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Novus Biologicals antibodies against elk3

a – c Determination of <t>ELK3</t> degradation pathways. The cell lysates extracted from HeLa cells treated with MG132 ( a ), chloroquine ( b ), or MLN4924 ( c ) were used to visualize ELK3 by WB. d ELK3 interaction with Cullin3. The cell lysates extracted from HEK293T cells transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the interaction between ELK3 and each of Cullins by IP and WB. e Cullin3 decreases ELK3 half-life. The cell lysates extracted from HEK293T cells transfected with indicated plasmids and treated with cycloheximide (CHX, 10 μg/ml) were used to evaluate the half-life of ELK3 by WB. f Knockdown of Cullin3 increases ELK3. The cell lysates extracted from HeLa cells stably expressing sh-mock or sh-CUL3 were used to evaluate the ELK3 protein levels by WB. g Cullin3 knockdown inhibits ELK3 destabilization. The cell lysates extracted from PC-3 cells stably expressing sh-mock or sh-Cul3 and treated with CHX (10 μg/ml) were used to evaluate the ELK3 half-life by WB. h Cullin3 induces ELK3 ubiquitination. The cell lysates extracted from HEK293T cells transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the ELK3 ubiquitination by IP and WB. Graphs in e and g The band intensity ELK3 bands measured using NIH image J computer program and normalized by β-actin intensity were plotted. Data, three independent experiments; Significance, * p < 0.05 obtained by Student t test.

Antibodies Against Elk3, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+elk3/ELK3+Antibody+(OTI1H3)/pmc11024157-86-7-15
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antibodies against elk3 - by Bioz Stars, 2026-09

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1) Product Images from "ELK3 destabilization by speckle-type POZ protein suppresses prostate cancer progression and docetaxel resistance"

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

Journal: Cell Death & Disease

doi: 10.1038/s41419-024-06647-0

a – c Determination of ELK3 degradation pathways. The cell lysates extracted from HeLa cells treated with MG132 ( a ), chloroquine ( b ), or MLN4924 ( c ) were used to visualize ELK3 by WB. d ELK3 interaction with Cullin3. The cell lysates extracted from HEK293T cells transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the interaction between ELK3 and each of Cullins by IP and WB. e Cullin3 decreases ELK3 half-life. The cell lysates extracted from HEK293T cells transfected with indicated plasmids and treated with cycloheximide (CHX, 10 μg/ml) were used to evaluate the half-life of ELK3 by WB. f Knockdown of Cullin3 increases ELK3. The cell lysates extracted from HeLa cells stably expressing sh-mock or sh-CUL3 were used to evaluate the ELK3 protein levels by WB. g Cullin3 knockdown inhibits ELK3 destabilization. The cell lysates extracted from PC-3 cells stably expressing sh-mock or sh-Cul3 and treated with CHX (10 μg/ml) were used to evaluate the ELK3 half-life by WB. h Cullin3 induces ELK3 ubiquitination. The cell lysates extracted from HEK293T cells transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the ELK3 ubiquitination by IP and WB. Graphs in e and g The band intensity ELK3 bands measured using NIH image J computer program and normalized by β-actin intensity were plotted. Data, three independent experiments; Significance, * p < 0.05 obtained by Student t test.

Figure Legend Snippet: a – c Determination of ELK3 degradation pathways. The cell lysates extracted from HeLa cells treated with MG132 ( a ), chloroquine ( b ), or MLN4924 ( c ) were used to visualize ELK3 by WB. d ELK3 interaction with Cullin3. The cell lysates extracted from HEK293T cells transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the interaction between ELK3 and each of Cullins by IP and WB. e Cullin3 decreases ELK3 half-life. The cell lysates extracted from HEK293T cells transfected with indicated plasmids and treated with cycloheximide (CHX, 10 μg/ml) were used to evaluate the half-life of ELK3 by WB. f Knockdown of Cullin3 increases ELK3. The cell lysates extracted from HeLa cells stably expressing sh-mock or sh-CUL3 were used to evaluate the ELK3 protein levels by WB. g Cullin3 knockdown inhibits ELK3 destabilization. The cell lysates extracted from PC-3 cells stably expressing sh-mock or sh-Cul3 and treated with CHX (10 μg/ml) were used to evaluate the ELK3 half-life by WB. h Cullin3 induces ELK3 ubiquitination. The cell lysates extracted from HEK293T cells transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the ELK3 ubiquitination by IP and WB. Graphs in e and g The band intensity ELK3 bands measured using NIH image J computer program and normalized by β-actin intensity were plotted. Data, three independent experiments; Significance, * p < 0.05 obtained by Student t test.

Techniques Used: Transfection, Knockdown, Stable Transfection, Expressing, Ubiquitin Proteomics

a , b ELK3 interacts with SPOP. The cell lysates extracted from HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to determine ELK3 interaction to E3 ligases by IP and WB. c Evaluation of endogenous interaction between ELK3 and SPOP. The cell lysates extracted from HeLa cells treated with MG132 (10 μM) for 8 h were used to evaluate the ELK3 and SPOP interaction by IP and WB. d Confirmation of ELK3 and SPOP interaction. The cell lysates extracted from HEK293T cells transiently transfected with His-ELK3 plasmid were used to confirm the ELK3 and partially purified bacterial His-SPOP by pulldown assay (PD) and WB. e SPOP induces ELK3 destabilization. The cell lysates extracted from HEK293T cells transiently transfected with His-ELK3 and dose-increasing Myc-SPOP plasmids were used to evaluate ELK3 protein level by WB. GFP was used as an internal control for equal transfection. f SPOP accelerates ELK3 destabilization. The cell lysates extracted from HEK293T cells transiently transfected with indicated plasmids and treated with CHX (10 μg/ml) were used to evaluate the ELK3 half-life by WB. g SPOP knockdown increases ELK3 protein levels. The cell lysates extracted from HeLa cells stably expressing sh-mock or sh-SPOP were used to evaluate the ELK3 protein levels by WB. h Depletion of SPOP did not affect ELK3 RNA expression. The levels of ELK3 mRNA extracted from HeLa cells stably expressing sh-mock or sh-SPOP were assessed by quantitative real-time RT-PCR using TaqMan RNA-to-CT 1-step kit. i SPOP knockdown prolongs ELK3 half-life. The cell lysates extracted from PC-3 cells stably expressing sh-mock or sh-SPOP and treated with CHX (10 μg/ml) were used to evaluate ELK3 half-life by WB. j , k SPOP induces ELK3 ubiquitination in cell and in vitro ubiquitination assay system. The cell lysates extracted from HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate ELK3 ubiquitination by IP and WB ( j ). In vitro ubiquitination assay was conducted using commercially available E1, E2, and ubiquitin (Ubi) proteins, along with His-tagged ELK3 purified from E. coli and Myc-tagged SPOP isolated from HEK293T cells overexpressing SPOP ( k ). Graphs in f and i The band intensity of ELK3 quantified by NIH image J computer program normalized by β-actin intensity was plotted. Data, three independent experiments; Significance, * p < 0.05 obtained by Student t test.

Figure Legend Snippet: a , b ELK3 interacts with SPOP. The cell lysates extracted from HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to determine ELK3 interaction to E3 ligases by IP and WB. c Evaluation of endogenous interaction between ELK3 and SPOP. The cell lysates extracted from HeLa cells treated with MG132 (10 μM) for 8 h were used to evaluate the ELK3 and SPOP interaction by IP and WB. d Confirmation of ELK3 and SPOP interaction. The cell lysates extracted from HEK293T cells transiently transfected with His-ELK3 plasmid were used to confirm the ELK3 and partially purified bacterial His-SPOP by pulldown assay (PD) and WB. e SPOP induces ELK3 destabilization. The cell lysates extracted from HEK293T cells transiently transfected with His-ELK3 and dose-increasing Myc-SPOP plasmids were used to evaluate ELK3 protein level by WB. GFP was used as an internal control for equal transfection. f SPOP accelerates ELK3 destabilization. The cell lysates extracted from HEK293T cells transiently transfected with indicated plasmids and treated with CHX (10 μg/ml) were used to evaluate the ELK3 half-life by WB. g SPOP knockdown increases ELK3 protein levels. The cell lysates extracted from HeLa cells stably expressing sh-mock or sh-SPOP were used to evaluate the ELK3 protein levels by WB. h Depletion of SPOP did not affect ELK3 RNA expression. The levels of ELK3 mRNA extracted from HeLa cells stably expressing sh-mock or sh-SPOP were assessed by quantitative real-time RT-PCR using TaqMan RNA-to-CT 1-step kit. i SPOP knockdown prolongs ELK3 half-life. The cell lysates extracted from PC-3 cells stably expressing sh-mock or sh-SPOP and treated with CHX (10 μg/ml) were used to evaluate ELK3 half-life by WB. j , k SPOP induces ELK3 ubiquitination in cell and in vitro ubiquitination assay system. The cell lysates extracted from HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate ELK3 ubiquitination by IP and WB ( j ). In vitro ubiquitination assay was conducted using commercially available E1, E2, and ubiquitin (Ubi) proteins, along with His-tagged ELK3 purified from E. coli and Myc-tagged SPOP isolated from HEK293T cells overexpressing SPOP ( k ). Graphs in f and i The band intensity of ELK3 quantified by NIH image J computer program normalized by β-actin intensity was plotted. Data, three independent experiments; Significance, * p < 0.05 obtained by Student t test.

