Journal: Acta Pharmaceutica Sinica. B

Article Title: The FAP α -activated prodrug Z-GP-DAVLBH inhibits the growth and pulmonary metastasis of osteosarcoma cells by suppressing the AXL pathway

doi: 10.1016/j.apsb.2021.08.015

Figure Lengend Snippet: Z-GP-DAVLBH inhibits the AXL/AKT/GSK-3 β / β -catenin pathway in osteosarcoma cells. (A)–(B) Osteosarcoma cells were treated with vehicle (0.1% DMSO) or Z-GP-DAVLBH (6 nmol/L) for 48 h. (A) The levels of p-AXL (Tyr779), AXL, p-AKT (Ser473), AKT, p-GSK-3 β (Ser9), and GSK-3 β in SJSA-1 and 143B cells treated with Z-GP-DAVLBH were determined by Western blotting analysis. (B) Representative blots of β -catenin, β -catenin (Ser552), and non-phosphorylated (active) β -catenin (Ser33/37/Thr41) in osteosarcoma cells. (C) and (D) Osteosarcoma cells after transfection with either NC siRNA or AXL siRNA were treated vehicle (0.1% DMSO) or Z-GP-DAVLBH (6 nmol/L) for 48 h. (C) Western blotting analysis was conducted to evaluate the effect of Z-GP-DAVLBH on the AXL/AKT/GSK-3 β / β -catenin pathway components and (D) EMT-related markers in osteosarcoma cells. (E) and (F) Osteosarcoma cells were treated with Z-GP-DAVLBH (6 nmol/L) for 24 h. Transwell assays were conducted to evaluate the effect of Z-GP-DAVLBH on the (E) migration and (F) invasion capacities of SJSA-1 and 143B cells transfected with the indicated siRNAs. Scale bar: 200 μm. Data are presented as mean±SEM, n = 3; ∗ P <0.05, ∗∗ P <0.01, and ∗∗∗ P <0.001 vs. the indicated groups.

Article Snippet: AXL (catalog 8661), p-AKT (Ser473) (catalog 4060), AKT (catalog 4691), p-histone H3 (Ser10) (catalog 53348), CDC2 (catalog 9116), CDC25C (5H9) (catalog 4688), CHK2 (catalog 3440), cyclin B1 (catalog 12231), Ki67 (catalog 9449), GAPDH (catalog 5174), E-cadherin (catalog 3195), N-cadherin (catalog 4061), ZO-1 (catalog 13663), Slug (catalog 9585), PARP (catalog 9532), cleaved PARP (catalog 5625), caspase-3 (catalog 14220), cleaved caspase-3 (catalog 9664), caspase-9 (catalog 9508), cleaved caspase-9 (catalog 20750), GSK-3 β (catalog 9315), p-GSK-3 β (Ser9) (catalog 5558), β -catenin (catalog 8480), active β -catenin (catalog 8814), p- β -catenin (Ser552) (catalog 5651), and GAPDH (catalog 5174) were purchased from Cell Singling Technology (Danvers, MA, USA).

Techniques: Western Blot, Transfection, Migration

Journal: Acta Pharmaceutica Sinica. B

Article Title: The FAP α -activated prodrug Z-GP-DAVLBH inhibits the growth and pulmonary metastasis of osteosarcoma cells by suppressing the AXL pathway

doi: 10.1016/j.apsb.2021.08.015

Figure Lengend Snippet: Ectopic expression of AXL attenuates the effect of Z-GP-DAVLBH on the malignant behaviors of osteosarcoma cells. (A) and (B) Osteosarcoma cells were treated with vehicle (0.1% DMSO) or Z-GP-DAVLBH (6 nmol/L) for 48 h. (A) Western blotting analysis of p-AXL (Tyr779), AXL, p-AKT (Ser473), AKT, p-GSK-3 β (Ser9), GSK-3 β , β -catenin, p- β -catenin (Ser552), and non-phosphorylated (active) β -catenin (Ser33/37/Thr41), and (B) EMT-related markers in SJSA-1 and 143B cells. (C) Osteosarcoma cells were treated with Z-GP-DAVLBH (6 nmol/L) for 24 h. Representative image of migrated (upper) and invaded (lower) osteosarcoma cells. Scale bar: 200 μm. Quantification of the number of migrated and invaded cells is shown. (D) and (E) SJSA-1 and 143B were treated with vehicle (0.1% DMSO) or Z-GP-DAVLBH (50 nmol/L for SJSA-1 cells and 100 nmol/L for 143B cells) for 48 h. (D) An MTT assay was conducted to evaluate the effect of Z-GP-DAVLBH on osteosarcoma cell viability. (E) An Annexin V-FITC apoptosis detection kit was used to evaluate the effect of Z-GP-DAVLBH on osteosarcoma cell apoptosis. Data are presented as mean±SEM, n = 3; ∗ P <0.05, ∗∗ P <0.01, and ∗∗∗ P <0.001 vs. the indicated group.

