Journal: iScience

Article Title: Morphological remodeling of Coxiella burnetii during its biphasic developmental cycle revealed by cryo-electron tomography

doi: 10.1016/j.isci.2023.107210

Figure Lengend Snippet: Multilayered cytoplasmic membrane invaginations in C. burnetii SCVs (A–E) Slices through electron cryotomograms of C. burnetii cells (purified from infected vero cells 28 dpi) highlighting the presence of multilayered cytoplasmic membrane invaginations (orange arrows) with zoom-ins of these invaginations shown in the enlargements at the lower right corner of each panel. The white arrows in panel C point to a membrane of presumably the host vacuole still attached to the OM of C. burnetii cell. Scale bar is 100 nm. (F) Lower panel: average density profile taken along the area indicated between the yellow lines in the upper panel highlighting the equidistant spacing of ∼10 nm between the different layers of the inner membrane invaginations.

Article Snippet: African green monkey kidney (Vero) fibroblasts (CCL-81) , American Type Culture Collection , https://www.atcc.org/products/ccl-81.

Techniques: Membrane, Purification, Infection

Journal: iScience

Article Title: Morphological remodeling of Coxiella burnetii during its biphasic developmental cycle revealed by cryo-electron tomography

doi: 10.1016/j.isci.2023.107210

Figure Lengend Snippet:

Article Snippet: African green monkey kidney (Vero) fibroblasts (CCL-81) , American Type Culture Collection , https://www.atcc.org/products/ccl-81.

Techniques: Mutagenesis, Membrane, Software, Tomography

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: Current Biology

Article Title: Presynaptic and postsynaptic determinants of claustro-cortical connectivity

doi: 10.1016/j.cub.2025.05.056

Figure Lengend Snippet: Figure 3. CLA projections selectively target interneuron populations (A) Illustration depicting experimental paradigm. Briefly, ChR2 was injected using an anterograde viral vector into the CLA (100 nL, 300 nL/min) into different transgenic mouse lines with fluorescent reporters to identify interneuron populations. Paired whole-cell patch-clamp recordings were made in the ACC, and CLA axons were photostimulated to investigate postsynaptic responses. Differential interference contrast (DIC) image of paired recordings of an interneuron and a putative pyramidal neuron. (B) Postsynaptic responses recorded in a layer 5/6 neuron in the presence of gabazine. The postsynaptic response was subsequently removed on bath application of TTX and recovered with TTX and 4AP, confirming that the input was monosynaptic. The subsequent postsynaptic response is abolished with AMPA and NMDA receptor antagonists, NBQX and APV, confirming that the input is excitatory. (C) Illustration of paired recordings of PV interneurons and pyramidal neurons. Confocal image of the ACC shows that PV interneurons are in layers 2/3 and 5/6 of the ACC. A total of 75 PV interneurons (n = 42 in layer 2/3, n = 33 in layer 5/6) were studied. Example trace of a PV interneuron. Representative postsynaptic responses in paired recordings between PV (orange trace) and pyramidal neuron populations (black trace). 69% of all recorded PV neurons were found to be responsive. (D) SOM: confocal image of the ACC shows that SOM interneurons are in layers 2/3 and 5/6 with neurites in layer 1. A total of 48 SOM interneurons (n = 1 in layer 1, n = 14 in layer 2/3, n = 33 in layer 5/6) were studied. Example trace of a SOM interneuron. Representative postsynaptic responses in paired recordings between SOM (purple trace) and pyramidal neuron populations (black trace). 35% of all recorded SOM neurons were found to be responsive.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Biocytin (ε-Biotinoyl-L-Lysine) ThermoFisher Cat. # B1592 Cy5-conjugated streptavidin Jackson ImmunoResearch Laboratories RRID: AB_2337245 DAPI (4′,6-diamidino-2-fenilindol, dihidrocloruro) ThermoFisher Cat. # D1306; RRID AB_2629482 Bacterial and virus strains pAAV2-CaMKIIa-hChR2(H134R)-EYFP Karl Deisseroth Cat. # 26969-AAV2; RRID:Addgene_26969 AAV5.EF1.dflox.hChR2(H134R)-mCherry.WPRE.hGH Karl Deisseroth Cat. # 20297; RRID: Addgene_20297 AAV5.EF1.dflox.hChR2(H134R)-eYFP.WPRE.hGH Karl Deisseroth Cat. # 20298; RRID: Addgene_20298 pAAV-hSyn-DIO-mCherry Addgene Cat. # 50459; RRID:Addgene_50459 Red Retrobeads Lumafluor https://lumafluor.com/ Chemicals, peptides, and recombinant proteins Isoflurane, Forene AbbVie AB (Solna, Sweden) Cat. #: 506949 Sodium pentobarbital APL AB (Stockholm, Sweden) Cat. #: 338327 Temgesic 0.3mg/ml Indivior Europe Limited (Apoteket) Cat. # 521634 Viscotears 2mg/g Thea (Apoteket) Cat. #: 569173 Xylocain Spray 100 mg/ml AstraZeneca Cat. #: 469429 Adhese Universal Refill Bottle 2x5g IvoClar Vivadent Cat. #: 663721WW Dental cement, Tetric EvoFlow, 1x 2 g IvoClar Vivadent Cat. #: 595953WW Silicone sealant Kwik-Cast WPI Cat. #: KWIK-CAST D-APV Tocris Cat. # 79055-68-8 NBQX disodium salt Tocris Cat. # 479347-86-9 Tetrodotoxin citrate (TTX) Tocris Cat. # 18660-81-6 4-Aminopyridine (4AP) Tocris Cat. # 504-24-5 SR-95531 (Gabazine) Merck Cat. # 104104-50-9 DiI (1,1′-dioctadecyl-3,3,3′3’tetramethylindocarbocyanine perchlorate) Invitrogen, ThermoFisher Cat. # D282 Critical commercial assays RNAscope Multiplex Fluorescent v2 ACD Cat. # 323100 Probe- Mm-Slc17a6-C1 ACD Cat. # 319171 Probe- Mm-CamkIIa-C2 ACD Cat. # 445231 TSA Cyanine 3 Akoya Biosciences Cat. #NEL744001KT TSA Cyanine 5 Akoya Biosciences Cat. #NEL745001KT Experimental models: Organisms/strains Mouse, C57BL/6J The Jackson Laboratory RRID: IMSR_JAX: 000664 Mouse, PV-Cre The Jackson Laboratory RRID: IMSR_JAX:017320 Mouse, SOM-Cre The Jackson Laboratory RRID: IMSR_JAX:018973 Mouse, Ai9 (RCL-tdT) The Jackson Laboratory RRID: IMSR_JAX:007909 Mouse, NPY-Cre The Jackson Laboratory RRID: IMSR_JAX:027851 Mouse, 5Ht3a-Cre The Rockefeller University, GENSAT RRID: MMRRC_036680-UCD Mouse, Vglut2-ires-cre The Jackson Laboratory RRID: IMSR_JAX:016963 Software and algorithms ImageJ FIJI NIH https://imagej.nih.gov/ij QuickNII NITRC https://quicknii.readthedocs.io/ VisuAlign NITRC https://visualign.readthedocs.io Python Python Software Foundation https://www.python.org/ (Continued on next page) Current Biology 35, 3174–3190.e1–e5, July 7, 2025 e1

Techniques: Injection, Plasmid Preparation, Transgenic Assay, Patch Clamp

Journal: eLife

Article Title: Specific Eph receptor-cytoplasmic effector signaling mediated by SAM–SAM domain interactions

doi: 10.7554/eLife.35677

Figure Lengend Snippet: ( A ) Selected images showing the morphologies of DU145 cells expressing the wild-type or various mutants of EphA2 before and after ephrin A1-Fc treated. Cells were stained for F-actin with fluorescent phallodin (red), and nuclei were labelled with DAPI (blue). ( B ) Quantification of the cell areas of DU145 cells expressing various constructs. Data represents the mean ±SEM from four independent experiments with each experiment of at least 400 cells (***, p<0.001 by Two-way ANOVA with multiple comparisons test). ( C ) Western blot analysis showing the expression levels of different EphA2 (anti-Flag) constructs.

Article Snippet: Cell line (Human) , DU145 , ATCC , Cat #HTB-81; RRID: CVCL_0105.

Techniques: Expressing, Staining, Construct, Western Blot

Journal: eLife

Article Title: Specific Eph receptor-cytoplasmic effector signaling mediated by SAM–SAM domain interactions

doi: 10.7554/eLife.35677

Figure Lengend Snippet: ( A ) Selected images showing the morphologies of DU145 cells infected with lenti-virus expressing EphA2 or the GFP control, with or without SHIP2-SAM co-expression. These cells were further divided into two groups, with or without ephrin A1-Fc treatment. SHIP2-SAM was detected by antibody against Myc-tag (green). Cells were stained for F-actin with fluorescent phalloidin (red) and nuclei were labeled with DAPI (blue). ( B ) Quantification of the cell sizes of DU145 cells expressing various proteins. Data represent the mean ± SEM from four independent experiments with each experiment of at least 500 cells (***, p<0.001 by Two-way ANOVA with multiple comparisons test). ( C ) Western blot analysis showing the expression levels of EphA2 (anti-Flag) and SHIP2-SAM (anti-Myc) in transfected cells in each group. GAPDH serves as the loading control.

Article Snippet: Cell line (Human) , DU145 , ATCC , Cat #HTB-81; RRID: CVCL_0105.

Techniques: Infection, Virus, Expressing, Control, Staining, Labeling, Western Blot, Transfection

Journal: eLife

Article Title: Specific Eph receptor-cytoplasmic effector signaling mediated by SAM–SAM domain interactions

doi: 10.7554/eLife.35677

Figure Lengend Snippet:

Article Snippet: Cell line (Human) , DU145 , ATCC , Cat #HTB-81; RRID: CVCL_0105.

Techniques: Transfection, Construct, Plasmid Preparation, Recombinant, Cloning, Software