Techniques Used: Transfection, Plasmid Preparation, Purification, Control, Knockdown, Stable Transfection, Expressing, RNA Expression, Quantitative RT-PCR, Ubiquitin Proteomics, In Vitro, Isolation

a ELK3 interacted with MATH domain of SPOP. Upper panel Schematic diagram of SPOP domains. The numbers at the MATH domain indicates SPOP mutations observed in PCa patients. Bottom panels The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated MG132 (10 μM) for 4 h were used to determine the SPOP’s binding domain by IP and WB. b MATH domain deletion of SPOP suppressed ELK3 ubiquitination. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated MG132 (10 μM) for 4 h were used to evaluate the SPOP-dependent ELK3 ubiquitination by IP and WB. c The deletion of the MATH or BTB domain of SPOP abolishes ELK3 destabilization. The cell lysates of HEK293T cells transiently transfected with indicated plasmids were used to evaluate ELK3 protein levels by WB. GFP was used as an internal control for equal transfection. d MATH domain mutation of SPOP abrogates interaction with ELK3. The cell lysates of HEK293T cells transiently transfected with the indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the interaction between SPOP and ELK3 by IP and WB. e MATH-F133V mutation of SPOP critically affects ELK3 stability. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with CHX (10 μg/ml) for indicated period were used to evaluate ELK3 protein levels by WB. The band intensity of ELK3 quantified by NIH image J computer program normalized by β-actin intensity was plotted. GFP was used an internal control for equal transfection. Data, three independent experiments; Significance, * p < 0.05 obtained by Student t test. f SPOP mutations at MATH domain inhibits endogenous ELK3 destabilization. The cell lysates of 22Rv1 cells stably expressing SPOP-wt or each of SPOP mutants (Y87C, W131G, and F133V) were used to evaluate ELK3 protein levels by WB. g SPOP mutations at MATH domain abolishes ELK3 ubiquitination. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate ELK3 ubiquitination by IP and WB.

Figure Legend Snippet: a ELK3 interacted with MATH domain of SPOP. Upper panel Schematic diagram of SPOP domains. The numbers at the MATH domain indicates SPOP mutations observed in PCa patients. Bottom panels The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated MG132 (10 μM) for 4 h were used to determine the SPOP’s binding domain by IP and WB. b MATH domain deletion of SPOP suppressed ELK3 ubiquitination. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated MG132 (10 μM) for 4 h were used to evaluate the SPOP-dependent ELK3 ubiquitination by IP and WB. c The deletion of the MATH or BTB domain of SPOP abolishes ELK3 destabilization. The cell lysates of HEK293T cells transiently transfected with indicated plasmids were used to evaluate ELK3 protein levels by WB. GFP was used as an internal control for equal transfection. d MATH domain mutation of SPOP abrogates interaction with ELK3. The cell lysates of HEK293T cells transiently transfected with the indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the interaction between SPOP and ELK3 by IP and WB. e MATH-F133V mutation of SPOP critically affects ELK3 stability. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with CHX (10 μg/ml) for indicated period were used to evaluate ELK3 protein levels by WB. The band intensity of ELK3 quantified by NIH image J computer program normalized by β-actin intensity was plotted. GFP was used an internal control for equal transfection. Data, three independent experiments; Significance, * p < 0.05 obtained by Student t test. f SPOP mutations at MATH domain inhibits endogenous ELK3 destabilization. The cell lysates of 22Rv1 cells stably expressing SPOP-wt or each of SPOP mutants (Y87C, W131G, and F133V) were used to evaluate ELK3 protein levels by WB. g SPOP mutations at MATH domain abolishes ELK3 ubiquitination. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate ELK3 ubiquitination by IP and WB.

