PTK7 CAR modified aβ T cells against PTK7-expressing osteosarcoma and rhabdomyosarcoma cell lines compared with mock T cell controls. Percentage cytotoxicity = the sum of 7AAD + , annexin V + , and 7AAD + annexin V + cells, gated on target cells. n = 2 donors with n = 3 biological replicates. Data represented as mean and error bars as SD.

Article Snippet: Membranes were blocked (5% Blotting-Grade Blocker w/v in TBS-T, BioRad) and probed overnight at 4°C using rabbit anti-PTK7 primary antibody (1:1000).

Techniques: Expressing, Whisker Assay, Immunohistochemistry, Western Blot, Control, Flow Cytometry, Cytotoxicity Assay, Modification

Journal: Cell Reports Medicine

Article Title: Identification and targeting of protein tyrosine kinase 7 (PTK7) as an immunotherapy candidate for neuroblastoma

doi: 10.1016/j.xcrm.2023.101091

Figure Lengend Snippet:

Article Snippet: Membranes were blocked (5% Blotting-Grade Blocker w/v in TBS-T, BioRad) and probed overnight at 4°C using rabbit anti-PTK7 primary antibody (1:1000).

Techniques: Virus, Microarray, Recombinant, Cell Isolation, Mass Spectrometry, CRISPR, Negative Control, Software, Lysis, Protease Inhibitor, Membrane, Bradford Protein Assay, BIA-KA, Binding Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 2. YAP1 and TAZ regulate expression of SLC38A1 and SLC7A5 in HCC cells. (A) Expression pattern of SLC family genes from microarray data. Amino acid transporters are shown in red. (B-E) The indicated cells were transfected with the indicated siRNAs. Cells were used for quanti- tative RT-PCR to measure SLC38A1 and SLC7A5. Expression level was normalized with siLuc samples. (F) Quantitative RT-PCR was done with Mst1/2 knockout samples to measure connective tissue growth factor, SLC38A1, and SLC7A5 messenger RNA expression level. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. Abbreviations: CTGF, connective tissue growth factor; Wt, wild type.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Expressing, Microarray, Transfection, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Knock-Out, RNA Expression

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 4. Amino acid transporters are important for proliferation of HCC cells. (A-D) SK-Hep1 and SNU-449 HCC cells were transiently trans- fected with the indicated siRNAs, and cell proliferation rates were measured by an MTT assay at the indicated time points. Values shown were normalized to siLuc-treated cells and represent mean 6 standard deviation. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. (E) Recovered cell growth by exogenous SLC38A1 and SLC7A5 in YAP1/TAZ-depleted SK-Hep1 cells. Myc-tagged exogenous SLC38A1 and SLC7A5 were expressed in SK-Hep1 cells. Empty expression vector pCMV6 was used as a control. Cell proliferation rates were measured by the MTT assay at 72 hours after transfection of expression vectors. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. (F,G) Tumor weight after treat- ment with siRNA-1,2-dioleoyl-sn-glycero-3-phosphatidylcholine in a subcutaneous xenograft model (F) or an orthotopic xenograft model (G) with SK-Hep1 HCC cells. At 6 weeks after siRNA-1,2-dioleoyl-sn-glycero-3-phosphatidylcholine injection, mice were killed and tumor weights measured (n 5 10 per group). Data are presented as mean 6 standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: MTT Assay, Standard Deviation, Expressing, Plasmid Preparation, Control, Transfection, Injection

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 5. YAP1 and TAZ directly bind to the promoters of amino acid transporters. (A) Schematic representation of SLC38A1 promoter regions for ChIP assay (top) and ChIP assay results (bottom). (B) Schematic representation of SLC7A5 promoter regions for ChIP assay (top) and ChIP assay results (bottom). ChIP assay was done in SK-Hep1 cells with indicated antibodies. Recruitment of YAP1, TAZ, and TEAD1 proteins to the SLC38A1 or SLC7A5 promoter was analyzed using primers specific to the SLC38A1 or SLC7A5 promoter. Immunoglobulin G was used as an internal control. (C,D) ChIP samples were used for quantitative RT-PCR for quantification of binding in indicated cell lines. Data are presented as mean 6 standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. (E,F) SK-Hep1 and SNU-449 cells were transi- ently transfected with indicated complementary DNA and reporter plasmids, and luciferase activity was measured by a luminometer (Promega). Data are presented as mean 6 standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. Abbreviations: IgG, immunoglobulin G; IP, immunoprecipitation; TBS, TEAD binding site.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Control, Quantitative RT-PCR, Binding Assay, Transfection, Luciferase, Activity Assay, Immunoprecipitation

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 6. mTORC1 is regulated by SLC38A1 and SLC7A5 in HCC cells. (A) SK-Hep1 and SNU-449 HCC cells were transiently trans- fected with the indicated siRNAs for 96 hours. Phosphorylation of S6K1 was assessed by western blotting with the indicated antibodies. (B) SK-Hep1 cells were treated with esterified glutamine (Gln, 4 mM) for 30 minutes after silencing of SLC38A1 with specific siRNA. Expression and phosphorylation of S6K1 and S6 were assessed in cell lysates by western blotting with indicated antibodies. (C,D) Xeno- grafted HCC tissues treated with the indicated siRNAs were used for western blot analysis with the indi- cated antibodies.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Phospho-proteomics, Western Blot, Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 7. YAP1 and TAZ activate mTORC1 through regulation of SLC38A1 and SLC7A5 (A,B). SK- Hep1 (A) and SNU-449 (B) HCC cells were transiently transfected with the indicated siRNA for 96 hours. Expression of YAP1 and TAZ and phosphorylation of S6K1 and S6 were assessed by western blot- ting with the indicated antibodies. (C) SK-Hep1 cells were transfected with SLC38A1 and SLC7A5 com- plementary DNA after silencing YAP1 and TAZ with specific siRNAs. Expression and phosphorylation of S6K1 and S6 were assessed in cell lysates by western blotting with the indicated antibodies. (D) Expres- sion of Mst1, Mst2, S6K1, and S6 and phosphorylation of S6K1 and S6 were assessed in liver tissues from wild-type and Mst1/2-/- mice (4 weeks old) by western blotting with indicated antibodies. Abbrevia- tion: Wt, wild type.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Transfection, Expressing, Phospho-proteomics, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 8. Significance of YAP1/TAZ-mediated regulation of mTORC1 in human HCC and mouse HCC models. (A,B) Kaplan-Meier plots of overall survival of HCC patients in cohort 1. Patients were stratified according to expression level of SLC38A1 (A) or SLC7A5 (B). P values were calculated with the log-rank test. (C) Liver tumor numbers after rapamycin treatment in Mst1/2-/- mice. At 62 days after rapamycin treatment, mice were killed and tumors counted. Data are presented as mean 6 standard error of the mean. (D) Representative images of liver tissue treated with rapamycin or vehicle. (E) Expression and phosphorylation of mTOR1 downstream targets in HCC tissues assessed by western blotting with the indicated antibodies after rapamycin or vehicle treatment in Mst1/2-/-

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Expressing, Phospho-proteomics, Western Blot