Journal: eLife

Article Title: Arbidol inhibits human esophageal squamous cell carcinoma growth in vitro and in vivo through suppressing ataxia telangiectasia and Rad3-related protein kinase

doi: 10.7554/eLife.73953

Figure Lengend Snippet: ( a ) Computational docking was used to identify the binding site of Arbidol and ATR. (b-d) Arbidol directly binds to ATR. The ATR pure protein (1 μg) ( b ) and esophageal squamous cell carcinoma (ESCC) cell lysate (500 μg) ( c , d ) were incubated with sepharose 4B beads conjugated with Arbidol or sepharose 4B beads alone. Western blot analysis was used to analyze the pull-down protein. ( e ) The Arbidol pull-down assay and amino acid site mutation of ATR protein. ( f ) In vitro kinase assay. Arbidol attenuated the phosphorylation of minichromosome maintenance 2 (MCM2) by inhibiting the kinase activity of ATR. Purified MCM2 protein was used as the substrate for the in vitro kinase assay with 200 ng active ATR (SignalChem) at room temperature for 15 min. ( g ) ATP competition assay. ATR pure protein (1 μg) was incubated for 16 hr with sepharose 4B beads conjugated with Arbidol, and with ATP (10 μg or 100 μg) or sepharose 4B beads alone. Western blot analysis was used to analyze the pull-down protein. Figure 3—source data 1. The original data of western blot in .

Article Snippet: ATR protein was purchased from SignalChem (No. A27-35G).

Techniques: Binding Assay, Incubation, Western Blot, Pull Down Assay, Mutagenesis, In Vitro, Kinase Assay, Phospho-proteomics, Activity Assay, Purification, Competitive Binding Assay

Journal: eLife

Article Title: Arbidol inhibits human esophageal squamous cell carcinoma growth in vitro and in vivo through suppressing ataxia telangiectasia and Rad3-related protein kinase

doi: 10.7554/eLife.73953

Figure Lengend Snippet: ( a ) Coomassie blue-stained gel of purified minichromosome maintenance 2 (MCM2), the black arrow indicates MCM2. ( b ) Mutant ataxia telangiectasia and Rad3-related (ATR) protein purified by coomassie bright blue staining gel; wild type protein was loaded into the first lane, mutant ATR protein was loaded into the the second and third lanes.

Article Snippet: ATR protein was purchased from SignalChem (No. A27-35G).

Techniques: Staining, Purification, Mutagenesis

Journal: Molecular Cancer Research

Article Title: Phosphoproteomics Reveals the Role of Constitutive KAP1 Phosphorylation by B-cell Receptor Signaling in Chronic Lymphocytic Leukemia

doi: 10.1158/1541-7786.mcr-21-0722

Figure Lengend Snippet: Figure 3. KAP1 S473 phosphorylation is regulated by BCR signaling. A, Immunoblotting showing the levels of KAP1 S473 phosphorylation and total KAP1 in CD19þ B cells isolated from peripheral blood of four patients with CLL and two healthy donors. Immunoblotting showing the levels of KAP1 S473 phosphorylation and total KAP1 after anti-human IgM (25 mg/mL) treatment of peripheral B cells from a healthy donor (B) and a patient with CLL (C). D, Immunoblotting showing the levels of KAP1 S473 phosphorylation and KAP1 protein at different time points after anti-human IgM stimulation (25 mg/mL) of multiple CLL cell lines. E, Immunoblotting showing the levels of phosphorylation of BTK in Mec1 and JVM-3 CLL cell lines after ibrutinib treatment. F, Immunoblotting showing the levels of phosphorylation of KAP1 in Mec1 and JVM-3 CLL cell lines after ibrutinib treatment. G, Immunoblotting showing the levels of indicated proteins at different time points in anti-IgM (25 mg/mL) stimulated CLL cells from patients in the absence or presence of ibrutinib (10 mmol/L) treatment. H, Immunoblotting showing the levels of KAP1 S473 phosphorylation and KAP1 protein in Mec1 CLL cells after treatment with the indicated inhibitors at the following concentration: ibrutinib (10 mmol/L), JNK inhibitor (SP600126, 20 mmol/L), Chk1 inhibitor (LY260318, 20 nmol/L), Chk2 inhibitor (DV1019, 1 mmol/L), and PKC inhibitor (G€o6983, 30 nmol/L). The ratio of phosphorylated KAP1 at S473 to total KAP1, as well as the ratio of phosphorylated BTK to total BTK in each line of blots in A, B, C, D, F, G, and H were indicated. Actin served as the protein loading control and was not used for the calculation of the ratio of KAP1 phosphorylation. H, healthy donors.

Article Snippet: CLL cell lines including Mec1 (RRID: CVCL_1870), JVM-2 (RRID: CVCL_1319), JVM-3 (RRID: CVCL_1320), JVM-13 (RRID: CVCL_1318) were purchased from DSMZ (Leibniz, Germany) and were passaged for experiments within 3 months after thawing.

Techniques: Phospho-proteomics, Western Blot, Isolation, Concentration Assay, Control

Journal: Molecular Cancer Research

Article Title: Phosphoproteomics Reveals the Role of Constitutive KAP1 Phosphorylation by B-cell Receptor Signaling in Chronic Lymphocytic Leukemia

doi: 10.1158/1541-7786.mcr-21-0722

Figure Lengend Snippet: Figure 4. KAP1 S473 phosphorylation promotes cell-cycle progression of CLL cells. A, Cell proliferation, as determined by CellVue dye dilution, of Mec1 and JVM-3 CLL cell lines following indicated treatments. B, Immunoblotting showing the knockdown of KAP1 expression in Mec1 cells by two independent shKAP1s. C, Schematic flowchart of coculture of primary CLL cells with L4.5puro feeder cells. CLL cells were transduced with a lentiviral vector carrying a puromycin-resistant gene either plus shRNA against KAP1 or not. Cell-cycle distribution, including cells in the apoptotic and dead phase (A/D), was determined by PI staining at days 0, 3, and 5. D, Hoechst 33342 staining showing the cell-cycle distribution of vector-transduced or KAP1- knockdown Mec1 cells. E, Knockdown of KAP1 caused increased mRNA levels of WEE1, CDC2, and CDC25A in Mec1 cells. qRT-PCR showing the relative levels of indicated mRNA in the sorted G2–M stage of KAP1-knockdown and control knockdown Mec1 cells. F, Immunoblotting showing that knockdown of KAP1 in Mec1 cells caused increased WEE1 protein levels. G, Expression of exogenous Flag- tagged WT KAP1 and KAP1(S473E) in vector-transduced or KAP1-knockdown Mec1 cells. H, Hoechst 33342 staining showing cell-cycle distribution of cells described in G. I, Statistics analysis of the result in H.J,Immunoblotting showing the equal levels of exogenous WT KAP1, KAP1-S473A, and KAP1-S473E in Mec1 cells. K, Cell proliferation analysis of WT KAP1, KAP1-S473A, and KAP1-S473E in vector control-transduced or KAP1-knockdown Mec1 cells. Results in the right panel of D and I are mean SEM (n ¼ 3). Actin served as the internal control in B, F, G, and J. , P < 0.05, , P < 0.01, and P < 0.001. D0, day 0; D1, day 1; D2, day 2; D3; day 3; D5, day 5.

Article Snippet: CLL cell lines including Mec1 (RRID: CVCL_1870), JVM-2 (RRID: CVCL_1319), JVM-3 (RRID: CVCL_1320), JVM-13 (RRID: CVCL_1318) were purchased from DSMZ (Leibniz, Germany) and were passaged for experiments within 3 months after thawing.

Techniques: Phospho-proteomics, Western Blot, Knockdown, Expressing, Transduction, Plasmid Preparation, shRNA, Staining, Quantitative RT-PCR, Control