Journal: The Journal of Clinical Investigation

Article Title: A pathogenic variant of AMOT leads to isolated X-linked congenital hydrocephalus due to N-terminal truncation

doi: 10.1172/JCI179438

Figure Lengend Snippet: ( A ) Illustration of the second position amino acid of AMOT130, AMOT130 ΔN , and rescue construct E2R_AMOT130 ΔN ; TNKS inhibition via XAV-939; and RNF146-mediated ubiquitylation of AMOT130. ( B ) Representative Western blot depicting AMOT130 ΔN and AMOT130 protein levels after TNKS inhibition by XAV-939 in primary skin fibroblasts derived from a female control, a male patient, or a male control. ( C ) Quantification of 5 independent Western blots shows that the protein levels of AMOT130 in male and female control primary skin fibroblasts are increased but AMOT130 ΔN level is not affected in patient primary skin fibroblasts upon XAV-939 treatment. The SD is reported. * P < 0.05, 2-way-ANOVA with Šídák’s multiple comparisons tests. ( D ) Representative Western blot depicting overexpressed AMOT130 ΔN and AMOT130 protein levels after XAV-939, JW 55, and WIKI4 (10 μM, for 12 hours) addition to MCF7 cells. ( E ) Quantification of 5 independent Western blots shows that overexpressed AMOT130 protein level is increased, but overexpressed AMOT130 ΔN protein level is unaffected after XAV-939, JW 55, and WIKI4 treatment in MCF7 cells. The SD is reported. ** P ≤ 0.01, *** P < 0.001, 2-way ANOVA with Šídák’s multiple comparisons tests. ( F ) Representative Western blot demonstrating protein levels of AMOT130, AMOT130 ΔN , E2R_AMOT130 ΔN , and TBD_AMOT130 ΔN . E2R_AMOT130 ΔN mimics the destabilized N-terminus of AMOT130 by 1 amino acid substitution in the second position: glutamic acid to arginine (E2R) in AMOT130 ΔN . TBD_AMOT130 ΔN contains TBD 77–84 of wild-type AMOT130 at the N-terminus of AMOT130 ΔN . ( G ) Quantification of 5 independent Western blots shows that the protein level of overexpressed E2R_AMOT130 ΔN and TBD_AMOT130 ΔN is significantly decreased compared with AMOT130 ΔN but is still higher than AMOT130. The SD is reported. * P ≤ 0.05, ** P ≤ 0.01, 1-way ANOVA with Dunnett’s multiple comparisons test. (AMOT130 ΔN was taken as a control group.)

Article Snippet: MCF7 cells were seeded at the density of 2 × 10 –5 /well in 12-well plates for (a) AMOT130, AMOT130 ΔN , and chimeric protein overexpression; (b) XAV-939 (10 μM; Selleckchem), JW 55, and WIKI4 (10 μM; MedChemExpress) treatment; (c) BMP6 stimulation; and (d) IF staining (cells seeded on coverslips) experiments.

Techniques: Construct, Inhibition, Western Blot, Derivative Assay, Control

Journal: Nature Communications

Article Title: Sufu - and Spop -mediated downregulation of Hedgehog signaling promotes beta cell differentiation through organ-specific niche signals

doi: 10.1038/s41467-019-12624-5

Figure Lengend Snippet: WNT signaling inhibition boosts Insulin + cell development in epithelial organoids. a , b Representative brightfield ( a ) and fluorescence microscopy ( b ) images of a forming organoid established from E10.5 Bapx1 Cre/+ ; ROSA26 mT/mG mesenchymal reporters demonstrates exclusion of GFP + Bapx1 -expressing mesenchyme. c , d Representative varieties of organoids at day 7 of culture are shown in c , with an extensively branched structure (left) and a more typical organoid (right). An undifferentiated spheroid is shown in d . e , f Organoid ( e ) and spheroid ( f ) structures formed in culture predominantly express epithelial marker, EPCAM, and pancreas marker, PDX1. g – i Morphology of organoids cultured in the presence of DMSO ( g ) vs. 2 µM WNT activator, CHIR ( h ), or 2.5 µM WNT inhibitor, WIKI4 ( i ), from day 0 to day 8. Arrows mark examples of epithelial branching projections. j – l Representative images of DMSO- ( j ), CHIR- ( k ), and WIKI4-treated ( l ) organoids stained for INS and AMY. m Quantification of organoids demonstrating extensive branching morphology (example in h ), as a proportion of all organoids ( n = 168 total organoids). n Immunofluorescent analysis of the ratio of AMY + area to DAPI + area for organoids cultured with or without 2.5 µM WIKI4 ( n = 42 DMSO-treated organoids, n = 63 WIKI4-treated organoids). o Immunofluorescent analysis of the ratio of INS + cell number to DAPI + cell area ( n = 42 DMSO-treated organoids, n = 63 WIKI4-treated organoids). Data are means ± SEM. n.s. denotes not significant, **** denotes p < 0.0005 by Student’s un-paired, two tailed t -test. Scale bars: Brightfield- 100 µm, Immunofluorescence- 90 µm

Article Snippet: Final concentrations of WIKI4 (Tocris) tested were 2.5 μM and 9 μM .

Techniques: Inhibition, Fluorescence, Microscopy, Expressing, Marker, Cell Culture, Staining, Two Tailed Test, Immunofluorescence

Journal: Nature Communications

Article Title: Sufu - and Spop -mediated downregulation of Hedgehog signaling promotes beta cell differentiation through organ-specific niche signals

doi: 10.1038/s41467-019-12624-5

Figure Lengend Snippet: Inhibition of WNT signaling improves human stem cell to beta-like cell differentiation. a Schematic of hESC differentiation to beta-like cell protocol. b Representative flow cytometry plot of day 13 cultures differentiated with DMSO, 9 µM WIKI4, or 2 µm CHIR at stage 4. Cells were stained with anti-PDX1 and anti-NKX6-1. c Kinetics of NKX6-1 + /PDX1 + pancreatic progenitor development from day 8 to day 13 of differentiation in the presence of DMSO, WIKI4, or CHIR ( n = 3 independent experiments). Data are means ± SEM. **** denotes p < 0.0001 by two-way ANOVA with Tukey’s multiple comparison test. d Representative flow cytometry plot of day 23 cultures differentiated at stage 4 with DMSO, WIKI4 or CHIR. Cells were stained with anti-PDX1, anti-NKX6-1 (upper panel) and anti-C-peptide (lower panel). e , f Quantification of NKX6-1 + /PDX1 + ( e ) and NKX6-1 + /C-Peptide + ( f ) percentages at day 23 of differentiation, in cultures treated with DMSO, WIKI4 or CHIR at stage 4. Data are means ± SEM. * denotes p < 0.05, ** denotes p < 0.01 by one-way ANOVA with Tukey’s multiple comparison test. g Down-regulation of mesenchymal Hedgehog (Hh) signaling by Sufu and Spop allows for the establishment of a pancreas-specific mesenchymal niche that properly supports epithelial development (left). Improper activation of Hh signaling due to loss of mesenchymal Sufu and Spop leads to a more gastrointestinal-like niche that secretes gastrointestinal factors such as WNT ligands (right). These WNT ligands can then act on the developing pancreatic epithelium to over-activate WNT signaling and impair beta cell differentiation

Article Snippet: Final concentrations of WIKI4 (Tocris) tested were 2.5 μM and 9 μM .

Techniques: Inhibition, Cell Differentiation, Flow Cytometry, Staining, Comparison, Activation Assay

Journal: Nature Communications

Article Title: Tankyrase disrupts metabolic homeostasis and promotes tumorigenesis by inhibiting LKB1-AMPK signalling

doi: 10.1038/s41467-019-12377-1

Figure Lengend Snippet: Tankyrases Regulate AMPK Activation through ribosylating LKB1. a Identification of the tankyrase inhibitor JW55 as an AMPK activator. A drug screen for regulators of AMPK was done by treating U2OS cells with a set of 406 drugs (the small-molecule Informer-Set contains 481 drugs, but we can only get 406 drugs from commercial sources) and metformin/AICAR. Both JW55 and JW-55 were the same compounds from two different commercial sources. b Tankyrase inhibitors specifically induce AMPK activation. HEK293A cells were treated with indicated inhibitors and proteins were detected by western blotting. c Double-knockdown of TNKS1/2 induces AMPK activation. HEK293A cells were transduced with the indicated shRNA and subjected to western blotting. d Depletion of TNKS1/2 suppresses expression of AMPK downstream glycolytic genes. Relative expression levels of PDK1, FAS SREBP-1c, and TNKS1/2 were detected by qPCR. e Interactions between endogenous LKB1 and TNKS1. Immunoprecipitation/western blotting were performed with the indicated antibodies. f LKB1 is ribosylated in vivo. Lysates from HEK293A cells were subjected to immunoprecipitation/western blotting assays. g LKB1 is ribosylated by TNKS1 in vitro. In vitro ribosylation was assessed by using recombinant TNKS1/LKB1 (E.coli purified GST-LKB1 and enzymatic active form of TNKS1, amino acids 1000–1328 with GST-tag). h E130 and E138 on LKB1 are key residues for TNKS1-induced ribosylation of LKB1. In vitro ribosylation was assessed by using the indicated proteins. i Structure view of LKB1/STRAD/MO25 complex. The LKB1-E130 residue is located close to the interface between LKB1 and STRAD. j Structure view of LKB1. The LKB1-E138 residue is located close to the ATP-binding pocket of LKB1. Statistical significance was determined by a two-tailed, unpaired Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Olaparib, XAV939, JW55, and WIKI4 were from Selleck Chemicals, G007-LK and AICAR were purchased from ApexBio, and the neutral lipid dye BODIPY 493/503 was obtained from Invitrogen.

Techniques: Activation Assay, Western Blot, Knockdown, Transduction, shRNA, Expressing, Immunoprecipitation, In Vivo, In Vitro, Recombinant, Purification, Residue, Binding Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Tankyrase disrupts metabolic homeostasis and promotes tumorigenesis by inhibiting LKB1-AMPK signalling

doi: 10.1038/s41467-019-12377-1

Figure Lengend Snippet: Tankyrase-RNF146 axis is involved in regulation of LKB1 complex integrity and LKB1 phosphorylation. a Tankyrase inhibitor treatment enhances LKB1/STRAD/MO25 complex formation. HEK293A cells were treated with DMSO or the tankyrase inhibitors JW55. WIKI4, XAV939, or G007-LK, and subjected to immunoprecipitation/western blotting. We use the ImageJ to calculate the band densitometry of western blots, and get the relative ratio by comparing the densitometry of MO25/LKB1 or STRAD/LKB1, and we get three relative ratios from three independent experiments. The relative ratio data was analyzed by the One-way ANOVA test and we used an F-test to compare variances, a P value of <0.05 was considered to indicate statistically significant differences, ** means P -value of <0.01 and *** means P -value of <0.001 (shown in Supplementary Data ). b TNKS1/2 depletion increases LKB1 complex formation. HEK293A cells were transduced with the indicated shRNAs and cell lysates were subjected to immunoprecipitation/western blotting. c Expression of TNKS1 but not the TNKS1-PD mutant disrupts LKB1 complex formation. shRNA-resistant inducible TNKS1 or TNKS1-PD were transducted into TNKS1/2-knockdown HEK293A cells, and cell lysates were subjected to immunoprecipitation. d Loss of RNF146 promotes LKB1 complex formation. HEK293A cells were transduced with the indicated shRNAs and cell lysates were subjected to immunoprecipitation/western blotting. e G007-LK treatment promotes complex formation of WT but not E130/138 A mutant of LKB1 with STRAD/MO25. Indicated cells were treated without or with G007-LK and cell lysates were subjected to pull-down assay. f Overexpression of RNF146 inhibits complex formation of WT but not E130/138 A mutant of LKB1 with STRAD/MO25. HeLa cells stable expressed wild-type or the E130/138 A mutant of LKB1 was transfected without or with Myc-RNF146. Cell lysates were subjected to pull-down assay. g TNKS1/2 depletion induces LKB1 phosphorylation at Ser428. HEK293A cells were transduced with the indicated shRNAs, and phosphorylation of LKB1 at different sites was assessed with the corresponding antibodies. h Knockdown of RNF146 promotes LKB1 phosphorylation. HEK293A cells were transduced with the indicated shRNAs and subjected to western blotting. Statistical significance was determined by a two-tailed, unpaired Student’s t- test. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Olaparib, XAV939, JW55, and WIKI4 were from Selleck Chemicals, G007-LK and AICAR were purchased from ApexBio, and the neutral lipid dye BODIPY 493/503 was obtained from Invitrogen.

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Transduction, Expressing, Mutagenesis, shRNA, Knockdown, Pull Down Assay, Over Expression, Transfection, Two Tailed Test

Journal: Journal of Cellular and Molecular Medicine

Article Title: Anticancer effects of melatonin via regulating lncRNA JPX‐Wnt/β‐catenin signalling pathway in human osteosarcoma cells

doi: 10.1111/jcmm.16894

Figure Lengend Snippet: Wnt/β‐catenin signalling pathway was involved in lncRNA JPX mediated tumour progression in OS cells. (A) The protein levels of Wnt/β‐catenin signalling target genes were measured by western blot in MG63 cells transfected with lncRNA NC or lncRNA JPX. (B) The protein expression of the core factors of Wnt/β‐catenin signalling pathway was examined by western blot assay in MG63 cells treated with Wiki4. (C) The proliferative ability of MG63 cells treated with lncRNA JPX or lncRNA JPX + Wiki4 was examined with colony formation assay. (D) The invasion increased by lncRNA JPX was decreased in the presence of Wiki4. (E) The migratory ability of MG63 cells treated with lncRNA JPX or lncRNA JPX + Wiki4 was detected by wound healing assay (F‐H) The roles of Wnt/β‐catenin signalling in the inhibition of lncRNA JPX by melatonin. Scale bar = 250 μm. Significant difference relative to control group was presented as * p < 0.05; ** p < 0.01 and *** p < 0.001

Article Snippet: Wiki4 was purchased from Chembridge, USA.

Techniques: Western Blot, Transfection, Expressing, Colony Assay, Wound Healing Assay, Inhibition, Control