Journal: Cell Death & Disease

Article Title: ELK3 destabilization by speckle-type POZ protein suppresses prostate cancer progression and docetaxel resistance

doi: 10.1038/s41419-024-06647-0

Figure Lengend Snippet: a Dephosphorylation of ELK3 prevent the interaction between ELK3 and SPOP. The cell lysates of HEK293T cells transiently transfected with indicated plasmids were treated with/without λ-phosphatase. The interaction between ELK3 and SPOP was evaluated by IP and WB. b CHK1/2 inhibition suppresses ELK3 destabilization. The cell lysates of HeLa cells treated with 5 μM of indicated inhibitor and CHX (10 μg/ml) for 12 h were used to evaluate the ELK3 protein levels by WB. c CHK1/2 inhibition abolishes ELK3 and SPOP interaction. The cell lysates of HEK293T cells transfected with indicated plasmids treated with 5 μM of indicated inhibitor for 12 h and MG132 (10 μM) for 4 h were used to evaluate the interaction between ELK3 and SPOP by IP and WB. d ELK3 interacts with CHK1 and CHK2. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the interaction between ELK3 and CHK1 or CHK2 by IP and WB. e CHK2 facilitates ELK3 ubiquitination by SPOP. cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the ELK3 ubiquitination by IP and WB. f CHK1/2 inhibition abrogates SPOP-mediated ELK3 ubiquitination. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with 5 μM of AZD7762 for 12 h and MG132 (10 μM) for 4 h were used to evaluate the SPOP-mediated ELK3 ubiquitination by IP and WB. g CHK1 knockdown increases ELK3 protein levels. The cell lysates of HeLa cells stably expressing sh-mock or sh-CHK1 or CHK2 were used to evaluate ELK3 protein levels by WB. h CHK1 and CHK2 phosphorylates ELK3. In vitro kinase assay using partially purified His-ELK3 and active CHK1 or CHK2 was conducted. ELK3 phosphorylation by CHK1 or CHK2 was evaluated RxxS/T antibody by WB. i ELK3 deg1 deletion abolishes CHK1- or CHK2-mediated phosphorylation. In vitro kinase assay using partial purified His-ELK3-wt or -∆Deg1 and active CHK1 or CHK2 was conducted. ELK3 phosphorylation by CHK1 or CHK2 was evaluated RxxS/T antibody by WB. j , k CHK2 phosphorylates ELK3 at Ser133 in cell system. The phosphorylation of ELK3 mediated by CHK2 in cell culture ( j ) and in vitro kinase assay system ( k ) was evaluated using phos-tag immunoblot analysis. Treatment with AZD7762 ( j ) and mutation of ELK3 Ser133 to Ala ( l ) abolished the phosphorylation of ELK3 induced by CHK2. l ELK3 phosphorylation at Ser133 is indispensable to interact with SPOP. ELK3 phosphorylation requirement for the interaction with SPOP was evaluated by IP and western blotting using cell lysates transiently expressing mock, His-ELK3-wt, His-ELK3-S133D, or His-ELK3-S133A.

Article Snippet: Subsequently, the partially purified ELK3-wt (1 μg) and ELK3-ΔDeg1 (1 μg) were combined with active CHK1 (cat. no.: C47-10H, SignalChem, Richmond, BC, Canada) and CHK2 (cat. no.: C48-10G, SignalChem) and cold ATP.

Techniques: De-Phosphorylation Assay, Transfection, Inhibition, Ubiquitin Proteomics, Knockdown, Stable Transfection, Expressing, In Vitro, Kinase Assay, Purification, Phospho-proteomics, Cell Culture, Western Blot, Mutagenesis

Journal: Nature Communications

Article Title: Aberrant cytoplasmic localization of MLH1 characterizes a cell population that seeds breast cancer recurrence

doi: 10.1038/s41467-025-67257-8

Figure Lengend Snippet: Representative immunofluorescence photomicrographs showing pChk2 ( A ) in cells grown in 2D or PCNA ( B ) in xenografted tumors (red) with a DAPI (blue) nuclear counterstain in indicated cells with indicated treatments. Accompanying quantification is presented as bar graphs from three biological replicates ( A ) and three tumors in each group ( B ). For A Fulvs (WT vs G55V p = 0.0042). For B WT (Veh vs Fulvs p = 0.00012), G55V (Veh vs Fulvs p = 0.038) and Fulvs (WT vs G55V p = 0.0058). Associated data in T47D cells in Fig. . C Bar graphs from three biological replicates showing growth of indicated cells after indicated treatments for 36 h relative to vehicle-treated controls. For Fulvs (WT vs G55V p = 0.0020). Associated data in T47D cells is presented in Fig. . D Representative images of T47D WT and T47D G55V spheroids after indicated treatments with accompanying quantification. Each dot represents an individual spheroid from a biological replicate (n = 5). For Fulvs/Veh (WT vs G55V p = 0.00030) and Fulvs+DIM/Fulvs (WT vs G55V p = 0.0027). E Regression analysis of cytoplasmic MLH1 positivity plotted against nuclear pChk2 positivity in HCI and BCM PDX lines p = 0.0003. F Dot plot representing quantification of cells positive for cytoplasmic localization of MLH1 and nuclear localization of pChk2 in each PDX tumor of the ST panel p = 0.0007. Each symbol represents an individual PDX. Experiments were performed 3 times. Associated representative images in Fig. . Two-sided Student’s t test determined all p-values. *** p ≤ 0.0001, ** p ≤ 0.01 and * p ≤ 0.05. For all graphs, data are presented as mean values ± SD. Vertical line depicts median value in ( F ). Scale bars = 20 µm ( A , B ) and 50 µM ( D ). Abbreviations: WT wild type, DAPI 4’,6-diamidino-2-phenylindole, pChk2 phosphorylated Chk2, PCNA proliferating cell nuclear antigen, cMLH1 cytoplasmic MLH1, nMLH1 nuclear Mlh1, PDXs patient derived xenografts, HCI PDXs from Huntsman Cancer Institute, BCM PDXs from Baylor College of Medicine, Rel. relative, A.U. arbitrary unit, Treatments, Veh vehicle, Fulvs fulvestrant, DIM di-indolyl methane. Source data for all figures available with the paper.

Article Snippet: CHK2 activator (3, 3;-diindolyl methane, cat# sc-204624; Santa Cruz Biotechnology) was used at 100 μmol/L concentrations.

Techniques: Immunofluorescence, Derivative Assay

Journal: Nature Communications

Article Title: Aberrant cytoplasmic localization of MLH1 characterizes a cell population that seeds breast cancer recurrence

doi: 10.1038/s41467-025-67257-8

Figure Lengend Snippet: A Schematic showing how MLH1 activates Chk2 and inhibits CDK4/6 activity. B qRT-PCR analysis of MCF7 cells of indicated genotypes for relative expression of specified genes following fulvestrant treatment from three biological replicates. For CDK6 (WT vs G55V p = 0.012), CDKN1B (WT vs G55V p = 0.0028), CCND1 (WT vs G55V p = 0.010). C Western blotting confirming CDK6 knockdown in MCF7 cells. Representative blot from three biological replicate. D , E Bar graph showing growth of MCF7 WT , MCF7 G55V and MCF7 shMLH1cells with respective treatments from three independent biological replicate. Bar graphs describing relative spheroid size grown from T47D WT , T47D G55V and T47D shMLH1 cells ( F ) and specified PDxO ( G ) in response to palbociclib treatment. For F each dot represents spheroid from an independent biological replicate (n = 5). For G each dot represents a biological replicate (n = 4). All experiments were performed >2 times. For D Fulvs (WT vs G55V p = 0.00002) and (WT vs shMLH1 p = 0.0019). For E Fulvs (WT vs G55V p = 0.00084, WT vs shMLH1 p = 0.0014), Palbo (WT vs G55V p = 0.0022, WT vs shMLH1 p = 0.010). For F WT (Veh vs Palbo p = 0.00013), G55V (Veh vs Palbo p = 0.000019), Palbo (WT vs G55V p = 0.000004). For G (HCI003 vs HCI017 p = 0.000012, HCI007 vs HCI017 p = 0.000012). H , I Line graphs of tumor volume over time for indicated groups with and without palbociclib treatment each line represents an individual tumor. Waterfall plots depicting CDK4/6 inhibitor response expressed as percent growth of treated tumors relative to vehicle-treated controls in PDX lines in response to ( J ) palbociclib (p = 0.011) ( K ) abemaciclib (p = 0.06). For graphs, data are presented as mean values ± SD. Schematic was made using BioRender https://BioRender.com/aok2eve . Two-sided Student’s t test determined p values for ( B – I ) and Fisher’s exact test determined p values for ( J , K ). Abbreviations: WT wild type, nMLH1 nuclear MLH1, cMLH1 cytoplasmic MLH1, pChk2 phosphorylated Chk2, Tx treatments, si small interfering, sh short hairpin, n.s. not significant, rel relative, vol volume, mm 3 millimeter cubed, PDXs patient-derived xenografts, HCI PDXs from Huntsman Cancer Institute, ST PDXs from Xenostart, T/C treatment/control, Treatments, Fulvs fulvestrant, Palbo palbociclib. Source data for all figures available with the paper.

Article Snippet: CHK2 activator (3, 3;-diindolyl methane, cat# sc-204624; Santa Cruz Biotechnology) was used at 100 μmol/L concentrations.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Western Blot, Knockdown, Derivative Assay, Control

Journal: Cell reports

Article Title: Epinephrine inhibits PI3Kα via the Hippo kinases

doi: 10.1016/j.celrep.2023.113535

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CHK2 , SignalChem , C48–10G.

Techniques: Recombinant, Fluorescence, Isolation, Double Knockout, Mutagenesis, Plasmid Preparation, Software