Techniques Used: Transfection, Binding Assay, Ubiquitin Proteomics, Control, Mutagenesis, Stable Transfection, Expressing

a Identification of ELK3 degron motifs for SPOP. ELK3 amino acid sequences were aligned to search putative degron motifs for SPOP. b Determination of ELK3 degron motif interacting to SPOP. 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 ELK3 degron motif by IP and WB. c Involvement of ELK3 Deg1 in its stabilization. The cell lysates of HEK293T cells transiently transfected with indicated plasmids were used to determine the ELK3 degron motif by WB. GFP was used an internal control for equal transfection. d ELK3 Deg1 deletion abrogates SPOP-mediated destabilization. Left panels The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with CHX (10 μg/ml) for an indicated time were used to evaluate the ELK3 protein levels by WB. GFP was used as an internal control for equal transfection. Graph The band intensity of ELK3 quantified by NIH image J computer program normalized by β-actin intensity was plotted. GFP was used an internal control for equal transfection. Data, three independent experiments; Significance, * p < 0.05 obtained by Student t test. e ELK3 Deg1 deletion abolishes SPOP-mediated ubiquitination. The cell lysate of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate SPOP-mediated ELK3 ubiquitination by IP and WB. f , g Protein–protein docking of ELK3 and SPOP. The SPOP structure (PDB ID: 3HQI) and ELK3 built by amino acid similarly modeling using Discovery Studio were used to predict SPOP and ELK3 interaction interface. Magenta, ELK3; Green, SPOP. The amino acids and interaction types involved in interaction between ELK3 and SPOP are provided in Supplementary Fig. .

Figure Legend Snippet: a Identification of ELK3 degron motifs for SPOP. ELK3 amino acid sequences were aligned to search putative degron motifs for SPOP. b Determination of ELK3 degron motif interacting to SPOP. 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 ELK3 degron motif by IP and WB. c Involvement of ELK3 Deg1 in its stabilization. The cell lysates of HEK293T cells transiently transfected with indicated plasmids were used to determine the ELK3 degron motif by WB. GFP was used an internal control for equal transfection. d ELK3 Deg1 deletion abrogates SPOP-mediated destabilization. Left panels The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with CHX (10 μg/ml) for an indicated time were used to evaluate the ELK3 protein levels by WB. GFP was used as an internal control for equal transfection. Graph The band intensity of ELK3 quantified by NIH image J computer program normalized by β-actin intensity was plotted. GFP was used an internal control for equal transfection. Data, three independent experiments; Significance, * p < 0.05 obtained by Student t test. e ELK3 Deg1 deletion abolishes SPOP-mediated ubiquitination. The cell lysate of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate SPOP-mediated ELK3 ubiquitination by IP and WB. f , g Protein–protein docking of ELK3 and SPOP. The SPOP structure (PDB ID: 3HQI) and ELK3 built by amino acid similarly modeling using Discovery Studio were used to predict SPOP and ELK3 interaction interface. Magenta, ELK3; Green, SPOP. The amino acids and interaction types involved in interaction between ELK3 and SPOP are provided in Supplementary Fig. .

Techniques Used: Transfection, Control, Ubiquitin Proteomics

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.

Figure Legend 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.

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

a , b SPOP inhibits ELK3-mediated c-fos promoter activity. HEK293T cells transiently transfected with indicated plasmids were used to evaluate c-fos promoter activity by luciferase assay. c SPOP knockdown increases c-fos promoter activity. HEK293T cells stably expressing sh-SPOP were transfected with c-fos -luc reporter plasmid and evaluated c-fos promoter activity by luciferase assay. d Confirmation of increased c-Fos protein by SPOP knockdown. The cell lysates of 22Rv1 cells stably expressing sh-SPOP were used to evaluate the c-Fos protein level by WB. e Establishment of ELK3 and SPOP double knockdown cells. The cell lysates of 22Rv1 cells stably expressing sh-SPOP and/or sh-ELK3 as indicated were used to evaluate the SPOP and c-Fos protein levels by WB. f Increased cell proliferation by SPOP knockdown suppresses cell proliferation by ELK3. The 22Rv1 cells in e were used to evaluate the effects of cell proliferation by CCK-8 assay. g Increased cell invasion by SPOP knockdown abrogated by ELK3 knockdown. The 22Rv1 cells in e were used to evaluate the effects of cell invasion by cell invasion assay using Matrigel Invasion Chamber. a – c and f , g Data, three independent experiments; Significance, * p < 0.05 obtained by Student t -test.

Figure Legend Snippet: a , b SPOP inhibits ELK3-mediated c-fos promoter activity. HEK293T cells transiently transfected with indicated plasmids were used to evaluate c-fos promoter activity by luciferase assay. c SPOP knockdown increases c-fos promoter activity. HEK293T cells stably expressing sh-SPOP were transfected with c-fos -luc reporter plasmid and evaluated c-fos promoter activity by luciferase assay. d Confirmation of increased c-Fos protein by SPOP knockdown. The cell lysates of 22Rv1 cells stably expressing sh-SPOP were used to evaluate the c-Fos protein level by WB. e Establishment of ELK3 and SPOP double knockdown cells. The cell lysates of 22Rv1 cells stably expressing sh-SPOP and/or sh-ELK3 as indicated were used to evaluate the SPOP and c-Fos protein levels by WB. f Increased cell proliferation by SPOP knockdown suppresses cell proliferation by ELK3. The 22Rv1 cells in e were used to evaluate the effects of cell proliferation by CCK-8 assay. g Increased cell invasion by SPOP knockdown abrogated by ELK3 knockdown. The 22Rv1 cells in e were used to evaluate the effects of cell invasion by cell invasion assay using Matrigel Invasion Chamber. a – c and f , g Data, three independent experiments; Significance, * p < 0.05 obtained by Student t -test.

Techniques Used: Activity Assay, Transfection, Luciferase, Knockdown, Stable Transfection, Expressing, Plasmid Preparation, CCK-8 Assay, Invasion Assay

a Inverse correlation of ELK3 and phospho-CHK1/2 protein levels under docetaxel treatment. The cell lysates of 22Rv1 cells treated with docetaxel (300 nM) for an indicated time were used to evaluate the indicated protein levels by WB. b Rescue of docetaxel-mediated decreased ELK3 protein level by MG132 treatment. The 22Rv1 cell lysates treated with docetaxel (300 nM) in the presence or absence of MG132 (10 μM) were used to evaluate ELK3 protein levels by WB. c Docetaxel-mediated decreased ELK3 protein is rescued by SPOP knockdown. The cell lysates of 22Rv1 cells stably expressing sh-SPOP and treated with docetaxel (300 nM) were used to evaluate ELK3 protein level by WB. d Docetaxel induces SPOP and ELK3 interaction. The cell lysates of HEK293T cells transfected with indicated plasmids and treated with docetaxel (300 nM) were used to evaluate the SPOP and ELK3 interaction by IP and WB. e Docetaxel induces ELK3 ubiquitination. The cell lysates of HEK293T cells transfected with indicated plasmids and treated with docetaxel (300 nM) were used to evaluate ELK3 ubiquitination by IP and WB. f Diminished interaction between SPOP and ELK3 increases PCa cell survivability. 22Rv1 cells stably expressing SPOP-WT or each of SPOP mutants treated with docetaxel (1 μM) for 48 h were used to cell survivability by CCK-8 assay. g SPOP and ELK3 double knockdown inhibits cell survivability by docetaxel in SPOP knockdown cells. 22Rv1 cells stably expressing sh-SPOP and/or sh-ELK3 treated with docetaxel (1 μM) for 48 h were used to evaluate cell survivability by CCK-8 assay. h SPOP and ELK3 double knockdown enhances apoptosis signaling by docetaxel. The cell lysates of 22Rv1 cells stably expressing sh-SPOP and/or sh-ELK3 treated with docetaxel (300 nM) were used to evaluate the protein levels of apoptosis-related signaling molecules by WB. i , j The SPOP-ELK3 axis plays a critical role in docetaxel-induced cell death. The cell lysates of 22Rv1 cells stably expressing sh-SPOP and/or SPOP were used to evaluate the protein levels of ELK3 and SPOP by WB ( i ), and to assess cell viability using the CCK-8 assay ( j ). k Increased ELK3 protein levels in PCa cancer tissues. IHC images of ELK3 in prostate cancer tissues ( n = 117) stained by DAB staining were analyzed by TissueFAXS system. Data, DAB intensity in normal vs. cancer tissues; Significance was obtained by Anova t test. l Increased ELK3 protein levels in PCa patient harboring SPOP-F133V mutation. Representative IHC image of ELK3 protein levels by DAB staining in prostate cancer tissues harboring SPOP-WT or SPOP-F133V. f , g Data, three independent experiments; Significance, * p < 0.05 obtained by Student t test.

Figure Legend Snippet: a Inverse correlation of ELK3 and phospho-CHK1/2 protein levels under docetaxel treatment. The cell lysates of 22Rv1 cells treated with docetaxel (300 nM) for an indicated time were used to evaluate the indicated protein levels by WB. b Rescue of docetaxel-mediated decreased ELK3 protein level by MG132 treatment. The 22Rv1 cell lysates treated with docetaxel (300 nM) in the presence or absence of MG132 (10 μM) were used to evaluate ELK3 protein levels by WB. c Docetaxel-mediated decreased ELK3 protein is rescued by SPOP knockdown. The cell lysates of 22Rv1 cells stably expressing sh-SPOP and treated with docetaxel (300 nM) were used to evaluate ELK3 protein level by WB. d Docetaxel induces SPOP and ELK3 interaction. The cell lysates of HEK293T cells transfected with indicated plasmids and treated with docetaxel (300 nM) were used to evaluate the SPOP and ELK3 interaction by IP and WB. e Docetaxel induces ELK3 ubiquitination. The cell lysates of HEK293T cells transfected with indicated plasmids and treated with docetaxel (300 nM) were used to evaluate ELK3 ubiquitination by IP and WB. f Diminished interaction between SPOP and ELK3 increases PCa cell survivability. 22Rv1 cells stably expressing SPOP-WT or each of SPOP mutants treated with docetaxel (1 μM) for 48 h were used to cell survivability by CCK-8 assay. g SPOP and ELK3 double knockdown inhibits cell survivability by docetaxel in SPOP knockdown cells. 22Rv1 cells stably expressing sh-SPOP and/or sh-ELK3 treated with docetaxel (1 μM) for 48 h were used to evaluate cell survivability by CCK-8 assay. h SPOP and ELK3 double knockdown enhances apoptosis signaling by docetaxel. The cell lysates of 22Rv1 cells stably expressing sh-SPOP and/or sh-ELK3 treated with docetaxel (300 nM) were used to evaluate the protein levels of apoptosis-related signaling molecules by WB. i , j The SPOP-ELK3 axis plays a critical role in docetaxel-induced cell death. The cell lysates of 22Rv1 cells stably expressing sh-SPOP and/or SPOP were used to evaluate the protein levels of ELK3 and SPOP by WB ( i ), and to assess cell viability using the CCK-8 assay ( j ). k Increased ELK3 protein levels in PCa cancer tissues. IHC images of ELK3 in prostate cancer tissues ( n = 117) stained by DAB staining were analyzed by TissueFAXS system. Data, DAB intensity in normal vs. cancer tissues; Significance was obtained by Anova t test. l Increased ELK3 protein levels in PCa patient harboring SPOP-F133V mutation. Representative IHC image of ELK3 protein levels by DAB staining in prostate cancer tissues harboring SPOP-WT or SPOP-F133V. f , g Data, three independent experiments; Significance, * p < 0.05 obtained by Student t test.

Techniques Used: Knockdown, Stable Transfection, Expressing, Transfection, Ubiquitin Proteomics, CCK-8 Assay, Staining, Mutagenesis

A schematic diagram illustrates the working model in which SPOP mutants enhance ELK3 stabilization, leading to increased resistance to docetaxel-induced cell death. Consequently, the SPOP-ELK3 axis plays a pivotal role in the progression of prostate cancer (PCa).

Figure Legend Snippet: A schematic diagram illustrates the working model in which SPOP mutants enhance ELK3 stabilization, leading to increased resistance to docetaxel-induced cell death. Consequently, the SPOP-ELK3 axis plays a pivotal role in the progression of prostate cancer (PCa).

Techniques Used:

Chromatin Immunoprecipitation:

Article Title: The Elk-3 target Abhd10 ameliorates hepatotoxic injury and fibrosis in alcoholic liver disease
Article Snippet: Oligonucleotides used to amplify the ABHD10 and GAPDH promoter regions were designed and synthesized by Shanghai Sangon Biotech (China). .. All ChIP-PCR steps were conducted with an EZ-ChIP kit (Millipore) with the overnight immunoprecipitation step performed with anti-ELK3 (5 μg, #NBP1-83960, Novus) or control rabbit IgG (5 μg, #NBP2-50261, Novus). .. RIPA buffer (0.1% SDS, 150 mM NaCl, 50 mM Tris pH 7.5, 1% Triton X-100) supplemented with freshly added cOmplete EDTA-free protease inhibitors (Sigma), 40 mM DTT, and phosphatase inhibitors (2 mM NaF, 1 mM Na 3 VO 4 , 2 mM β-glycerophosphate) was used to lyse cells and tissue samples.

Article Title: The Elk-3 target Abhd10 ameliorates hepatotoxic injury and fibrosis in alcoholic liver disease.
Article Snippet: Oligonucleotides used to amplify the ABHD10 and GAPDH promoter regions were designed and synthesized by Shanghai Sangon Biotech (China). .. All ChIP-PCR steps were conducted with an EZ-ChIP kit (Millipore)17 with the overnight immunoprecipitation step performed with anti-ELK3 (5 μg, #NBP1-83960, Novus) or control rabbit IgG (5 μg, #NBP250261, Novus). ..

Immunoprecipitation:

Article Title: Early Progression of Abdominal Aortic Aneurysm is Decelerated by Improved Endothelial Barrier Function via ALDH2‐LIN28B‐ELK3 Signaling
Article Snippet: .. The lysates were pre-cleaned with a mixture of salmon sperm DNA-protein A/G, followed by immunoprecipitation with an anti-ELK3 (Novus Biologicals, NBP1-83960) or normal IgG (Novus Biologicals, NBP2-24891) antibody overnight at 4°C. .. Then, single-strand salmon sperm DNA saturated with protein A/G plus-agarose beads (Santa Cruz Biotechnology, sc-2003) was added and incubated for 1 hours.

Control:

other:

Article Title: ELK3 destabilization by speckle-type POZ protein suppresses prostate cancer progression and docetaxel resistance
Article Snippet: Anti-ELK3 (cat. no.: NBP2-01264) antibody was purchased from Novus (St. Louis, MO, USA).

Article Title: Circular RNA circTmem241 drives group III innate lymphoid cell differentiation via initiation of Elk3 transcription
Article Snippet: Anti-Elk3 (Cat# NBP1-83960) was from Novus Biologicals.

Western Blot:

Article Title: Circular RNA circTmem241 drives group III innate lymphoid cell differentiation via initiation of Elk3 transcription
Article Snippet: For ChIP experiments: Anti-H3K4me3 (Cell Signalling Technology, #9751S, C42D8, 2microgram/sample), anti-H3K36me3 (Bioworld, #BS7239, 2microgram/sample) and anti-Ash1l (Bioss, #bs-9671R, 2 microgram/sample). .. For western blot: Anti-Elk3 (Novus Biologicals, #NBP1-83960, 1:500), anti-Tmem241 (Sinobiological, #203644-T32, 1:400), anti-Ash1l (Bioss, #bs-9671R, 1:500), anti-Nono (Proteintech, #66361-1-Ig, 2A2B10, 1:200), anti-Flag tag antibody (Abcam, #ab205606, 1:1000), anti-"-actin (Beijing Ray Antibody Biotech, #RM2001, 1:1000), HRP goat anti-mouse IgG (Beijing Ray Antibody Biotech, #RM3001, 1:3000) and HRP goat anti-rabbit IgG (Beijing Ray Antibody Biotech, #RM3002, 1:3000). .. For FACS experiments: Anti-Lineage cocktail (#88-7772-72, 1:300), Anti-CD127 (#46-1273-82, A7R34, 1:200), anti-Sca-1 (#11-5981-85, D7, 1:200), anti-Flt3 (#12-1351-82, A2F10, 1:300), anti-!4"7 (#17-5887-82, DATK32, 1:200), anti-Id2 (#12-9475-80, ILCID2, 1:300), anti-PLZF (#12-9320-80, Mags.21F7, 1:200), anti-Eomes (#53-4875-82, Dan11mag, 1:200), anti-NKp46 (#11-3351-80, 29A1.4, 1:200), anti-NK1.1 (#17-5941-82, PK136, 1:300), anti-CD45.1 (#45-0453-80, A20, 1:500), anti-CD45.2 (#12-0454-82, 30-F11, 1:500), anti-CD25 (#12-0251-81, PC61.5, 1:300), anti-Gata3 (#25-9966-41, TWAJ, 1:200), anti-ROR#t (#17-6981-80, AFKJS-9, 1:200), anti-CD19 (#12-0193-81, 1D3, 1:500), anti-KLRG1 (#17-5893-82, 2F1, 1:200), anti-CD90 (#11-0903-81, HIS51, 1:500), anti-IL-22 (# 17-7222-80, IL22JOP, 1:200), anti-Ki67 (#11-5698-80, SolA15, 1:300), anti-BrdU (#11-5071-41, BU20A, 1:200) and anti-PD1 (#25-9985-80, J43, 1:300) were purchased from eBiosciences (San Diego, USA).

Incubation:

Article Title: ELK3-CXCL16 axis determines natural killer cell cytotoxicity via the chemotactic activity of CXCL16 in triple negative breast cancer
Article Snippet: Total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to poly(vinylidene fluoride) PVDF membranes (Bio-Rad). .. The membranes were blocked with 5% nonfat milk and then probed overnight at 4°C with anti-ELK3 (Novus Biologicals, NBP2-01264), anti-CXCL16 (Santa Cruz Biotechnology, sc-376395), and anti-GAPDH (Santa Cruz Biotechnology, sc‐166574, Dallas, Texas, USA) antibodies, followed by washing with TBS-T. After washing, the membranes were incubated for 1 h at RT with a secondary antibody (antimouse, GeneTex, GTX213111-01). .. Immunoreactivity was detected using an ECL kit (Thermo Scientific, Rochester, NY, USA) and ImageQuant Las 4000 (GE Healthcare, Chicago,

Image Search Results

( A ) Heatmap showing differentially expressed genes in KOPTK1 cells with or without FTO shRNA expression. Differential genes are derived from RNA-seq data and defined as |fold change| ≥ 1.5, P value <0.05. ( B ) KEGG enrichment analysis of down-regulated transcripts in FTO -depleted KOPTK1 cells. ( C ) Cumulative distribution function representing the log2 peak intensity of m 6 A-modified sites in KOPTK1 cells with or without FTO knockdown. The P value was calculated using two-tailed Kolmogorov-Smirnov test. ( D ) Top consensus sequence motif identified within m 6 A peaks by hypergeometric optimization of motif enrichment (HOMER) in KOPTK1 cells with or without FTO knockdown. ( E ) Metagene profiles of m 6 A peak distribution along a normalized transcript composed of three rescaled nonoverlapping segments: 5′UTR, CDS, and 3′UTR in KOPTK1 cells with or without FTO knockdown. ( F ) Distribution of genes exhibiting a significant change in both m 6 A level and overall transcript level in FTO -depleted KOPTK1 cells. ( G ) KEGG pathway enrichment analysis with the 634 hyperdown genes shown in (F). ( H ) Bubble-rank plot of hyperdown transcripts in (F). The genes encoding transcription factors are highlighted in red. ( I ) Growth curves of FTO -depleted KOPTK1 cells with or without the ectopic expression of ELK3 (left). Immunoblots of FTO and ELK3 are shown on the right. Data are presented as means (±SD) from two biological replicates, each performed with three technical replicates. Data were analyzed by two-way ANOVA with Bonferroni’s multiple comparisons. *** P < 0.001.

Journal: Science Advances

Article Title: FTO regulates ELK3-mediated metabolic rewiring and represents a unique therapeutic target in T cell leukemia

doi: 10.1126/sciadv.adq3052

Figure Lengend Snippet: ( A ) Heatmap showing differentially expressed genes in KOPTK1 cells with or without FTO shRNA expression. Differential genes are derived from RNA-seq data and defined as |fold change| ≥ 1.5, P value <0.05. ( B ) KEGG enrichment analysis of down-regulated transcripts in FTO -depleted KOPTK1 cells. ( C ) Cumulative distribution function representing the log2 peak intensity of m 6 A-modified sites in KOPTK1 cells with or without FTO knockdown. The P value was calculated using two-tailed Kolmogorov-Smirnov test. ( D ) Top consensus sequence motif identified within m 6 A peaks by hypergeometric optimization of motif enrichment (HOMER) in KOPTK1 cells with or without FTO knockdown. ( E ) Metagene profiles of m 6 A peak distribution along a normalized transcript composed of three rescaled nonoverlapping segments: 5′UTR, CDS, and 3′UTR in KOPTK1 cells with or without FTO knockdown. ( F ) Distribution of genes exhibiting a significant change in both m 6 A level and overall transcript level in FTO -depleted KOPTK1 cells. ( G ) KEGG pathway enrichment analysis with the 634 hyperdown genes shown in (F). ( H ) Bubble-rank plot of hyperdown transcripts in (F). The genes encoding transcription factors are highlighted in red. ( I ) Growth curves of FTO -depleted KOPTK1 cells with or without the ectopic expression of ELK3 (left). Immunoblots of FTO and ELK3 are shown on the right. Data are presented as means (±SD) from two biological replicates, each performed with three technical replicates. Data were analyzed by two-way ANOVA with Bonferroni’s multiple comparisons. *** P < 0.001.

Article Snippet: Antibodies used in the experiments include FTO (Proteintech, 27226-1-AP), ELK3 (OriGene, TA503603; NOVUS, NBP2-01264), ALKBH5 (Abcam, ab195377), and β-ACTIN (Abclonal, AC026).

Techniques: shRNA, Expressing, Derivative Assay, RNA Sequencing, Modification, Knockdown, Two Tailed Test, Sequencing, Western Blot

( A ) