Journal: Cellular and Molecular Life Sciences

Article Title: FBXW2 inhibits prostate cancer proliferation and metastasis via promoting EGFR ubiquitylation and degradation

doi: 10.1007/s00018-022-04320-3

Figure Lengend Snippet: FBXW2 is down-regulated in highly-metastasis PCa cells and tissues, and enhanced FBXW2 expression inhibits cancer growth and metastasis. a Both mRNA and protein levels of FBXW2 were significantly decreased in highly-progressive PCa tissues. We collected 9 clinical samples and divided them into three groups: prostatic hyperplasia (Nor), Non-metastatic PCa (PCa, Gleason score ≤ 7) and metastatic prostate cancer (M-PCa, Gleason score > 7). Then, the mRNA and protein levels of FBXW2 in clinical samples were analyzed by qPCR analysis and immunoblotting (IB), respectively ( *p < 0.05, **p < 0.01, ***p < 0.001; n = 3; one-way ANOVA). b FBXW2 protein levels in prostate cell lines were analyzed by IB analysis (ns, no significance, **p < 0.01, ***p < 0.001; n = 3; one-way ANOVA). c – e Overexpression of FBXW2 in PC3 cells inhibited cell proliferation ( c ), invasion ( d ), and migration ( e ). Scale bar:100 µm ( d ) and 200 µm ( e ). Cell viability was detected with Cell Counting Kit-8 (CCK-8) assay ( ***p < 0.001; n = 3; two-way ANOVA). The cell migration ability and invasion ability were determined using the wound-healing and transwell assays, respectively ( ***p < 0.001; n = 3; Student’s two-tailed t test). f Overexpression of FBXW2 induced G1-phase cell cycle arrest in PC3 cells. The PC3 cells were transfected with vector or HA-FBXW2, and cell cycle distributions were then analyzed by flow cytometry ( **p < 0.01, ***p < 0.001; n = 3; Student’s two-tailed t test). g Effects of FBXW2 overexpression on the expression of cell progression-related proteins in PC3 cells. GAPDH levels served as the control for equal loading ( ***p < 0.001; n = 3; Student’s two-tailed t test)

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:1000 overnight, 4 °C); rabbit-FBXW2 (11499-1-AP, proteintech; 1:500 overnight, 4 °C); rabbit-EGFR (#4267, CST; 1:1000 overnight, 4 °C); rabbit-caspase 3 (19677–1-AP, proteintech, 1:1000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science, 1:2000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma, 1:2000 overnight, 4 °C); mouse-His-Tag (66005-1-Ig, proteintech, 1:1000 overnight, 4 °C); rabbit-AR (#5153, CST, 1:1000 overnight, 4 °C); rabbit-p-AKT (#4060S, CST, 1:1000 overnight, 4 °C); rabbit-AKT (#4691S, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin B1 (#12231S, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin D1 (#2978, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin E1 (11554-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p-STAT3 (#9145, CST, 1:1,000 overnight, 4 °C); mouse-STAT3 (ab119352, Abcam, 1:1,000 overnight, 4 °C); rabbit-BAX (50599-2-Ig, proteintech, 1:1000 overnight, 4 °C); rabbit-BCL2 (12789-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-PARP1 (13371-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p27 (25614-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p21 (103551-1-AP, proteintech, 1:1000 overnight, 4 °C); mouse-GAPDH (60004-1-Ig, proteintech, 1:2000 overnight, 4 °C); mouse-β-Actin (60008-1-Ig, proteintech, 1:5000 overnight, 4 °C); mouse-α-Tubulin (66031-1-Ig, proteintech, 1:5000 overnight, 4 °C).

Techniques: Expressing, Western Blot, Over Expression, Migration, Cell Counting, CCK-8 Assay, Two Tailed Test, Transfection, Plasmid Preparation, Flow Cytometry, Control

Journal: Cellular and Molecular Life Sciences

Article Title: FBXW2 inhibits prostate cancer proliferation and metastasis via promoting EGFR ubiquitylation and degradation

doi: 10.1007/s00018-022-04320-3

Figure Lengend Snippet: Augmented FBXW2 inhibits PCa tumor growth and attenuates osteolytic bone tumor tumorigenesis. a–c Overexpression of FBXW2 inhibited PCa tumor growth in vivo. PC3 cells stably expressing Vector and HA-FBXW2 (1 × 10 6 cells) were inoculated s.c. in both flanks of nude mice. After indicated time, the tumors were harvested and photographed ( a ) ( ***p < 0.001; n = 5; Student’s two-tailed t test; Scale bars, 200 μm). The tumor growth was monitored twice a week for up to 30 days and growth curve plotted ( b ) ( ***p < 0.001; n = 5; two-way ANOVA). Body weight was measured and plotted ( c ) (ns, no significance; n = 5; two-way ANOVA). d Overexpression of FBXW2 attenuated osteolytic bone tumor tumorigenesis in vivo. 2.0 × 10 6 PC3 stable cells (Vector or HA-FBXW2) were re-suspended in 100 µL PBS, and 10 µL of cell solution was slowly injected into NOD/SCID mice. Representative radiographical images of osteolytic bone tumor in the indicated tibia of the mice (left). Bars, 4 mm. Representative H&E-stained sections of the indicated tibia of the mice (right). Scale bar, 500 µm and 50 µm. The sum of bone metastasis scores in the tibia of the Vector or HA-FBXW2 mice groups ( ***p < 0.001; n = 10; Student’s two-tailed t test)

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:1000 overnight, 4 °C); rabbit-FBXW2 (11499-1-AP, proteintech; 1:500 overnight, 4 °C); rabbit-EGFR (#4267, CST; 1:1000 overnight, 4 °C); rabbit-caspase 3 (19677–1-AP, proteintech, 1:1000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science, 1:2000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma, 1:2000 overnight, 4 °C); mouse-His-Tag (66005-1-Ig, proteintech, 1:1000 overnight, 4 °C); rabbit-AR (#5153, CST, 1:1000 overnight, 4 °C); rabbit-p-AKT (#4060S, CST, 1:1000 overnight, 4 °C); rabbit-AKT (#4691S, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin B1 (#12231S, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin D1 (#2978, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin E1 (11554-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p-STAT3 (#9145, CST, 1:1,000 overnight, 4 °C); mouse-STAT3 (ab119352, Abcam, 1:1,000 overnight, 4 °C); rabbit-BAX (50599-2-Ig, proteintech, 1:1000 overnight, 4 °C); rabbit-BCL2 (12789-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-PARP1 (13371-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p27 (25614-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p21 (103551-1-AP, proteintech, 1:1000 overnight, 4 °C); mouse-GAPDH (60004-1-Ig, proteintech, 1:2000 overnight, 4 °C); mouse-β-Actin (60008-1-Ig, proteintech, 1:5000 overnight, 4 °C); mouse-α-Tubulin (66031-1-Ig, proteintech, 1:5000 overnight, 4 °C).

Techniques: Over Expression, In Vivo, Stable Transfection, Expressing, Plasmid Preparation, Two Tailed Test, Injection, Staining

Journal: Cellular and Molecular Life Sciences

Article Title: FBXW2 inhibits prostate cancer proliferation and metastasis via promoting EGFR ubiquitylation and degradation

doi: 10.1007/s00018-022-04320-3

Figure Lengend Snippet: FBXW2 is inversely correlated with EGFR in PCa, and regulates EGFR protein level. a Inverse correlation at the protein levels of EGFR versus FBXW2 in PCa cell lines ( *p < 0.05, **p < 0.01, ***p < 0.001; n = 3; one-way ANOVA). b Expression of FBXW2 and EGFR in PCa tissues. PCa tissue microarrays were stained with FBXW2 and EGFR, and then photographed (Scale bars, 100 μm). Association analysis of FBXW2 and EGFR in PCa tissues. Data were analyzed using SPSS software ( P = 0.001, n = 42, Pearson’s test). c , d Overexpression of FBXW2 reduced EGFR protein level, but not EGFR mRNA levels. PC3 cells were transfected with vector or HA-FBXW2, and followed by IB ( c ) or qPCR ( d ) ( ***p < 0.001; n = 3; Student’s two-tailed t test). e FBXW2 could bind to endogenous EGFR: Cell lysates from PC3 cells were pulled down with anti-FBXW2 Abs, followed by IB with indicated Abs. f Overexpression of FBXW2, but not its ΔF mutant, shortened protein half-life of exogenous EGFR. After transfection with relevant plasmids for 48 h, 293 T cells were switched to fresh medium (10% FBS) containing cycloheximide (CHX) and incubated for indicated time periods before being harvested for IB. The band density was quantified using ImageJ software and plotted ( **p < 0.01; ***p < 0.001; n = 3; two-way ANOVA). g Protein levels of EGFR downstream were markedly decreased by FBXW2 overexpression. PC3 cells were transfected with vector or HA-FBXW2, and followed by IB (ns, no significance, ***p < 0.001; n = 3; Student’s two-tailed t test). h , i Transfection of FBXW2 significantly abrogated invasion ability ( h ) and proliferation ( i ) caused by EGF. Scale bar: 100 µm ( h ). Transfected the vector control or plasmid expressing HA-FBXW2 for 48 h in PC3 cells were treated with or without EGF (10 ng/mL). The cell ability and invasion ability were detected by CCK-8 assay ( *p < 0.1, ***p < 0.001; n = 3; two-way ANOVA) and transwell assays (* **p < 0.001; n = 3; one-way ANOVA), respectively

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:1000 overnight, 4 °C); rabbit-FBXW2 (11499-1-AP, proteintech; 1:500 overnight, 4 °C); rabbit-EGFR (#4267, CST; 1:1000 overnight, 4 °C); rabbit-caspase 3 (19677–1-AP, proteintech, 1:1000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science, 1:2000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma, 1:2000 overnight, 4 °C); mouse-His-Tag (66005-1-Ig, proteintech, 1:1000 overnight, 4 °C); rabbit-AR (#5153, CST, 1:1000 overnight, 4 °C); rabbit-p-AKT (#4060S, CST, 1:1000 overnight, 4 °C); rabbit-AKT (#4691S, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin B1 (#12231S, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin D1 (#2978, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin E1 (11554-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p-STAT3 (#9145, CST, 1:1,000 overnight, 4 °C); mouse-STAT3 (ab119352, Abcam, 1:1,000 overnight, 4 °C); rabbit-BAX (50599-2-Ig, proteintech, 1:1000 overnight, 4 °C); rabbit-BCL2 (12789-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-PARP1 (13371-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p27 (25614-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p21 (103551-1-AP, proteintech, 1:1000 overnight, 4 °C); mouse-GAPDH (60004-1-Ig, proteintech, 1:2000 overnight, 4 °C); mouse-β-Actin (60008-1-Ig, proteintech, 1:5000 overnight, 4 °C); mouse-α-Tubulin (66031-1-Ig, proteintech, 1:5000 overnight, 4 °C).

Techniques: Expressing, Staining, Software, Over Expression, Transfection, Plasmid Preparation, Two Tailed Test, Mutagenesis, Incubation, Control, CCK-8 Assay

Journal: Cellular and Molecular Life Sciences

Article Title: FBXW2 inhibits prostate cancer proliferation and metastasis via promoting EGFR ubiquitylation and degradation

doi: 10.1007/s00018-022-04320-3

Figure Lengend Snippet: FBXW2 binds to EGFR via its consensus degron motif, and degrades EGFR. a Two possible binding sites on EGFR, MU1 (446/447, 452) and MU2 (1041/1042, 1045/1046). b Loss of FBXW2-EGFR binding site in mutant: EGFR or its mutants was co-transfected with HA-FBXW2, followed by IP with HA Abs and IB. c Overexpression of FBXW2 reduced basal level of EGFR and EGFR-MU1 in a dose manner, but not EGFR-MU2. (ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001; n = 3; one-way ANOVA). d FBXW2 shortened the protein half-life of EGFR and EGFR-MU1, but not EGFR-MU2. Stable 293 T cells were incubated with CHX for indicated time periods and harvested for IB (ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001; n = 3; two-way ANOVA). e CK1 kinase mediated EGFR phosphorylation at FBXW2 binding motif. PC3 cells were transfected HA-FBXW2, and treated with or without CK1 inhibitor IC261 (10 mM), and harvested for IB. GAPDH levels served as the control for equal loading (ns, no significance, ***p < 0.001; n = 3; two-way ANOVA). f , g Under EGF stimulation, transfected FBXW2 alone or co-transfection with wild-type EGFR suppressed and invasion ( f ) and cell proliferation ( g ) in PC3 cells, but was abrogated by simultaneous transfection of EGFR-MU2. Scale bar:100 µm ( f ). Transfected HA-FBXW2 alone or in combinations with FLAG-EGFR (WT versus MU2) into PC3 cells and then treated with EGF (10 ng/mL). The cell proliferation and invasion ability were detected by CCK-8 assay ( **p < 0.01, ***p < 0.001; n = 3; two-way ANOVA) and transwell assays ( **p < 0.01, ***p < 0.001; n = 3; one-way ANOVA), respectively

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:1000 overnight, 4 °C); rabbit-FBXW2 (11499-1-AP, proteintech; 1:500 overnight, 4 °C); rabbit-EGFR (#4267, CST; 1:1000 overnight, 4 °C); rabbit-caspase 3 (19677–1-AP, proteintech, 1:1000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science, 1:2000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma, 1:2000 overnight, 4 °C); mouse-His-Tag (66005-1-Ig, proteintech, 1:1000 overnight, 4 °C); rabbit-AR (#5153, CST, 1:1000 overnight, 4 °C); rabbit-p-AKT (#4060S, CST, 1:1000 overnight, 4 °C); rabbit-AKT (#4691S, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin B1 (#12231S, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin D1 (#2978, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin E1 (11554-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p-STAT3 (#9145, CST, 1:1,000 overnight, 4 °C); mouse-STAT3 (ab119352, Abcam, 1:1,000 overnight, 4 °C); rabbit-BAX (50599-2-Ig, proteintech, 1:1000 overnight, 4 °C); rabbit-BCL2 (12789-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-PARP1 (13371-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p27 (25614-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p21 (103551-1-AP, proteintech, 1:1000 overnight, 4 °C); mouse-GAPDH (60004-1-Ig, proteintech, 1:2000 overnight, 4 °C); mouse-β-Actin (60008-1-Ig, proteintech, 1:5000 overnight, 4 °C); mouse-α-Tubulin (66031-1-Ig, proteintech, 1:5000 overnight, 4 °C).

Techniques: Binding Assay, Mutagenesis, Transfection, Over Expression, Incubation, Phospho-proteomics, Control, Cotransfection, CCK-8 Assay

Journal: Cellular and Molecular Life Sciences

Article Title: FBXW2 inhibits prostate cancer proliferation and metastasis via promoting EGFR ubiquitylation and degradation

doi: 10.1007/s00018-022-04320-3

Figure Lengend Snippet: Putative kinases for FBXW2 phosphorylation at the EGFR binding motif (TSNNST)

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:1000 overnight, 4 °C); rabbit-FBXW2 (11499-1-AP, proteintech; 1:500 overnight, 4 °C); rabbit-EGFR (#4267, CST; 1:1000 overnight, 4 °C); rabbit-caspase 3 (19677–1-AP, proteintech, 1:1000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science, 1:2000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma, 1:2000 overnight, 4 °C); mouse-His-Tag (66005-1-Ig, proteintech, 1:1000 overnight, 4 °C); rabbit-AR (#5153, CST, 1:1000 overnight, 4 °C); rabbit-p-AKT (#4060S, CST, 1:1000 overnight, 4 °C); rabbit-AKT (#4691S, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin B1 (#12231S, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin D1 (#2978, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin E1 (11554-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p-STAT3 (#9145, CST, 1:1,000 overnight, 4 °C); mouse-STAT3 (ab119352, Abcam, 1:1,000 overnight, 4 °C); rabbit-BAX (50599-2-Ig, proteintech, 1:1000 overnight, 4 °C); rabbit-BCL2 (12789-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-PARP1 (13371-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p27 (25614-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p21 (103551-1-AP, proteintech, 1:1000 overnight, 4 °C); mouse-GAPDH (60004-1-Ig, proteintech, 1:2000 overnight, 4 °C); mouse-β-Actin (60008-1-Ig, proteintech, 1:5000 overnight, 4 °C); mouse-α-Tubulin (66031-1-Ig, proteintech, 1:5000 overnight, 4 °C).

Techniques: Phospho-proteomics, Binding Assay

Journal: Cellular and Molecular Life Sciences

Article Title: FBXW2 inhibits prostate cancer proliferation and metastasis via promoting EGFR ubiquitylation and degradation

doi: 10.1007/s00018-022-04320-3

Figure Lengend Snippet: FBXW2 promotes EGFR ubiquitylation via its consensus degron motif (TSNNST) on EGFR for subsequent degradation. a FBXW2 promoted ubiquitylation of EGFR in vivo: PC3 cells were transfected with indicated plasmids, lysed under denatured condition at 6 M guanidinium solution, followed by Ni-beads pull-down. b FBXW2, but not its ΔF mutant, promoted ubiquitylation of EGFR in vivo: 293 T cells were transfected with indicated plasmids, lysed under denatured condition at 6 M guanidinium solution, followed by Ni-beads pull-down. c EGFR was conjugated with lysine (K) 48- or 63-linked polyubiquitin chains: 293 T cells transfected with different ubiquitin mutants were co-overexpressed both HA-FBXW2 and FLAG-EGFR. After 48 h transfection, cells were treated with MG132 for 4 h followed by IP and IB. d FBXW2 promoted ubiquitylation of wild-type EGFR and EGFR-MU1, not EGFR-MU2 in vivo: 293 T cells were transfected with indicated plasmids, lysed under denatured condition at 6 M guanidinium solution, followed by Ni-beads pull-down. GAPDH levels served as the control for equal loading

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:1000 overnight, 4 °C); rabbit-FBXW2 (11499-1-AP, proteintech; 1:500 overnight, 4 °C); rabbit-EGFR (#4267, CST; 1:1000 overnight, 4 °C); rabbit-caspase 3 (19677–1-AP, proteintech, 1:1000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science, 1:2000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma, 1:2000 overnight, 4 °C); mouse-His-Tag (66005-1-Ig, proteintech, 1:1000 overnight, 4 °C); rabbit-AR (#5153, CST, 1:1000 overnight, 4 °C); rabbit-p-AKT (#4060S, CST, 1:1000 overnight, 4 °C); rabbit-AKT (#4691S, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin B1 (#12231S, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin D1 (#2978, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin E1 (11554-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p-STAT3 (#9145, CST, 1:1,000 overnight, 4 °C); mouse-STAT3 (ab119352, Abcam, 1:1,000 overnight, 4 °C); rabbit-BAX (50599-2-Ig, proteintech, 1:1000 overnight, 4 °C); rabbit-BCL2 (12789-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-PARP1 (13371-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p27 (25614-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p21 (103551-1-AP, proteintech, 1:1000 overnight, 4 °C); mouse-GAPDH (60004-1-Ig, proteintech, 1:2000 overnight, 4 °C); mouse-β-Actin (60008-1-Ig, proteintech, 1:5000 overnight, 4 °C); mouse-α-Tubulin (66031-1-Ig, proteintech, 1:5000 overnight, 4 °C).

Techniques: In Vivo, Transfection, Mutagenesis, Ubiquitin Proteomics, Control

Journal: Cellular and Molecular Life Sciences

Article Title: FBXW2 inhibits prostate cancer proliferation and metastasis via promoting EGFR ubiquitylation and degradation

doi: 10.1007/s00018-022-04320-3

Figure Lengend Snippet: Schematic model for FBXW2-induced EGFR degradation inhibiting growth and metastasis of PCa cells. During tumorigenesis, FBXW2, as a tumor suppressor, promotes ubiquitylation and degradation of EGFR, leading to repression of EGFR downstream, eventually to control growth and metastasis of PCa cells

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:1000 overnight, 4 °C); rabbit-FBXW2 (11499-1-AP, proteintech; 1:500 overnight, 4 °C); rabbit-EGFR (#4267, CST; 1:1000 overnight, 4 °C); rabbit-caspase 3 (19677–1-AP, proteintech, 1:1000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science, 1:2000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma, 1:2000 overnight, 4 °C); mouse-His-Tag (66005-1-Ig, proteintech, 1:1000 overnight, 4 °C); rabbit-AR (#5153, CST, 1:1000 overnight, 4 °C); rabbit-p-AKT (#4060S, CST, 1:1000 overnight, 4 °C); rabbit-AKT (#4691S, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin B1 (#12231S, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin D1 (#2978, CST, 1:1000 overnight, 4 °C); rabbit-Cyclin E1 (11554-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p-STAT3 (#9145, CST, 1:1,000 overnight, 4 °C); mouse-STAT3 (ab119352, Abcam, 1:1,000 overnight, 4 °C); rabbit-BAX (50599-2-Ig, proteintech, 1:1000 overnight, 4 °C); rabbit-BCL2 (12789-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-PARP1 (13371-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p27 (25614-1-AP, proteintech, 1:1000 overnight, 4 °C); rabbit-p21 (103551-1-AP, proteintech, 1:1000 overnight, 4 °C); mouse-GAPDH (60004-1-Ig, proteintech, 1:2000 overnight, 4 °C); mouse-β-Actin (60008-1-Ig, proteintech, 1:5000 overnight, 4 °C); mouse-α-Tubulin (66031-1-Ig, proteintech, 1:5000 overnight, 4 °C).

Techniques: Control

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a ) β-TrCP1 binds to endogenous FBXW2: Cell lysates from H1299 cells were pulled down with anti-FBXW2 or anti-β-TrCP1 Abs, followed by IB with indicated Abs. ( b ) β-TrCP1 binds to phosphor-FBXW2 peptide: Cell lysates from A549 cells were incubated with bead-conjugated FBXW2 non-phosphorylated or phosphor-peptide containing β-TrCP binding motif. Beads were washed and subjected to IB with indicated Abs. ( c – e ) Overexpression of β-TrCP1, but not its ΔF mutant, decreases the levels of FBXW2 protein: H1299 and H358 cells were co-transfected with F-FBXW2 (FLAG-tagged FBXW2) and increasing amounts of β-TrCP1, or transfected with increasing amounts of β-TrCP1 or β-TrCP1ΔF alone, followed by IB with indicated Abs. The band density was quantified. ( f ) β-TrCP1 overexpression has no effect on FBXW2 mRNA: H1299 cells were transfected with β-TrCP1 or vector control, followed by qRT-PCR for mRNA expression. Error bars indicate mean +s.d. of three repeats. ( g ) β-TrCP silencing increases the endogenous levels of FBXW2 protein, but not mRNA: A549 and H23 cells were transfected with siRNA targeting both β-TrCP1 and β-TrCP2, along with scrambled siRNA, followed by IB (top panels) or qRT-PCR (bottom panel). Error bars indicate mean +s.d. of three repeats. ( h , i ) FBXW2 half-life is shortened by β-TrCP1, but not by β-TrCP1ΔF: FLAG-FBXW2 was transfected into H1299 cells, along with the vector control or plasmid expressing HA-β-TrCP1 or HA-β-TrCP1ΔF. Cells were switched to fresh medium (10% FBS) containing cycloheximide (CHX) 48 h post transfection for indicated time periods and harvested for IB. The band density was quantified. ( j ) β-TrCP1 RNAi silencing extends protein half-life of endogenous FBXW2. H23 cells were transfected with either control RNAi, or RNAi targeting both β-TrCP1 and β-TrCP2 for 48 h. Cells were cultured in fresh medium containing CHX and incubated for indicated time periods before being harvested for IB.

Article Snippet: The following primary antibodies were used: goat-FBXW2 (sc-160326, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (ab83467, Abcam; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (#11499-1-AP, Proteintech; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, cellsignal; 1:1,000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science; 1:2,000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma; 1:2,000 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-P27 (#554069, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-β-Actin (sc-47778, SANTA CRUZ; 1:10,000 overnight, 4 °C); rabbit-Cleaved Notch1 (Val1744) (#4147, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-GRK2 (C-15) (sc-562, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-VRK2 (H-255)(sc-98733, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL11 (H-120)(sc-135126, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL3 (GTX110755, GeneTex; 1:1,000 overnight, 4 °C); mouse-p130 (A-10) (sc-374521, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW11 (#13149-1-AP, Proteintech; 1:1,000 overnight, 4 °C); rabbit-wee1 (#4936, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-c-jun (sc-1694, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-cul-1(sc-11384, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FOXO1 (#2880, Cellsignal; 1:1,000 overnight, 4 °C); mouse-cdh1 (#CC43, Calbiochem; 1:1,000 overnight, 4 °C); rabbit-rbx1 (made by our lab; 1:1,000 overnight, 4 °C).

Techniques: Incubation, Binding Assay, Over Expression, Mutagenesis, Transfection, Plasmid Preparation, Control, Quantitative RT-PCR, Expressing, Cell Culture

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a ) β-TrCP1 has no effects on FBXW2 binding mutant: H1299 cells were transfected with indicated plasmids, switched 48 h later to fresh medium containing CHX and harvested at indicated periods for IB. The band density was quantified. ( b ) Loss of β-TrCP1-FBXW2 binding in degron site mutant: FBXW2 or its 3A mutant at the degron site was co-transfected with HA-β-TrCP1, followed by IP with FLAG Ab and IB. ( c ) β-TrCP1 shortens the protein half-life of FBXW2, triggered by serum addition to serum-starved (SS) cells: H1299 cells were transfected with indicated plasmids, switched 48 h later to fresh medium containing10% FBS (fetal bovine serum) and CHX and harvested at indicated periods for IB. The band density was quantified. ( d , e ) CK1 kinase mediates FBXW2 phosphorylation at β-TrCP1 binding motif: H1299 cells were transfected of siRNA targeting GRK2, or treated by CK1 inhibitor IC-261 (10 μM) ( d ), or transfected with siRNA targeting VRK2 ( e ), followed by transfected with FLAG-β-TrCP1 for 48 h. Cells were harvested at indicated points after CHX treatment for IB. The band density was quantified. ( f , g ) β-TrCP1 promotes FBXW2 ubiquitylation in vivo : 293 ( f ) or H1299 ( g ) cells were transfected with indicated plasmids, lysed under denatured condition at 6 M guanidinium solution, followed by Ni-beads pull-down. Washed beads were boiled for IB to detect polyubiquitylation of exogenous FBXW2 ( f ) or endogenous FBXW2 ( g ). ( h ) β-TrCP1 promotes FBXW2 ubiquitylation in vitro : β-TrCP1 (E3) was prepared by transfecting HA-β-TrCP1 or HA-β-TrCP1ΔF into 293 cells, followed by HA-bead IP and 3 × 3 HA peptide elution. FBXW2 and FBXW2 mutant were prepared by transfecting FLAG-FBXW2 or FLAG-FBXW2-3A into 293 cells, followed by FLAG-bead IP. β-TrCP1 (E3) and FBXW2 (substrate) were added into a reaction mixture containing ATP, ubiquitin, E1 and E2, followed by constant mixing for 60 min. The reaction mixture was then loaded onto PAGE gel for IB using anti-FLAG Ab. ( i ) β-TrCP1 promotes FBXW2 ubiquitylation via K48 linkage: H1299 cells were transfected with indicated plasmids, lysed under denatured condition at 6 M guanidinium solution, followed by Ni-beads pull-down and IB for FBXW2.

Article Snippet: The following primary antibodies were used: goat-FBXW2 (sc-160326, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (ab83467, Abcam; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (#11499-1-AP, Proteintech; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, cellsignal; 1:1,000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science; 1:2,000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma; 1:2,000 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-P27 (#554069, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-β-Actin (sc-47778, SANTA CRUZ; 1:10,000 overnight, 4 °C); rabbit-Cleaved Notch1 (Val1744) (#4147, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-GRK2 (C-15) (sc-562, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-VRK2 (H-255)(sc-98733, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL11 (H-120)(sc-135126, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL3 (GTX110755, GeneTex; 1:1,000 overnight, 4 °C); mouse-p130 (A-10) (sc-374521, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW11 (#13149-1-AP, Proteintech; 1:1,000 overnight, 4 °C); rabbit-wee1 (#4936, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-c-jun (sc-1694, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-cul-1(sc-11384, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FOXO1 (#2880, Cellsignal; 1:1,000 overnight, 4 °C); mouse-cdh1 (#CC43, Calbiochem; 1:1,000 overnight, 4 °C); rabbit-rbx1 (made by our lab; 1:1,000 overnight, 4 °C).

Techniques: Binding Assay, Mutagenesis, Transfection, Phospho-proteomics, In Vivo, In Vitro, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a ) Fluctuation of the levels of F-box proteins and VRK2 kinase during cell cycle progression: H358 cells were serum starved for 48 h, followed by serum addition. Cells were harvested at indicated time points and subjected to FACS and IB analyses using indicated Abs. ( b – f ) FBXW2-3A mutant rescues growth-promoting phenotype induced by β-TrCP1 overexpression ( b – d ), and wt FBXW2 rescues growth-promoting phenotype induced by SKP2 overexpression ( b , e , f ): H1299 cells were co-transfected with the indicated plasmids, followed by IB ( b ), ATP-lite proliferation assay ( n =3) ( c , e ) and clonogenic survival assay ( n =3) ( d , f ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01. ( g – i ) FBXW2 depletion stimulates cell growth, which is abrogated by simultaneous SKP2 depletion: H1299 cells were transfected with shRNAs targeting FBXW2 alone or in combination with shRNA targeting SKP2, along with scramble control, and then harvested for IB ( g ), ATP-lite proliferation assay ( n =3) ( h ) and clonogenic survival assay ( n =3) ( i ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01; *** P <0.001. ( j , k ) FBXW2 depletion stimulates tumour growth, which is abrogated by simultaneous SKP2 depletion: H1299 cells were transfected with shRNAs targeting FBXW2 alone or in combination with shRNA targeting SKP2, along with scramble control, followed by injection (1 × 10 6 cells) into nude mice. Tumour growth was observed for 33 days ( j ). Tumours were then harvested, photographed and weighted ( k ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: The following primary antibodies were used: goat-FBXW2 (sc-160326, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (ab83467, Abcam; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (#11499-1-AP, Proteintech; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, cellsignal; 1:1,000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science; 1:2,000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma; 1:2,000 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-P27 (#554069, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-β-Actin (sc-47778, SANTA CRUZ; 1:10,000 overnight, 4 °C); rabbit-Cleaved Notch1 (Val1744) (#4147, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-GRK2 (C-15) (sc-562, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-VRK2 (H-255)(sc-98733, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL11 (H-120)(sc-135126, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL3 (GTX110755, GeneTex; 1:1,000 overnight, 4 °C); mouse-p130 (A-10) (sc-374521, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW11 (#13149-1-AP, Proteintech; 1:1,000 overnight, 4 °C); rabbit-wee1 (#4936, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-c-jun (sc-1694, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-cul-1(sc-11384, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FOXO1 (#2880, Cellsignal; 1:1,000 overnight, 4 °C); mouse-cdh1 (#CC43, Calbiochem; 1:1,000 overnight, 4 °C); rabbit-rbx1 (made by our lab; 1:1,000 overnight, 4 °C).

Techniques: Mutagenesis, Over Expression, Transfection, Proliferation Assay, Clonogenic Cell Survival Assay, shRNA, Control, Injection

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a – c ) Expression levels among three F-box proteins in lung cancer cell lines and tissues: Cell lysates from eight lung cancer cell lines and one immortalized line (BEAS-2B) were subjected to IB ( a ); SE: Short exposure; LE: Long exposure. Lung cancer tissue microarrays were stained with indicated Abs and photographed ( b , Scale bars, 100 μm), and data were then analysed using SPSS software to obtain coefficient ( c ; P <0.001, Pearson's test). ( d – f ) Protein expression of three F-box proteins in lung cancer and their relationship with patient survival: Continuous protein expression values were classified into the low and high groups with equal number of patients, and 5-year survival time was used for Kaplan-Meier survival analysis ( d – f ). Kaplan-Meier survival analysis indicated that patient with higher expression of FBXW2 was related to a better overall survival (log-rank test, P =0.032) ( e ); Higher expression of β-TrCP1 and SKP2 were related to a worse overall patient survival (log-rank test, P <0.001 and 0.001, respectively) ( d , f ).

Article Snippet: The following primary antibodies were used: goat-FBXW2 (sc-160326, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (ab83467, Abcam; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (#11499-1-AP, Proteintech; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, cellsignal; 1:1,000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science; 1:2,000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma; 1:2,000 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-P27 (#554069, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-β-Actin (sc-47778, SANTA CRUZ; 1:10,000 overnight, 4 °C); rabbit-Cleaved Notch1 (Val1744) (#4147, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-GRK2 (C-15) (sc-562, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-VRK2 (H-255)(sc-98733, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL11 (H-120)(sc-135126, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL3 (GTX110755, GeneTex; 1:1,000 overnight, 4 °C); mouse-p130 (A-10) (sc-374521, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW11 (#13149-1-AP, Proteintech; 1:1,000 overnight, 4 °C); rabbit-wee1 (#4936, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-c-jun (sc-1694, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-cul-1(sc-11384, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FOXO1 (#2880, Cellsignal; 1:1,000 overnight, 4 °C); mouse-cdh1 (#CC43, Calbiochem; 1:1,000 overnight, 4 °C); rabbit-rbx1 (made by our lab; 1:1,000 overnight, 4 °C).

Techniques: Expressing, Staining, Software

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a ) FBXW2 mutants have reduced binding with SCF components: H1299 cells were transfected with indicated plasmids, followed by G418 selection. Resistant clones were pooled and subjected to FLAG-bead IP and IB with indicated Abs. ( b , c ) FBXW2 mutants MU-S84C ( b ) and MU-E269K ( c ) are unable to shorten SKP2 protein half-life: Stable H1299 cells were incubated with CHX for indicated time periods and harvested for IB. The band density was quantified. ( d , e ) Loss- or gain-of-function of FBXW2 mutants: H1299 cells stably expressing MU-S84C and MU-E269K mutants were subjected to ATP-lite proliferation assay ( n =3) ( d ), and clonogenic survival assay ( n =3) ( e ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01; *** P <0.001. ( f , g ) FBXW2 mutants either lose the tumour suppressor function or gain the oncogenic function in vivo : H1299 cells stably expressing MU-S84C and MU-E269K mutants (1 × 10 6 cells) were inoculated s.c. in both flanks of nude mice. The tumour growth was monitored twice a week for up to 28 days and growth curve plotted ( f ). Tumour tissues were harvested, photographed and weighed at 28 days ( g ). Student's t -test was used to compare each experimental group with the control group. Shown are mean±s.e.m., * P <0.05; ** P <0.01; *** P <0.001. ( h ) Immunohistochemical staining of xenograft tumour tissues. Tumour tissues from four groups of mice were fixed, sectioned and stained with indicated antibodies. Scale bars: 100 μm. Shown are mean±s.e.m., * P <0.05; ** P <0.01. ( i ) The oncogene-tumour suppressor-oncogene axis—a working model: During tumorigenesis, oncogenic β-TrCP1 is activated to promote ubiquitylation and degradation of FBXW2 tumour suppressor, resulting in abrogation of FBXW2-induced SKP2 degradation. Accumulated oncogenic SKP2 promotes ubiquitylation and degradation of tumour suppressors such as p21, p27, p130, FOXO1, and leading to activation of CDKs, E2F and mTOR, eventually to uncontrolled proliferation of cancer cells.

Article Snippet: The following primary antibodies were used: goat-FBXW2 (sc-160326, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (ab83467, Abcam; 1:1,000 overnight, 4 °C); rabbit-FBXW2 (#11499-1-AP, Proteintech; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, cellsignal; 1:1,000 overnight, 4 °C); rat-HA (#11867423001, Roche Life Science; 1:2,000 overnight, 4 °C); mouse-FLAG (#F1804, Sigma; 1:2,000 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-P27 (#554069, BD Pharmingen; 1:2,000 overnight, 4 °C); mouse-β-Actin (sc-47778, SANTA CRUZ; 1:10,000 overnight, 4 °C); rabbit-Cleaved Notch1 (Val1744) (#4147, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-GRK2 (C-15) (sc-562, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-VRK2 (H-255)(sc-98733, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL11 (H-120)(sc-135126, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXL3 (GTX110755, GeneTex; 1:1,000 overnight, 4 °C); mouse-p130 (A-10) (sc-374521, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FBXW11 (#13149-1-AP, Proteintech; 1:1,000 overnight, 4 °C); rabbit-wee1 (#4936, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-c-jun (sc-1694, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-cul-1(sc-11384, SANTA CRUZ; 1:1,000 overnight, 4 °C); rabbit-FOXO1 (#2880, Cellsignal; 1:1,000 overnight, 4 °C); mouse-cdh1 (#CC43, Calbiochem; 1:1,000 overnight, 4 °C); rabbit-rbx1 (made by our lab; 1:1,000 overnight, 4 °C).

Techniques: Binding Assay, Transfection, Selection, Clone Assay, Incubation, Stable Transfection, Expressing, Proliferation Assay, Clonogenic Cell Survival Assay, In Vivo, Control, Immunohistochemical staining, Staining, Activation Assay

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a ) FBXW2 binds to SKP2 in vivo : Lysates from H1299 cells were pulled down with anti-FBXW2 (left panel) or anti-SKP2 (right panel), followed by IB. ( b ) FBXW2 failed to bind to a SKP2 mutant: H1299 cells were tranfected with indicated plasmids, followed by FLAG IP and HA IB or direct IB. ( c , d ) Ectopically expressed FBXW2 reduces the endogenous levels of SKP2 protein, but not mRNA: Cells were transfected with HA-FBXW2, followed by IB ( c ) or qRT-PCR for SKP2 ( d ). Error bars indicate mean +s.d. of three repeats. ( e , f ) FBXW2 overexpression decreases the levels of the exogenous and endogenous SKP2 proteins: Cells were co-transfected with indicated plasmids, followed by IB 48 h post transfection. ( g ) FBXW2 depletion increases the levels of SKP2 protein, but not mRNA: Cells were transfected with FBXW2 siRNA and scramble siRNA, followed by IB or qRT-PCR for SKP2. Error bars indicate mean+s.d. of three repeats. ( h ) FBXW2-induced SKP2 degradation is independent of CDH1: Cells were transfected with FLAG-FBXW2 and/or siRNA against CDH1, and then harvested for IB. ( i ) FBXW2 shortens SKP2 half-life: Cells were transfected with HA-FBXW2, and switched 48 h post transfection to fresh medium containing CHX for indicated periods with or without MG132 treatment for last 2 h, and then harvested for IB. The band density was quantified. ( j ) FBXW2 knockdown extends SKP2 half-life: Cells were transfected with siRNA targeting FBXW2. Cells were switched 48 h later to fresh medium containing CHX for indicated periods and harvested for IB. The band density was quantified. ( k , l ) FBXW2 promotes ubiquitylation of SKP2 ( k ), but not SKP2-3A mutant ( l ): Cells were transfected with indicated plasmids, followed by Ni-beads pull-down and IB for SKP2. ( m ) FBXW2 promotes SKP2 ubiquitylation by in vitro assay: H1299 cells were transfected with indicated plasmids. Pull-down purified FBXW2 and FBXW2ΔF (E3s), Pull-down purified SKP2 (substrate), were added into a reaction mixture containing ATP, ubiquitin, E1 and E2, followed by IB using anti-FLAG Ab. ( n ) FBXW2 promotes SKP2 ubiquitylation via K48 linkage: Cells were transfected with indicated plasmids, lysed under denatured condition at 6 M guanidinium solution, followed by Ni-beads pull-down and IB for SKP2.

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, Cellsignal; 1:500 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:1,000 overnight, 4 °C); mouse-ki-67 (#550609, BD Pharmingen; 1:1,000 overnight, 4 °C); rabbit-Cleaved Caspase-3 (Asp175) (#9661, Cellsignal; 1:500 overnight, 4 °C).

Techniques: In Vivo, Mutagenesis, Transfection, Quantitative RT-PCR, Over Expression, Knockdown, In Vitro, Purification, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a ) Fluctuation of the levels of F-box proteins and VRK2 kinase during cell cycle progression: H358 cells were serum starved for 48 h, followed by serum addition. Cells were harvested at indicated time points and subjected to FACS and IB analyses using indicated Abs. ( b – f ) FBXW2-3A mutant rescues growth-promoting phenotype induced by β-TrCP1 overexpression ( b – d ), and wt FBXW2 rescues growth-promoting phenotype induced by SKP2 overexpression ( b , e , f ): H1299 cells were co-transfected with the indicated plasmids, followed by IB ( b ), ATP-lite proliferation assay ( n =3) ( c , e ) and clonogenic survival assay ( n =3) ( d , f ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01. ( g – i ) FBXW2 depletion stimulates cell growth, which is abrogated by simultaneous SKP2 depletion: H1299 cells were transfected with shRNAs targeting FBXW2 alone or in combination with shRNA targeting SKP2, along with scramble control, and then harvested for IB ( g ), ATP-lite proliferation assay ( n =3) ( h ) and clonogenic survival assay ( n =3) ( i ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01; *** P <0.001. ( j , k ) FBXW2 depletion stimulates tumour growth, which is abrogated by simultaneous SKP2 depletion: H1299 cells were transfected with shRNAs targeting FBXW2 alone or in combination with shRNA targeting SKP2, along with scramble control, followed by injection (1 × 10 6 cells) into nude mice. Tumour growth was observed for 33 days ( j ). Tumours were then harvested, photographed and weighted ( k ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, Cellsignal; 1:500 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:1,000 overnight, 4 °C); mouse-ki-67 (#550609, BD Pharmingen; 1:1,000 overnight, 4 °C); rabbit-Cleaved Caspase-3 (Asp175) (#9661, Cellsignal; 1:500 overnight, 4 °C).

Techniques: Mutagenesis, Over Expression, Transfection, Proliferation Assay, Clonogenic Cell Survival Assay, shRNA, Control, Injection

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a – c ) Expression levels among three F-box proteins in lung cancer cell lines and tissues: Cell lysates from eight lung cancer cell lines and one immortalized line (BEAS-2B) were subjected to IB ( a ); SE: Short exposure; LE: Long exposure. Lung cancer tissue microarrays were stained with indicated Abs and photographed ( b , Scale bars, 100 μm), and data were then analysed using SPSS software to obtain coefficient ( c ; P <0.001, Pearson's test). ( d – f ) Protein expression of three F-box proteins in lung cancer and their relationship with patient survival: Continuous protein expression values were classified into the low and high groups with equal number of patients, and 5-year survival time was used for Kaplan-Meier survival analysis ( d – f ). Kaplan-Meier survival analysis indicated that patient with higher expression of FBXW2 was related to a better overall survival (log-rank test, P =0.032) ( e ); Higher expression of β-TrCP1 and SKP2 were related to a worse overall patient survival (log-rank test, P <0.001 and 0.001, respectively) ( d , f ).

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, Cellsignal; 1:500 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:1,000 overnight, 4 °C); mouse-ki-67 (#550609, BD Pharmingen; 1:1,000 overnight, 4 °C); rabbit-Cleaved Caspase-3 (Asp175) (#9661, Cellsignal; 1:500 overnight, 4 °C).

Techniques: Expressing, Staining, Software

Journal: Nature Communications

Article Title: The β-TrCP-FBXW2-SKP2 axis regulates lung cancer cell growth with FBXW2 acting as a tumour suppressor

doi: 10.1038/ncomms14002

Figure Lengend Snippet: ( a ) FBXW2 mutants have reduced binding with SCF components: H1299 cells were transfected with indicated plasmids, followed by G418 selection. Resistant clones were pooled and subjected to FLAG-bead IP and IB with indicated Abs. ( b , c ) FBXW2 mutants MU-S84C ( b ) and MU-E269K ( c ) are unable to shorten SKP2 protein half-life: Stable H1299 cells were incubated with CHX for indicated time periods and harvested for IB. The band density was quantified. ( d , e ) Loss- or gain-of-function of FBXW2 mutants: H1299 cells stably expressing MU-S84C and MU-E269K mutants were subjected to ATP-lite proliferation assay ( n =3) ( d ), and clonogenic survival assay ( n =3) ( e ). Shown is mean±s.e.m. Student's t -test was performed, * P <0.05; ** P <0.01; *** P <0.001. ( f , g ) FBXW2 mutants either lose the tumour suppressor function or gain the oncogenic function in vivo : H1299 cells stably expressing MU-S84C and MU-E269K mutants (1 × 10 6 cells) were inoculated s.c. in both flanks of nude mice. The tumour growth was monitored twice a week for up to 28 days and growth curve plotted ( f ). Tumour tissues were harvested, photographed and weighed at 28 days ( g ). Student's t -test was used to compare each experimental group with the control group. Shown are mean±s.e.m., * P <0.05; ** P <0.01; *** P <0.001. ( h ) Immunohistochemical staining of xenograft tumour tissues. Tumour tissues from four groups of mice were fixed, sectioned and stained with indicated antibodies. Scale bars: 100 μm. Shown are mean±s.e.m., * P <0.05; ** P <0.01. ( i ) The oncogene-tumour suppressor-oncogene axis—a working model: During tumorigenesis, oncogenic β-TrCP1 is activated to promote ubiquitylation and degradation of FBXW2 tumour suppressor, resulting in abrogation of FBXW2-induced SKP2 degradation. Accumulated oncogenic SKP2 promotes ubiquitylation and degradation of tumour suppressors such as p21, p27, p130, FOXO1, and leading to activation of CDKs, E2F and mTOR, eventually to uncontrolled proliferation of cancer cells.

Article Snippet: The following primary antibodies were used: rabbit-FBXW2 (ab83467, Abcam; 1:500 overnight, 4 °C); rabbit-SKP2 (#2652, Cellsignal; 1:1,000 overnight, 4 °C); rabbit-β-TrCP1 (D13F10) (#4394, Cellsignal; 1:500 overnight, 4 °C); mouse-P21 (#556430, BD Pharmingen; 1:1,000 overnight, 4 °C); mouse-ki-67 (#550609, BD Pharmingen; 1:1,000 overnight, 4 °C); rabbit-Cleaved Caspase-3 (Asp175) (#9661, Cellsignal; 1:500 overnight, 4 °C).

Techniques: Binding Assay, Transfection, Selection, Clone Assay, Incubation, Stable Transfection, Expressing, Proliferation Assay, Clonogenic Cell Survival Assay, In Vivo, Control, Immunohistochemical staining, Staining, Activation Assay

Journal: Cell Reports Medicine

Article Title: Hemagglutinin stalk-binding antibodies enhance effectiveness of neuraminidase inhibitors against influenza via Fc-dependent effector functions

doi: 10.1016/j.xcrm.2022.100718

Figure Lengend Snippet:

Article Snippet: 16 hours after infection, the media was replaced with 50 μL of assay buffer (RPMI 1640 supplemented with 4% (vol/vol) low IgG FBS(Gibco)) containing oseltamivir carboxylate (Toronto Research Chemicals) or baloxavir marboxil (Selleck Chemicals) and serial dilutions of antibody or serum.

Techniques: Control, Virus, Recombinant, Construct, Cell Culture, Expressing, Plasmid Preparation, Bioassay, Variant Assay, Luciferase, Software