Journal: International Journal of Biological Sciences

Article Title: IC261, a specific inhibitor of CK1δ/ε, promotes aerobic glycolysis through p53-dependent mechanisms in colon cancer

doi: 10.7150/ijbs.40960

Figure Lengend Snippet: CK1ε and CK1δ are upregulated in colon cancer and associated with poor clinical outcome. (A, B) CK1ε and CK1δ mRNA were detected in colon cancer and paired adjacent normal tissues by using qRT-PCR. (C, D) CK1ε and CK1δ proteins were detected in colon cancer and paired adjacent normal tissues by using IHC. (E) Kaplan-Meier analysis of the CK1ε and CK1δ expression on overall survival of colon cancer patients (** p <0.01).

Article Snippet: The slides were incubated overnight at 4°C with CK1ε and CK1δ antibody (Cell Signaling Technology USA), followed by incubation with secondary antibodies (Abm, China).

Techniques: Quantitative RT-PCR, Expressing

Journal: International Journal of Biological Sciences

Article Title: IC261, a specific inhibitor of CK1δ/ε, promotes aerobic glycolysis through p53-dependent mechanisms in colon cancer

doi: 10.7150/ijbs.40960

Figure Lengend Snippet: Correlation between the clinicopathological features and CK1δ expression

Article Snippet: The slides were incubated overnight at 4°C with CK1ε and CK1δ antibody (Cell Signaling Technology USA), followed by incubation with secondary antibodies (Abm, China).

Techniques: Expressing

Journal: Nature Communications

Article Title: Neddylation inhibition induces glutamine uptake and metabolism by targeting CRL3 SPOP E3 ligase in cancer cells

doi: 10.1038/s41467-022-30559-2

Figure Lengend Snippet: a HEK293 cells were transfected with indicated plasmids, followed by immunoprecipitation with FLAG-agarose beads and immunoblotting. b HEK293 cells were transfected with various FLAG-CK1 isoforms, immunoprecipitated with FLAG-agarose beads and analyzed by immunoblotting. c Endogenous CK1δ protein in SUM159 cell lysates were pulled down with anti-S CK1δ antibody, and associated ASCT2 were detected by immunoblotting with anti-ASCT2 Ab (top, IP). Cell lysates were subjected to immunoblotting (bottom, input). d MDA-MB-231 cells were transfected with indicated CK1 isoforms, then analyzed by immunoblotting. e MDA-MB-231 cells were transfected siRNA targeting CK1δ for 48 h, followed by immunoblotting. f HEK293 cells were transfected with FLAG-SPOP and HA-ASCT2 and treated with D4476 for 12 h before harvesting, followed by immunoprecipitation with FLAG-agarose beads and analyzed by immunoblotting. g MDA-MB-231 cells were treated with different concentrations of CK1 inhibitor D4476 for 24 h, then analyzed by immunoblotting. h , i MDA-MB-231 cells were transfected with si-NC or siRNA targeting CK1δ (si-CK1δ#1), then incubated with CHX for various time points, and analyzed by immunoblotting ies ( h ), the band density of ASCT2 was quantified using ImageJ software and normalized to α-tubulin (mean ± SD, n = 3) ( i ). j HEK293 cells were transfected with indicated plasmids, lysed under denaturing conditions, followed by Ni-beads pulldown, and immunoblotting for ASCT2. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used: ASCT2 (Cell Signaling Technology, D7C12, 8057, dilution: 1:1000); ASCT2 (Abcam, ab84903, dilution: 1:800); β-actin (Sigma-Aldrich, A5441, dilution: 1:10000); CK1δ (Santa Cruz, sc-55553, dilution: 1:1000); CK1δ (Cell Signaling Technology, 12417 S, dilution: 1:1000); CUL-1 (Santa Cruz, sc-11384, dilution: 1:1000); CUL-2 (Abcam, ab166917, dilution: 1:1000); CUL-3 (Cell Signaling Technology, 2759 S, dilution: 1:1000); CUL-4A (Cell Signaling Technology, 2699 S, dilution: 1:1000); CUL-4B (Proteintech, 12916-1-AP, dilution: 1:1000); CUL-5 (Santa Cruz, sc-13014, dilution: 1:1000); FLAG, clone M2 (Sigma-Aldrich, F1804-500UG, dilution: 1:2000); FLAG M2 affinity gel (Sigma-Aldrich, A2220-5ML); GLUD1 (Abcam, ab89967, dilution: 1:1000); GLS (Abcepta, ap8809b, dilution: 1:1000); GOT2 (ProteinTech, 14800-1-AP, dilution: 1:1000); GRK2 (ProteinTech, 13990-1-AP, dilution: 1:1000); GRK2 (Cell Signaling Technology, 3982, dilution: 1:1000); HA (Sigma, H6908, dilution: 1:2000); Anti-HA High Affinity (3F10) (Roche, 11867423001); HIF-1α (Santa Cruz, sc-53546, dilution: 1:1000); NAEβ (Abcam, ab124728, dilution: 1:1000); NAE1 (Cell Signaling Technology, 14321S, dilution: 1:1000); NEDD8 (Abcam, ab81264, dilution: 1:1000); SNAT1 (Abcam, ab134268, dilution: 1:1000); SNAT2 (Abcam, ab90677, dilution: 1:1000); SPOP (Abcam, ab137537, dilution: 1:1000); SPOP (Affinit, DF12106, dilution: 1:800); SPOP (Santa Cruz, sc-377206, dilution: 1:1000); p-SPOP (Ser222) (dilution: 1:1000); α-tubulin (Sigma, Clone AA13, T8203, dilution: 1:10000); UBE2M (Santa Cruz, sc-390064, dilution: 1:1000); UBE2F (ProteinTech, 17056-1-AP, dilution: 1:1000); Peroxidase AffiniPure Goat Anti-Rabbit IgG (H + L) (Jackson, 11-035-144, dilution: 1:4000); Peroxidase AffiniPure Goat Anti-Mouse IgG (H + L) (Jackson, 115-035-146, dilution: 1:4000); Peroxidase AffiniPure Goat Anti-Rat IgG (H + L) (Jackson, 112-035-143, dilution: 1:4000).

Techniques: Transfection, Immunoprecipitation, Western Blot, Incubation, Software

Journal: EBioMedicine

Article Title: SPOP promotes ubiquitination and degradation of LATS1 to enhance kidney cancer progression

doi: 10.1016/j.ebiom.2020.102795

Figure Lengend Snippet: Cullin3 SPOP is the physiological E3 ubiquitin ligase for LATS1. (a) IB analysis of WCLs derived from 293T, 786-O and A498 cells treated with 10μM MG132 for 10 hr. (b) IB analysis of immunoprecipitates (IPs) and WCLs derived from 293T kidney cells transfected with indicated constructs. Cells were treated with MG132 (10 μM) before harvesting. (c) IB analysis of WCLs derived from A498 cells transfected with Cullin3 siRNA. (d) IB analysis of WCLs derived from 786-O cells after the specified duration of 100 μg/ml cycloheximide (CHX) transfected with Cullin3 siRNA. (e) The abundance of LATS1 protein in (d) was quantified and plotted. (f) IB analysis of WCLs and immunoprecipitates (IPs) derived from 293T cells transfected with indicated constructs. Cells were treated with MG132 (10μM) before harvesting. (g) IB analysis of WCLs derived from 293T kidney cells transfected with increasing doses of plasmid encoding SPOP. (h) IB analysis of WCLs derived from 786-O cells transfected with increasing doses of plasmid encoding SPOP. (i) IB analysis of WCLs derived from 293T kidney cells or A498 cells transfected with indicated constructs. Where indicated, cells were treated with 10μM MG132 before harvesting. (j) IB analysis of WCLs and products of ubiquitination derived from 293T cells transfected with indicated constructs.

Article Snippet: Anti-Cul3(2759), anti-LATS1(3477) and anti-CK1(12417) antibodies were purchased from Cell Signaling.

Techniques: Derivative Assay, Transfection, Construct, Plasmid Preparation

Journal: EBioMedicine

Article Title: SPOP promotes ubiquitination and degradation of LATS1 to enhance kidney cancer progression

doi: 10.1016/j.ebiom.2020.102795

Figure Lengend Snippet: Cullin3 SPOP E3 ubiquitin ligase negatively regulates the protein stability of LATS1. (a) IB analysis of WCLs derived from A498 or 786-O cells infected with the indicated lentiviral shRNA vectors. (b) IB analysis of WCLs derived from 293T or A498 cells transfected with HA-tagged SPOP plasmid. (c) Quantitative real-time PCR (qRT-PCR) analysis to detect LATS1 and SPOP mRNA levels derived from 786-O or A498 cells after depletion of SPOP. Data are shown as mean ±SD of three independent experiments. * P <0.05, Student's t test. (d) IB analysis of WCLs derived from 293T cells after the specified duration of cycloheximide (CHX) transfected with Myc-tagged SPOP plasmid. (e) The abundance of LATS1 protein in (d) was quantified and plotted. (f) IB analysis of WCLs derived from A498 cells after the specified duration of 100μg/ml cycloheximide (CHX) infected with indicated lentiviral shRNA vectors. (g) The abundance of LATS1 protein in (f) was quantified and plotted.

Article Snippet: Anti-Cul3(2759), anti-LATS1(3477) and anti-CK1(12417) antibodies were purchased from Cell Signaling.

Techniques: Derivative Assay, Infection, shRNA, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: EBioMedicine

Article Title: SPOP promotes ubiquitination and degradation of LATS1 to enhance kidney cancer progression

doi: 10.1016/j.ebiom.2020.102795

Figure Lengend Snippet: SPOP-mediated ubiquitination and degradation of LATS1 depends on the degron motif. (a) Amino acid sequence alignment of LATS1 with the SPOP–binding motif (degron) in known substrates of SPOP. (b) IB analysis of WCLs derived from 293T cells transfected with indicated plasmids. (c) IB analysis of WCLs and immunoprecipitates (IPs) derived from 293T cells transfected with indicated plasmids and treated with 10μM MG132 for 10 h before harvesting. (d) IB analysis of WCLs derived from 293T cells after the specified duration of 100μg/ml cycloheximide (CHX) transfected with indicated Flag-tagged LATS1 plasmids. (e) The abundance of LATS1 protein in (d) was quantified and plotted. (f) IB analysis of WCLs and immunoprecipitates (IPs) derived from 293T cells transfected with indicated plasmids and treated with 10μM MG132 for 10 hr before harvesting. (g) IB analysis of WCLs and immunoprecipitates (IPs) derived from 293T cells transfected with indicated plasmids and treated with 10μM MG132 for 10 h before harvesting. (h) IB analysis of WCLs derived from 293T cells transfected with indicated plasmids. (i) IB analysis of WCLs derived from 293T cells after the specified duration of 100μg/ml cycloheximide (CHX) transfected with indicated Flag-tagged LATS1 plasmids. (j) The abundance of LATS1 protein in (i) was quantified and plotted.

Article Snippet: Anti-Cul3(2759), anti-LATS1(3477) and anti-CK1(12417) antibodies were purchased from Cell Signaling.

Techniques: Sequencing, Binding Assay, Derivative Assay, Transfection

Journal: EBioMedicine

Article Title: SPOP promotes ubiquitination and degradation of LATS1 to enhance kidney cancer progression

doi: 10.1016/j.ebiom.2020.102795

Figure Lengend Snippet: CKΙδ promotes the interaction and degradation of LATS1 by SPOP. (a) IB analysis of WCLs derived from 293T cells transfected with indicated plasmids. (b) IB analysis of WCLs derived from 293T, A498 and 786-O cells incubated with CKΙ inhibitor IC261 (50μM) or D4476 (20μM) before harvesting. (c) IB analysis of WCLs derived from kidney derived 293T cells transfected with CKΙδ siRNA. (d) IB analysis of WCLs and immunoprecipitates (IPs) derived from 293T kidney cells transfected with indicated plasmids and treated with 10μM MG132 for 10 hr before harvesting. (e) IB analysis of WCLs derived from 293T cells transfected with indicated plasmids. (f) IB analysis of WCLs derived from 293T cells transfected with indicated plasmids. (g) IB analysis of WCLs and immunoprecipitates (IPs) derived from 293T cells transfected with indicated plasmids and treated with 10μM MG132 for 10 h before harvesting.

Article Snippet: Anti-Cul3(2759), anti-LATS1(3477) and anti-CK1(12417) antibodies were purchased from Cell Signaling.

Techniques: Derivative Assay, Transfection, Incubation

Journal: EBioMedicine

Article Title: SPOP promotes ubiquitination and degradation of LATS1 to enhance kidney cancer progression

doi: 10.1016/j.ebiom.2020.102795

Figure Lengend Snippet: SPOP promotes tumorigenesis. (a) 786-O-pcDNA3.1, 786-O-SPOP polyclonal stable kidney cancer cell lines were injected subcutaneously into the BALB/c-nu/nu mice. 40 days later, mice were anesthetic and taken a picture. (b) The tumours were dissected and taken a picture. (c) The weights of the dissected tumours in (b). (d) In vivo tumour growth was measured over the indicated time period. (e) The body weights of the BALB/c-nu/nu mice are measured over the indicated time period. (f) Immunohistochemistry staining of SPOP and LATS1 in tissue sections of xenografted tumours in BALB/c-nu/nu mice injected with the indicated cell lines. Scale bars, 20 μm. (g) IB analysis of the LATS1 protein levels in the dissected tumours. (h) Statistical analyses of the correlation between SPOP and LATS1 expression in cytoplasm of the kidney cancer tissue microarray. (i) Representative images of SPOP and LATS1 IHC from 89 cases of kidney cancer.

Article Snippet: Anti-Cul3(2759), anti-LATS1(3477) and anti-CK1(12417) antibodies were purchased from Cell Signaling.

Techniques: Injection, In Vivo, Immunohistochemistry, Staining, Expressing, Microarray