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: 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: Nature Communications

Article Title: Tankyrase inhibition promotes endocrine commitment of hPSC-derived pancreatic progenitors

doi: 10.1038/s41467-024-53068-w

Figure Lengend Snippet: a Schematic representation of the four stages of differentiation from human Pluripotent Stem Cells (hPSCs) to PDX1 + /NKX6-1 + pancreatic progenitors (PPs). Nicotinamide (NA) was substituted at stage 4 by either Histone Deacetylase (HDAC), Poly-ADP Ribose Polymerase (PARP) or Tankyrase (TNKS) inhibitors. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ). b Percentage of PDX1 + /NKX6-1 + cells measured by flow cytometry between day 8 and day 13 of differentiation after treatment with Noggin and EGF alone (-) or in combination with NA or the TNKSi XAV939, IWR-1, MN64 or WIKI4 ( n = 3 independent biological replicates. P -values are <0.0001 for NA, XAV939, IWR-1, MN64, WIKI4 when comparing day 12/13 to day 8. For control, P -value is 0.002 at day 12, and <0.0001 at day 13 when compared to day 8. Two-way ANOVA with Tukey’s multiple-comparison test between day 8 and day 12/13 of each condition. Error bar represents ± SEM). c , d Representative flow cytometry plots of NKX6-1 and PDX1 expression at day 13 and quantification of PDX1 + /NKX6-1 + cells ( n = 5 independent biological replicates, One-way ANOVA with Dunnett’s multiple-comparison test. All significant conditions produced P -values < 0.0001. Error bars represent ± SEM.). e , f Gene expression analysis of PDX1 and NKX6-1 on day 13. Data normalized to the human housekeeping gene TBP ( hTBP ) ( n = 3 independent biological replicates. Exact P -values are reported in the figure. One-way ANOVA with Dunnett’s multiple-comparison test. Error bars represent ± SEM).

Article Snippet: In addition, either 10 mM Nicotinamide (Sigma) or 9 μM WIKI4 (Tocris) were added in this stage, and the medium was replaced daily until d11.

Techniques: Histone Deacetylase Assay, Flow Cytometry, Control, Comparison, Expressing, Produced, Gene Expression

Journal: Nature Communications

Article Title: Tankyrase inhibition promotes endocrine commitment of hPSC-derived pancreatic progenitors

doi: 10.1038/s41467-024-53068-w

Figure Lengend Snippet: a Schematic representation of the six stages of differentiation from hPSCs to C-Peptide (C-PEP) + /NKX6-1 + β-like cells. NA was substituted at stage 4 by either DMSO or different small molecules targeting either the nicotinamide (XAV939) or the Adenosine subsite (JW74, WIKI4, JW55, G007-LK) of TNKS. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ). b , c Representative flow cytometry plots and quantification of the percentage of C-PEP + /NKX6-1 + β-like cells at stage 6 obtained after the different treatment groups at stage 4 ( n = 5 for DMSO, n = 3 for XAV939 and JW74, n = 8 for NA, n = 6 for WIKI4 and JW55, n = 7 for G007-LK. All replicates represent independent biological replicates. Exact P -values are reported in the figure. One-way ANOVA with Dunnett’s multiple-comparison test. Error bars represent ± SEM.).

Article Snippet: In addition, either 10 mM Nicotinamide (Sigma) or 9 μM WIKI4 (Tocris) were added in this stage, and the medium was replaced daily until d11.

Techniques: Flow Cytometry, Comparison

Journal: Nature Communications

Article Title: Tankyrase inhibition promotes endocrine commitment of hPSC-derived pancreatic progenitors

doi: 10.1038/s41467-024-53068-w

Figure Lengend Snippet: a Global distribution of genes by log-transformed False Discovery Rate (FDR) and log-transformed fold change in expression between NA- and WIKI4-derived stage 4 cells. b Heatmap of scaled and log-transformed expression data for genes differentially expressed between NA- and WIKI4-derived stage 4 populations. Two clusters were identified—genes upregulated by NA (cluster 1), and genes upregulated by WIKI4 (cluster 2). ( c Gene expression analysis of proliferation markers ( MKI67 , TOP2A ) and cell cycle regulators ( CDKN1A , CDKN2B ) by RT-qPCR on day 13. Data normalized to housekeeping gene hTBP ( n = 4 independent biological replicates. Exact P -values are reported in the figure. Two-tailed paired student’s t -test. Error bars represent SEM). d Cell count of NA- and WIKI4-derived stage 4 populations ( n = 6 independent biological replicates. The exact P -value is reported in the figure. Two-tailed paired student’s t -test. Error bars represent SEM). e , f Representative flow cytometry plots and quantification of NKX6-1/Ki67 expression profile ( n = 4 independent biological replicates. Exact P -values are reported in the figure. Two-tailed paired student’s t -test. Error bars represent SEM). g Gene set enrichment analysis (GSEA) of gene sets associated with the integrin signaling pathway, comparing NA- and WIKI4-derived stage 4 populations (Data analyzed by Kolmogorov–Smirnov test. No correction was performed). h Gene expression analysis of integrin subunits ( ITGB1, ITGA3, ITGA5, ITGAV ) by RT-qPCR in NA- and WIKI4-derived cells at stage 4. Data normalized to housekeeping gene hTBP ( n = 5 independent biological replicates. Exact P -values are reported in the figure. Two-tailed paired student’s t -test. Error bars are SEM). i Immunofluorescence staining of NA- and WIKI4-derived cells at stage 4 with ITGB1 (red) and ACTA2 (green). DAPI (gray) represents nuclei. The scale bar represents 125 µm.

Article Snippet: In addition, either 10 mM Nicotinamide (Sigma) or 9 μM WIKI4 (Tocris) were added in this stage, and the medium was replaced daily until d11.

Techniques: Transformation Assay, Expressing, Derivative Assay, Gene Expression, Quantitative RT-PCR, Two Tailed Test, Cell Counting, Flow Cytometry, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Tankyrase inhibition promotes endocrine commitment of hPSC-derived pancreatic progenitors

doi: 10.1038/s41467-024-53068-w

Figure Lengend Snippet: a Schematic representation of pancreatic differentiation highlighting reaggregation at day 20 and extended culture to day 33. PDX1 + cells were treated with either NA or WIKI4 at stage 4. On day 20, cells were dissociated into single cells and allowed to reaggregate in stage 6 media. On day 23, cells were transferred to a growth factor-free media until day 33. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ). b Immunofluorescence staining of NA- and WIKI4-derived cells in day 23 non-reaggregated, day 23 reaggregated, and day 33 reaggregated conditions. Sections were stained for β cell markers (NKX6-1/PDX1/C-PEP) and pancreatic hormones (GCG/INS/SST). DAPI represents nuclei. Scale bar represents 50 µm. The white box represents 500% digital magnification of specific areas. c Representative flow cytometry plots of C-PEP/NKX6-1 profile of non-reaggregated (day 23), reaggregated (day 23), and extended culture aggregates (day 33). d – f Quantification of C-PEP + /NKX6-1 + and C-PEP - /NKX6-1 - populations from NA- or WIKI4-treated reaggregated and extended cultures, respectively ( n = 4 for d , n = 5 for f , all replicates are independent biological replicates. Exact P -values are reported in the figure. Two-way ANOVA with Tukey’s multiple-comparison test. Error bar represents SEM). g Relative C-PEP Median Fluorescence Intensity (MFI) of NA- and WIKI4-treated β-like cells after extended culture ( n = 5 independent biological replicates. Exact P -values are reported in the figure. Two-way ANOVA with Tukey’s multiple-comparison test. Error bar represents SEM). h Static Glucose Stimulated Insulin Secretion assay was performed to calculate the stimulation index after extended culture of NA- and WIKI4-derived aggregates ( n = 5 independent biological replicates. Exact P -values are reported in the figure. One-way ANOVA with Dunnett’s multiple-comparison test. Error bar represents SEM).

Article Snippet: In addition, either 10 mM Nicotinamide (Sigma) or 9 μM WIKI4 (Tocris) were added in this stage, and the medium was replaced daily until d11.

Techniques: Immunofluorescence, Staining, Derivative Assay, Flow Cytometry, Comparison, Fluorescence

Journal: Nature Communications

Article Title: Tankyrase inhibition promotes endocrine commitment of hPSC-derived pancreatic progenitors

doi: 10.1038/s41467-024-53068-w

Figure Lengend Snippet: a Schematic of cell preparation and transplantation. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ). b Weekly fasting blood glucose measurements ( n = 8 for NA, from 7 independent differentiation cohorts; n = 9 for WIKI4, from 7 independent differentiation cohorts; n = 5 for non-diabetic control; n = 3 for STZ control. P -values listed: *STZ vs. non-diabetic—0.0077, 0.024 at week 13, 14; # NA vs. non-diabetic—0.0004, 0.0057 at week 15, 16; † NA vs WIKI4 – 0.0022, 0.0401 at week 15, 16. Two-way ANOVA with Tukey’s multiple-comparison test. Error bars represent SEM). c The percentage of NA- and WIKI4-transplanted mice that achieved euglycemia post-transplantation. Data was analyzed using chi-square test at every timepoint. d H&E staining of transplanted kidney grafts. The black box represents an area of magnification. Scale bar represents 500 µm. e Immunofluorescence staining of β-cell markers (C-PEP/NKX6-1/PDX1). White box areas were subjected to 500% digital magnification. Scale bar represents 100 µm. f IPGTT performed at 15 weeks post-transplantation ( n = 4 for NA; n = 5 for WIKI4; n = 4 for non-diabetic control; n = 5 for STZ control. All data points represent independent biological replicates. Exact P -values are reported in the figure. Two-way ANOVA with Tukey’s multiple-comparison test. Error bars represent SEM). g Glucose stimulated C-PEP secretion assay at 15 weeks post-transplantation ( n = 4 for NA; n = 6 for WIKI4. All data points represent independent biological replicates. Exact P -values are reported in the figure. One-tailed student’s t -test. Error bars represent SEM).

Article Snippet: In addition, either 10 mM Nicotinamide (Sigma) or 9 μM WIKI4 (Tocris) were added in this stage, and the medium was replaced daily until d11.

Techniques: Transplantation Assay, Control, Comparison, Staining, Immunofluorescence, One-tailed Test