Article Snippet: AXL (catalog 8661), p-AKT (Ser473) (catalog 4060), AKT (catalog 4691), p-histone H3 (Ser10) (catalog 53348), CDC2 (catalog 9116), CDC25C (5H9) (catalog 4688), CHK2 (catalog 3440), cyclin B1 (catalog 12231), Ki67 (catalog 9449), GAPDH (catalog 5174), E-cadherin (catalog 3195), N-cadherin (catalog 4061), ZO-1 (catalog 13663), Slug (catalog 9585), PARP (catalog 9532), cleaved PARP (catalog 5625), caspase-3 (catalog 14220), cleaved caspase-3 (catalog 9664), caspase-9 (catalog 9508), cleaved caspase-9 (catalog 20750), GSK-3 β (catalog 9315), p-GSK-3 β (Ser9) (catalog 5558), β -catenin (catalog 8480), active β -catenin (catalog 8814), p- β -catenin (Ser552) (catalog 5651), and GAPDH (catalog 5174) were purchased from Cell Singling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot, MTT Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: The FAP α -activated prodrug Z-GP-DAVLBH inhibits the growth and pulmonary metastasis of osteosarcoma cells by suppressing the AXL pathway

doi: 10.1016/j.apsb.2021.08.015

Figure Lengend Snippet: Z-GP-DAVLBH inhibits epithelial–mesenchymal transition and suppresses pulmonary metastasis of osteosarcoma cells in vivo . (A) BALB/c nude mice bearing 143B tumors were treated with vehicle (0.9% NaCl solution containing 1% DMSO) or Z-GP-DAVLBH (2 mg/kg, i.v.) every other day for 22 days. Lung tissues were collected and performed by hematoxylin-eosin (H&E) staining. Scale bar: 500 μm for low magnification images or 100 μm for high magnification images. (B) Quantification of the area and number of lung metastatic foci. (C) IHC staining of EMT-related markers in SJSA-1 and 143B tumor tissues. Quantification of IHC staining of EMT-related markers is shown. (D) IHC staining of p-AXL (Tyr779) and non-phosphorylated (active) β -catenin (Ser33/37/Thr41) in SJSA-1 and 143B tumor tissues. Scale bar: 50 μm. Quantification of IHC staining is shown. Data are presented as mean±SEM, n = 6; ∗ P <0.05, ∗∗ P <0.01, ∗∗∗ P <0.001 vs. the Vehicle group.

Article Snippet: AXL (catalog 8661), p-AKT (Ser473) (catalog 4060), AKT (catalog 4691), p-histone H3 (Ser10) (catalog 53348), CDC2 (catalog 9116), CDC25C (5H9) (catalog 4688), CHK2 (catalog 3440), cyclin B1 (catalog 12231), Ki67 (catalog 9449), GAPDH (catalog 5174), E-cadherin (catalog 3195), N-cadherin (catalog 4061), ZO-1 (catalog 13663), Slug (catalog 9585), PARP (catalog 9532), cleaved PARP (catalog 5625), caspase-3 (catalog 14220), cleaved caspase-3 (catalog 9664), caspase-9 (catalog 9508), cleaved caspase-9 (catalog 20750), GSK-3 β (catalog 9315), p-GSK-3 β (Ser9) (catalog 5558), β -catenin (catalog 8480), active β -catenin (catalog 8814), p- β -catenin (Ser552) (catalog 5651), and GAPDH (catalog 5174) were purchased from Cell Singling Technology (Danvers, MA, USA).

Techniques: In Vivo, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: Notch3-dependent β-catenin signaling mediates EGFR TKI drug persistence in EGFR mutant NSCLC

doi: 10.1038/s41467-018-05626-2

Figure Lengend Snippet: Identification and validation of regulators of EGFR TKI-mediated DPCs in EGFR mutant NSCLC cells. a qRT-PCR validation of PAI1 and MMP7 expression levels in HCC4006 cells. b HCC4006 cells expressing Tcf/Lef-GFP reporter were treated with DMSO or 0.1 μM erlotinib for 6 days and sorted by FACS for GFP fluorescence. c Histograms and quantification from triplicates was plotted and include a negative control with mutated Tcf/Lef consensus binding sites. The mutant reporter has no activity after erlotinib treatment. DMSO-treated cells were used as a base line control. d HCC827 (left) and HCC4006 (right) were stably infected with shRNAs targeting non-targeting control (shNTC) or β-catenin (shRNAs #3 and #5) were treated with DMSO or erlotinib and subjected to ALDH assay. For ( a ) and ( c ) error bars represent SD from biological triplicates. For ( d ) error bars represent SD from technical triplicates

Article Snippet: Following antibodies were used in this study: β-catenin (1:1000, 8480 and 9562) phospho-β-catenin (1:1000 dilution, S552), Non-phospho (active) β-catenin (1:1000, 8814 and 4270), Notch3 (1:1000, D11B8), Notch3 (1:1000, 8G5), β-actin total (1:1000), Oct4 (1:1000, 2750), Nanog (1:1000, 4903) were purchased from Cell Signaling Technologies.

Techniques: Biomarker Discovery, Mutagenesis, Quantitative RT-PCR, Expressing, Fluorescence, Negative Control, Binding Assay, Activity Assay, Control, Stable Transfection, Infection

Journal: Nature Communications

Article Title: Notch3-dependent β-catenin signaling mediates EGFR TKI drug persistence in EGFR mutant NSCLC

doi: 10.1038/s41467-018-05626-2

Figure Lengend Snippet: Notch3 associates with β-catenin in an EGFR TKI-dependent manner. a HCC4006 and b HCC827 cells were treated with DMSO or 0.1 μM erlotinib for 1 or 6 days and cell lysates were immunoprecipitated (IP) with an antibody recognizing Notch3 and blotted (WB) with antibodies recognizing β-catenin or Notch3. A reciprocal IP with an anti-β-catenin antibody was performed and blotted with anti-Notch3 and anti-β-catenin antibodies. c HCC827 cells were treated with erlotinib or GSI alone or in combination and cell lysates were immunoprecipitated with Notch3 and blotted with β-catenin antibody. Cell lysates were also analyzed for Notch3, β-catenin, and β-tubulin. d HCC4006 and e HCC827 cells were treated for 6 days with vehicle (DMSO) or 0.1 μM erlotinib and stained with DAPI and probed with anti-Notch3 and anti-β-catenin antibodies. Images were overlayed to show co-localization. For ( d ) and ( e ) scale bar is 30 μm. Error bars represent SD from technical triplicates

Article Snippet: Following antibodies were used in this study: β-catenin (1:1000, 8480 and 9562) phospho-β-catenin (1:1000 dilution, S552), Non-phospho (active) β-catenin (1:1000, 8814 and 4270), Notch3 (1:1000, D11B8), Notch3 (1:1000, 8G5), β-actin total (1:1000), Oct4 (1:1000, 2750), Nanog (1:1000, 4903) were purchased from Cell Signaling Technologies.

Techniques: Immunoprecipitation, Staining

Journal: Nature Communications

Article Title: Notch3-dependent β-catenin signaling mediates EGFR TKI drug persistence in EGFR mutant NSCLC

doi: 10.1038/s41467-018-05626-2

Figure Lengend Snippet: EGFR TKI treatment increase β-catenin protein stability. a Cells were treated as described in Fig. 3 and were analyzed for the expression of total and non-phospho (active) β-catenin by WB using an antibody that recognizes the active form of β-catenin. b Cells were treated as described in Fig. 4 and prior to harvesting, cells were further treated with cycloheximide for the indicated times. Cell lysates were analyzed for non-phospho (active) β-catenin by western blot. c Western blots were quantitated and data plotted (trend line) as percentage of non-phospho β-catenin remaining as a function of cycloheximide treatment. d HCC827 cells were treated with erlotinib or DMSO and subjected to cytoplasmic and nuclear fractionation. Equal amounts of total protein from each fraction was analyzed for β-catenin (active, inactive, and total), Notch3, β-tubulin, and HDAC2. Error bars represent SD from biological triplicates

Article Snippet: Following antibodies were used in this study: β-catenin (1:1000, 8480 and 9562) phospho-β-catenin (1:1000 dilution, S552), Non-phospho (active) β-catenin (1:1000, 8814 and 4270), Notch3 (1:1000, D11B8), Notch3 (1:1000, 8G5), β-actin total (1:1000), Oct4 (1:1000, 2750), Nanog (1:1000, 4903) were purchased from Cell Signaling Technologies.

Techniques: Expressing, Western Blot, Fractionation

Journal: Nature Communications

Article Title: Notch3-dependent β-catenin signaling mediates EGFR TKI drug persistence in EGFR mutant NSCLC

doi: 10.1038/s41467-018-05626-2

Figure Lengend Snippet: EGFR TKI treatment activates β-catenin signaling, which is responsible for the maintenance of DPCs. a HCC4006-Tcf/Lef-GFP reporter cells were treated with 0.1 μM erlotinib for 6 days and sorted for GFP expression by FACS into GFP high (high β-catenin activity) and GFP low (low β-catenin activity) cell populations, which were subsequently analyzed for Notch3, β-catenin, and stem cell markers by WB. b GFP high and GFP low cell populations were subjected to sphere formation assay. Quantification of total number of pulmospheres from both groups is shown. c EGFR TKI persistent pulmospheres are sensitive to β-catenin pathway inhibitors. HCC4006 cells were treated with erlotinib and TCF-GFP reporter positive cells were subjected to pulmosphere assay in the presence of DMSO or β-catenin inhibitors, XAV939, ICG-001. Quantification of total number of pulmospheres from all experimental conditions. d HCC4006-Tcf/Lef-GFP reporter cells were treated with 0.1 μM erlotinib for 6 days and cells with high and low GFP reporter activities were isolated by flow sorting. A limiting dilution assay was performed by injecting NSG mice with the number of GFP high and GFP low cells indicated. After 8 weeks the tumors were excised and tumor volumes and weights were measured. Solid-filled circles indicate individual tumor weights and open circles indicate no tumor grew at the site of injection. Red circles represent the group of tumors that were obtained by injection of GFP high HCC4006 cells. Green circles represent group of tumors that were obtained by injection of GFP low HCC4006 cells. Linear model was used to compare GFP high and GFP low groups within each group of cell dilution level. P values were adjusted for multiple comparisons by Holm’s procedure. For ( b ) and ( c ) error bars represent SD from biological triplicates

Article Snippet: Following antibodies were used in this study: β-catenin (1:1000, 8480 and 9562) phospho-β-catenin (1:1000 dilution, S552), Non-phospho (active) β-catenin (1:1000, 8814 and 4270), Notch3 (1:1000, D11B8), Notch3 (1:1000, 8G5), β-actin total (1:1000), Oct4 (1:1000, 2750), Nanog (1:1000, 4903) were purchased from Cell Signaling Technologies.

Techniques: Expressing, Activity Assay, Tube Formation Assay, Isolation, Limiting Dilution Assay, Injection

Journal: Nature Communications

Article Title: Notch3-dependent β-catenin signaling mediates EGFR TKI drug persistence in EGFR mutant NSCLC

doi: 10.1038/s41467-018-05626-2

Figure Lengend Snippet: In vivo demonstration of EGFR TKI-induced drug persistent cells with EMT phenotype. a Mice with HCC827 (top) and HCC4006 (bottom) tumor xenografts were treated with methylcellulose (control) or erlotinib for 21 days. After the drug treatments tumors were harvested and subjected to IHC analysis for the putative stem cell marker, ALDH1A. b Mice with HCC827 tumor xenografts were treated with methylcellulose (control) or erlotinib for 21 days. Tumors were harvested and disrupted into single cell preparations, which were then subjected to ALDH assay. c – e EGFR TKI treatment increases Vimentin expression and decreases E-cadherin expression in vivo. f EGFR TKI treatment increases Notch3 expression in vivo. g EGFR TKI treatment increases the non-phospho (activated) β-catenin levels in vivo. h EGFR TKI treatment increases β-catenin levels in vivo. Error bars represent SD from techincal triplicates

Article Snippet: Following antibodies were used in this study: β-catenin (1:1000, 8480 and 9562) phospho-β-catenin (1:1000 dilution, S552), Non-phospho (active) β-catenin (1:1000, 8814 and 4270), Notch3 (1:1000, D11B8), Notch3 (1:1000, 8G5), β-actin total (1:1000), Oct4 (1:1000, 2750), Nanog (1:1000, 4903) were purchased from Cell Signaling Technologies.

Techniques: In Vivo, Control, Marker, Expressing

Journal: Nature Communications

Article Title: Notch3-dependent β-catenin signaling mediates EGFR TKI drug persistence in EGFR mutant NSCLC

doi: 10.1038/s41467-018-05626-2

Figure Lengend Snippet: In vivo demonstration of EGFR TKI therapy-induced co-localization of Notch3 and β-catenin. a , b EGFR TKI treatment increases co-localization of Notch3 and β-catenin in vivo. Co-localization experiment was performed as in Fig. . For ( a ) and ( b ) scale bar is 50 μm

Article Snippet: Following antibodies were used in this study: β-catenin (1:1000, 8480 and 9562) phospho-β-catenin (1:1000 dilution, S552), Non-phospho (active) β-catenin (1:1000, 8814 and 4270), Notch3 (1:1000, D11B8), Notch3 (1:1000, 8G5), β-actin total (1:1000), Oct4 (1:1000, 2750), Nanog (1:1000, 4903) were purchased from Cell Signaling Technologies.

Techniques: In Vivo

Journal: Nature Communications

Article Title: Notch3-dependent β-catenin signaling mediates EGFR TKI drug persistence in EGFR mutant NSCLC

doi: 10.1038/s41467-018-05626-2

Figure Lengend Snippet: Increased Notch3 and β-catenin in patient biopsy samples at the time of acquired resistance to erlotinib. a , b Pre and post EGFR TKI therapy tumor biopsies were collected and IHC analysis of Notch3 ( a ) and β-catenin ( b ) was performed

Article Snippet: Following antibodies were used in this study: β-catenin (1:1000, 8480 and 9562) phospho-β-catenin (1:1000 dilution, S552), Non-phospho (active) β-catenin (1:1000, 8814 and 4270), Notch3 (1:1000, D11B8), Notch3 (1:1000, 8G5), β-actin total (1:1000), Oct4 (1:1000, 2750), Nanog (1:1000, 4903) were purchased from Cell Signaling Technologies.

Techniques:

Journal: Nature Communications

Article Title: Notch3-dependent β-catenin signaling mediates EGFR TKI drug persistence in EGFR mutant NSCLC

doi: 10.1038/s41467-018-05626-2

Figure Lengend Snippet: EGFR TKI treatment in combination with β-catenin inhibition strongly attenuates tumor onset in EGFR mutant NSCLC in vivo. a Human HCC4006 subcutaneous xenografts treated with erlotinib (50 mg/kg/day) or ICG-001 (150 mg/kg/day) alone or in combination for 5 days in a week for 9 weeks. b Overall survival analysis of HCC4006 xenograft mice that were treated with erlotinib or ICG-001 alone or in combination ( P = 0.0001). c Human HCC827 subcutaneous xenografts were grown in mice that were treated with erlotinib (50 mg/kg/day) alone or in combination with ICG-001 (150 mg/kg/day) for 5 days a week for 3 weeks. After treatments were stopped, tumor recurrence was compared between the two groups. d Overall survival analysis of HCC827 xenograft mice that were treated with erlotinib alone or in combination with ICG-001 ( P = 0.0001) for 21 days. Overall survival was determined after treatments were stopped. For ( a ) error bars represent SEM

Article Snippet: Following antibodies were used in this study: β-catenin (1:1000, 8480 and 9562) phospho-β-catenin (1:1000 dilution, S552), Non-phospho (active) β-catenin (1:1000, 8814 and 4270), Notch3 (1:1000, D11B8), Notch3 (1:1000, 8G5), β-actin total (1:1000), Oct4 (1:1000, 2750), Nanog (1:1000, 4903) were purchased from Cell Signaling Technologies.

Techniques: Inhibition, Mutagenesis, In Vivo

Journal: Oncotarget

Article Title: Acetyl-CoA carboxylase inhibitors attenuate WNT and Hedgehog signaling and suppress pancreatic tumor growth

doi: 10.18632/oncotarget.12650

Figure Lengend Snippet: A . Expression of β-catenin target genes in Capan-2 cells after treatment with DMSO or 10 μM BAY ACC002 for 72 h. RNA was extracted and the mRNA expression level of β-catenin target genes was determined by qRT-PCR. B . Capan-2 cells were treated 200ng recombinant human WNT3A, and grown in the presence of DMSO or 10 μM BAY ACC002 for 72 h. RNA was extracted and the mRNA expression level of AXIN2 was determined by qRT-PCR. C . HEK293-TOP cells were stimulated with 200ng recombinant human WNT3A, and grown in the presence of DMSO or 10 μM BAY ACC002 for 72 h. RNA was extracted and the mRNA expression level of AXIN2 was determined by qRT-PCR. D . Expression of HH target genes in Capan-2 cells, treated with BAY ACC002. Cells were treated as in (A) and mRNA expression levels were determined by qRT-PCR. E . Expression of AXIN2 and GLI1 in Capan-2 cells treated with DMSO or 10 μM BAY ACC002 for 96 h. The cells were then lysed and protein levels were detected by Western blot. GAPDH levels were monitored as a control. F . Growth curves of DanG cells, treated with varying concentrations of BAY ACC002 (arrow indicates time of drug addition). Cell growth was measured over time using the xCELLigence system. Experiments were performed in triplicate. G ., H . and I . Capan-2, BxPC-3 and Panc-1 cells were treated with BAY ACC002, and cell growth over time was determined as in (G). Each bar in (A) - (D) represents mean±SEM ( n = 3-4 for (A), n = 2-3 for (B)-(D), *, p < 0.05, **, p < 0.01, ***, p < 0.001, Student's t test).

Article Snippet: SHH (C9C5) Rabbit monoclonal antibody (2207), non-phospho β-catenin (Ser33/37/Thr41) (D13A1) rabbit monoclonal antibody (8814), and AXIN2 (76G6) rabbit monoclonal antibody (2151) were purchased from Cell Signaling (Danvers, MA, US).

Techniques: Expressing, Quantitative RT-PCR, Recombinant, Western Blot, Control

Journal: Oncotarget

Article Title: Acetyl-CoA carboxylase inhibitors attenuate WNT and Hedgehog signaling and suppress pancreatic tumor growth

doi: 10.18632/oncotarget.12650

Figure Lengend Snippet: A . AXIN2 and GLI1 expression in Capan-2 pancreatic cancer tumors, treated with vehicle or 30 mg/kg/day BAY ACC002 for 7 days. RNA was extracted from the tumors and expression of AXIN2 and GLI1 was determined by qRT-PCR. Each bar represents mean±SEM, with individual animals represented by dots ( n = 4, *, p < 0.05). B . IHC analysis of β-catenin expression in representative pancreatic cancer sections from mice, carrying Capan-2 or PAXF 2046 tumors, treated with vehicle or BAY ACC002 (35 mg/kg/day on days 1-5, and then 30 mg/kg/day on a 3 days ON/1 day OFF schedule until day 35 for the Capan-2 model; and 30 mg/kg/day for 29 days for the PAXF 2046 model). Arrows point to nuclear β-catenin expression. C . IHC analysis of GLI1 expression in representative pancreatic cancer sections from Capan-2 and PAXF 2046 tumors described in (B). Arrows point to GLI1 positive cells. D . H&E, Alcian blue, and E-cadherin IHC staining of representative sections from the PAXF 2046 tumors, described in (B). Scale bar for (B), (C), and the third set of (D) is 50 μm, and for the first two sets of (D), 100 μm.

Article Snippet: SHH (C9C5) Rabbit monoclonal antibody (2207), non-phospho β-catenin (Ser33/37/Thr41) (D13A1) rabbit monoclonal antibody (8814), and AXIN2 (76G6) rabbit monoclonal antibody (2151) were purchased from Cell Signaling (Danvers, MA, US).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry

Journal: EMBO Reports

Article Title: PAWS 1 controls Wnt signalling through association with casein kinase 1α

doi: 10.15252/embr.201744807

Figure Lengend Snippet: A–C Ectopic axis induction in Xenopus embryos following xPAWS1 mRNA injection. Xenopus embryos were injected at the one‐cell stage with 500 pg of either HA_xPAWS1 (B) or xPAWS_HA mRNA(C). A variety of dorsalised phenotypes were observed including enlarged cement glands (asterisk), partial (arrowhead) and complete secondary axis (arrow). Scale bars are 2 mm. D–I Dissociated animal caps injected with 50 pg of β‐catenin_GFP mRNA were imaged over 3 h following treatment with the GSK3β inhibitor CHIR99021. Maximum intensity projection of β‐catenin_GFP‐injected cells before (D) and 3 h (E) after CHIR99021 treatment, demonstrating stabilisation and nuclear localisation of β‐catenin_GFP in the absence of xPAWS1. Single z‐section of a β‐catenin_GFP expressing cell and corresponding fluorescence intensity profile across the nucleus before (F and G) and following 3 h of CHIR99021 treatment (H and I). Cells were imaged using a Zeiss LSM710 microscope, and intensity measurements from a single z‐section were taken using Zen Black software. Scale bars are 20 μm. J Expression level of Myc‐tagged(MT)xPAWS1 and MTxPAWS1 mutants at stage 10. Extracts from embryos injected with 250 pg of MTxPAWS1 and MTxPAWS1 mutants were immunoblotted with antibodies against Myc‐tag (green) and α‐tubulin (red). The image was captured with a Li‐Cor Odyssey scanner using Image Studio software (Li‐Cor). K Schematic illustration of the strategy employed to generate PAWS1‐GFP knock‐ins in U2OS cells. A pair of guide RNAs which recognise a genomic sequence upstream of the stop codon of PAWS1 gene was used in combination with a donor vector which inserts GFP in frame with the c‐terminus of PAWS1. L Cell extracts from PAWS1 GFP/GFP cells compared with the PAWS1 −/− , confirmed that the gene in the reverse DNA strand of PAWS1, SLC5A10 is not disturbed. M Mass fingerprinting analysis of PAWS1‐GFP interactors from PAWS1 GFP/GFP ‐knock‐in U2OS cells compared with PAWS1 −/− U2OS cells (from Fig A) identified CK1α as a major interactor. The table shows total spectral counts for PAWS1 and CK1α tryptic peptides identified in anti‐GFP IPs. N The highlighted tryptic peptides identified by mass spectrometry on CK1α indicate the overall protein coverage. The included image was obtained using Scaffold V4.3 analysis of the LC‐MS/MS data. O Stable U2OS Flp‐In Trex cells were subjected to 20 ng/ml doxycycline for inducing PAWS1‐GFP expression or GFP expression alone for 24 h. Wnt3A or control medium was added to the cells for 6 h before lysis. 20 mg of cell extract was subjected to GFP‐trap IP. Input (20 μg protein), 5% of the pull down and flow‐through extract (20 μg protein) were subjected to SDS–PAGE followed by Western blot analysis with the indicated antibodies. Source data are available online for this figure.

Article Snippet: Antibodies included PAWS1 (DU33022), SMAD1 (DU19291), CK1α (Bethyl Laboratories #Α301‐991A‐M), CK1ε (CST #12448), HA (Sigma HA‐7 #H3663), FLAG‐HRP (Sigma #A8592), GFP (Abcam, #ab6556), Myc tag (Sigma 9E10 #M5546), α‐tubulin (ThermoScientific #MA1‐80189, Sigma #T5168), AXIN2 (CST #76G6), AXIN1 (CST #2074), β‐catenin (CST #D10A8; Santa Cruz Biotechnology #H‐102), GSK3‐α/β (Santa Cruz #sc‐7291), GSK3β (CST #9315S), SLC5A10 (Abcam, #ab167156), active β‐catenin (Millipore #05‐665, CST D13A1 #8814), LRP6 (CST #C47E12), GAPDH (CST #D16H11), Lamin A/C (CST #2032) and HA‐HRP (Roche 2013819001).

Techniques: Injection, Expressing, Fluorescence, Microscopy, Software, Sequencing, Plasmid Preparation, Knock-In, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Lysis, SDS Page, Western Blot

Journal: EMBO Reports

Article Title: PAWS 1 controls Wnt signalling through association with casein kinase 1α

doi: 10.15252/embr.201744807

Figure Lengend Snippet: A xPAWS1 stabilises exogenous β‐catenin in whole embryos. Xenopus embryos were injected into the animal pole at the one‐cell stage with mRNAs encoding either 50 pg of β‐cat_GFP or with 50 pg of β‐cat_GFP and 250 pg xPAWS1_mCherryHA mRNAs. At stage 10, embryo extracts were immunoblotted with the indicated antibodies. Top panel: anti‐HA and anti‐GFP antibodies; middle panel: anti‐active β‐catenin (single white asterisk is active β‐catenin_GFP; double white asterisk is endogenous active β‐catenin) and anti‐α‐tubulin antibodies; bottom panel: anti‐GFP and anti‐α‐tubulin antibodies. B xPAWS1 stabilises endogenous β‐catenin in naïve animal caps. Embryos were injected at the one‐cell stage with 250 pg of xPAWS1_mCherryHA mRNA. At stage 8.5, animal caps were collected from injected and uninjected embryos and cultured until control embryos reached stage 10. Extracts were immunoblotted with the indicated antibodies. Top panel: anti‐active β‐catenin and anti‐α‐tubulin antibodies; bottom panel: anti‐HA, anti‐β‐catenin and anti‐α‐tubulin antibodies. C, D Quantification of (A and B), respectively. In (C), β‐catenin_GPF or active β‐catenin_GFP bands were normalised to α‐tubulin and then expressed as a fold change relative to the expression of β‐catenin_GFP and active β‐catenin_GFP respectively from embryos injected with β‐catenin_GFP alone. In (D), endogenous β‐catenin and active β‐catenin were normalised to α‐tubulin and then expressed as a fold change relative to the expression of β‐catenin and active β‐catenin (respectively) from uninjected cells. E Nuclear translocation of β‐catenin_GFP. Dissociated animal cap cells injected with either 50 pg of β‐catenin_GFP or with 50 pg β‐catenin_GFP and 250 pg xPAWS1_mCherryHA mRNAs were plated on coverslips and imaged by confocal microscopy. Only β‐catenin_GFP cells co‐injected with xPAWS1_mCherryHA mRNA accumulated robust levels of β‐catenin in the nucleus. Scale bars are 20 μm. F xPAWS1 induces expression of Siamois and Chordin transcripts in animal caps. Embryos were injected at the one‐cell stage with 250 pg of MT_xPAWS1 mRNA, and then at stage 8.5, animal caps were collected from injected and uninjected embryos and assessed for Chordin and Siamois expression by qPCR ( n = 3; error bars represent ± SD, ** P = 0.001, *** P = 0.0001; ordinary one‐way ANOVA with multiple comparisons, uninjected as control column). G–I The DUF1669 domain (G, in red) is necessary but not sufficient to induce a secondary axis (H) and activate Siamois expression (I) while the BMPR1 phosphorylation sites S 610 , S 613 and S 614 (yellow) are dispensable ( n = 3; error bars represent ± SD). 250 pg of MT_xPAWS1 mRNAs encoding N‐ and C‐terminal truncation fragments were injected into one ventral blastomere at the four‐cell stage. Axis induction was assessed at stage 28. In (I), embryos were injected at the one‐cell stage with 250 pg of MT_xPAWS1 mRNAs encoding N‐ and C‐terminal mutants, and then at stage 8.5, animal caps were collected and assessed for Siamois expression by qPCR. J HEK293 and U2OS cells were transfected with PAWS1 cDNA, or empty vector as a control and TOPFlash luciferase activity was measured after treatment with either conditioned medium (L‐CM), Wnt3A‐conditioned medium (L3‐CM) or 20 mM LiCl for 12 h. Data are normalised to Renilla internal control ( n = 4; error bars represent ± SEM). Source data are available online for this figure.

Article Snippet: Antibodies included PAWS1 (DU33022), SMAD1 (DU19291), CK1α (Bethyl Laboratories #Α301‐991A‐M), CK1ε (CST #12448), HA (Sigma HA‐7 #H3663), FLAG‐HRP (Sigma #A8592), GFP (Abcam, #ab6556), Myc tag (Sigma 9E10 #M5546), α‐tubulin (ThermoScientific #MA1‐80189, Sigma #T5168), AXIN2 (CST #76G6), AXIN1 (CST #2074), β‐catenin (CST #D10A8; Santa Cruz Biotechnology #H‐102), GSK3‐α/β (Santa Cruz #sc‐7291), GSK3β (CST #9315S), SLC5A10 (Abcam, #ab167156), active β‐catenin (Millipore #05‐665, CST D13A1 #8814), LRP6 (CST #C47E12), GAPDH (CST #D16H11), Lamin A/C (CST #2032) and HA‐HRP (Roche 2013819001).

Techniques: Injection, Cell Culture, Expressing, Translocation Assay, Confocal Microscopy, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: EMBO Reports

Article Title: PAWS 1 controls Wnt signalling through association with casein kinase 1α

doi: 10.15252/embr.201744807

Figure Lengend Snippet: U2OS wild‐type (WT) and PAWS1 −/− (KO) cells were treated with control‐conditioned medium or Wnt3A‐conditioned medium, and the extracts (0.5 mg protein) were subjected to immunoprecipitation using antibodies against the endogenous CK1α, AXIN1 and β‐catenin antibodies or anti‐rabbit pre‐immune IgG as a control (10 μg antibodies coupled to 10 μl packed protein‐G sepharose beads). IPs were resolved by SDS–PAGE and immunoblotted with the indicated antibodies. Source data are available online for this figure.

Article Snippet: Antibodies included PAWS1 (DU33022), SMAD1 (DU19291), CK1α (Bethyl Laboratories #Α301‐991A‐M), CK1ε (CST #12448), HA (Sigma HA‐7 #H3663), FLAG‐HRP (Sigma #A8592), GFP (Abcam, #ab6556), Myc tag (Sigma 9E10 #M5546), α‐tubulin (ThermoScientific #MA1‐80189, Sigma #T5168), AXIN2 (CST #76G6), AXIN1 (CST #2074), β‐catenin (CST #D10A8; Santa Cruz Biotechnology #H‐102), GSK3‐α/β (Santa Cruz #sc‐7291), GSK3β (CST #9315S), SLC5A10 (Abcam, #ab167156), active β‐catenin (Millipore #05‐665, CST D13A1 #8814), LRP6 (CST #C47E12), GAPDH (CST #D16H11), Lamin A/C (CST #2032) and HA‐HRP (Roche 2013819001).

Techniques: Immunoprecipitation, SDS Page

Journal: EMBO Reports

Article Title: PAWS 1 controls Wnt signalling through association with casein kinase 1α

doi: 10.15252/embr.201744807

Figure Lengend Snippet: U2OS wild‐type and PAWS1 −/− cells were exposed to either Wnt3A or control medium for the indicated time points. Cell extracts were subjected to SDS–PAGE followed by Western blot analysis with the indicated antibodies. U2OS wild‐type (WT), PAWS1 −/− (KO), PAWS1 WT (+WT) and PAWS1 F296A (F/A) rescue cells were exposed to either Wnt3A or control medium for 3 h followed by separation and preparation of cytoplasmic and nuclear fractions. The extracts were subjected to SDS–PAGE followed by Western blot analysis with the indicated antibodies. The lower panel represents the fold changes in active β‐catenin intensities in each fraction relative to those seen in the cytoplasmic fraction of control WT U2OS cells. The intensities of the active β‐catenin bands in each fraction were quantified by using the ImageJ software. Source data are available online for this figure.

Article Snippet: Antibodies included PAWS1 (DU33022), SMAD1 (DU19291), CK1α (Bethyl Laboratories #Α301‐991A‐M), CK1ε (CST #12448), HA (Sigma HA‐7 #H3663), FLAG‐HRP (Sigma #A8592), GFP (Abcam, #ab6556), Myc tag (Sigma 9E10 #M5546), α‐tubulin (ThermoScientific #MA1‐80189, Sigma #T5168), AXIN2 (CST #76G6), AXIN1 (CST #2074), β‐catenin (CST #D10A8; Santa Cruz Biotechnology #H‐102), GSK3‐α/β (Santa Cruz #sc‐7291), GSK3β (CST #9315S), SLC5A10 (Abcam, #ab167156), active β‐catenin (Millipore #05‐665, CST D13A1 #8814), LRP6 (CST #C47E12), GAPDH (CST #D16H11), Lamin A/C (CST #2032) and HA‐HRP (Roche 2013819001).

Techniques: SDS Page, Western Blot, Software

Journal: Journal of translational medicine

Article Title: PSAT1 positively regulates the osteogenic lineage differentiation of periodontal ligament stem cells through the ATF4/PSAT1/Akt/GSK3β/β-catenin axis.

doi: 10.1186/s12967-022-03775-z

Figure Lengend Snippet: Fig. 6 PSAT1 regulated Akt/GSK3β/β-catenin signaling pathway in PDLSCs. A The phosphorylation level of Erk, Akt, and GSK3β, and the protein level of active-β-catenin were analyzed in PDLSCs with PSAT1 overexpressed. B The phosphorylation level of Erk, Akt, and GSK3β, and the protein level of active-β-catenin were analyzed in PDLSCs with PSAT1 knocked down. C Western Blot analysis of β-Catenin protein in nucleus and cytoplasm after PSAT1 was overexpressed. D Western Blot analysis of β-Catenin protein in nucleus and cytoplasm after PSAT1 was knocked down. OEPSAT1: PDLSCs with PSAT1 overexpressed. OENC: control PDLSCs for OEPSAT1 PDLSCs. shPSAT1: PDLSCs with PSAT1 knocked down. shNC: control PDLSCs for shPSAT1 PDLSCs. *p < 0.05; **p < 0.01

Article Snippet: Primary antibodies included the following: PSAT1 (ab96136, Abcam, Cambridge, MA, USA); ALP (ab108337, Abcam); COL1A1 (#84336, CST, Danvers, MA, USA); RUNX2 (ab23981, Abcam); Akt (pan) (#4691, CST); p-Akt (Ser473) (p-Akt) (#4060, CST); p-GSK3β (#9323, CST); GSK-3β (#12456, CST); β-Catenin (#8480, CST); Nonphospho (Active) β-Catenin (#8814, CST); ATF4 (#11815, CST); β-Actin (sc-517582, CST); HistoneH3 (17168-1-AP, Proteintech, Chicago, IL, USA); and GAPDH (HRP-60,004, Proteintech).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Journal of translational medicine

Article Title: PSAT1 positively regulates the osteogenic lineage differentiation of periodontal ligament stem cells through the ATF4/PSAT1/Akt/GSK3β/β-catenin axis.

doi: 10.1186/s12967-022-03775-z

Figure Lengend Snippet: Fig. 7 LY294002 reversed the effects of overexpressing PSAT1 on the osteogenic differentiation of PDLSCs. A The phosphorylation level of Akt and GSK3β, and the protein level of active-β-catenin were analyzed in PDLSCs. B The quantitative analysis of ALP activity in PDLSCs after osteogenic induction. C ALP staining of PDLSCs after osteogenic induction for 7 days. D Alizarin red staining of PDLSCs after osteogenic induction for 21 days. E Quantitative analysis of mineralized matrix after osteogenic induction for 21 days. F The protein levels of COL1A1, ALP and RUNX2 in PDLSCs after osteogenic induction for 7 days. G The mRNA levels of COL1A1, ALP and RUNX2 in PDLSCs after osteogenic induction for 7 days. OEPSAT1: PDLSCs with PSAT1 overexpressed. OENC: control PDLSCs. *p < 0.05; **p < 0.01

Article Snippet: Primary antibodies included the following: PSAT1 (ab96136, Abcam, Cambridge, MA, USA); ALP (ab108337, Abcam); COL1A1 (#84336, CST, Danvers, MA, USA); RUNX2 (ab23981, Abcam); Akt (pan) (#4691, CST); p-Akt (Ser473) (p-Akt) (#4060, CST); p-GSK3β (#9323, CST); GSK-3β (#12456, CST); β-Catenin (#8480, CST); Nonphospho (Active) β-Catenin (#8814, CST); ATF4 (#11815, CST); β-Actin (sc-517582, CST); HistoneH3 (17168-1-AP, Proteintech, Chicago, IL, USA); and GAPDH (HRP-60,004, Proteintech).

Techniques: Phospho-proteomics, Activity Assay, Staining, Control

Journal: Journal of translational medicine

Article Title: PSAT1 positively regulates the osteogenic lineage differentiation of periodontal ligament stem cells through the ATF4/PSAT1/Akt/GSK3β/β-catenin axis.

doi: 10.1186/s12967-022-03775-z

Figure Lengend Snippet: Fig. 8 SC79 reversed the effects of knocking down PSAT1 on the osteogenic differentiation of PDLSCs. A The phosphorylation level of Akt and GSK3β, and the protein level of active-β-catenin were analyzed in PDLSCs. B The quantitative analysis of ALP activity in PDLSCs after osteogenic induction. C ALP staining of PDLSCs after osteogenic induction for 7 days. D Alizarin red staining of PDLSCs after osteogenic induction for 21 days. E Quantitative analysis of mineralized matrix after osteogenic induction for 21 days. F The protein levels of COL1A1, ALP and RUNX2 in PDLSCs after osteogenic induction for 7 days. G The mRNA levels of COL1A1, ALP and RUNX2 in PDLSCs after osteogenic induction for 7 days. shPSAT1: PDLSCs with PSAT1 knocked down. shNC: control PDLSCs. *p < 0.05; **p < 0.01

Article Snippet: Primary antibodies included the following: PSAT1 (ab96136, Abcam, Cambridge, MA, USA); ALP (ab108337, Abcam); COL1A1 (#84336, CST, Danvers, MA, USA); RUNX2 (ab23981, Abcam); Akt (pan) (#4691, CST); p-Akt (Ser473) (p-Akt) (#4060, CST); p-GSK3β (#9323, CST); GSK-3β (#12456, CST); β-Catenin (#8480, CST); Nonphospho (Active) β-Catenin (#8814, CST); ATF4 (#11815, CST); β-Actin (sc-517582, CST); HistoneH3 (17168-1-AP, Proteintech, Chicago, IL, USA); and GAPDH (HRP-60,004, Proteintech).

Techniques: Phospho-proteomics, Activity Assay, Staining, Control

Journal: Journal of translational medicine

Article Title: PSAT1 positively regulates the osteogenic lineage differentiation of periodontal ligament stem cells through the ATF4/PSAT1/Akt/GSK3β/β-catenin axis.

doi: 10.1186/s12967-022-03775-z

Figure Lengend Snippet: Fig. 10 Schematic diagram. PSAT1 could regulate the osteogenic differentiation of PDLSCs through Akt/GSK3β/β-catenin signaling pathway, and the transcription of PSAT1 is modulated by transcription factor ATF4

Article Snippet: Primary antibodies included the following: PSAT1 (ab96136, Abcam, Cambridge, MA, USA); ALP (ab108337, Abcam); COL1A1 (#84336, CST, Danvers, MA, USA); RUNX2 (ab23981, Abcam); Akt (pan) (#4691, CST); p-Akt (Ser473) (p-Akt) (#4060, CST); p-GSK3β (#9323, CST); GSK-3β (#12456, CST); β-Catenin (#8480, CST); Nonphospho (Active) β-Catenin (#8814, CST); ATF4 (#11815, CST); β-Actin (sc-517582, CST); HistoneH3 (17168-1-AP, Proteintech, Chicago, IL, USA); and GAPDH (HRP-60,004, Proteintech).

Techniques: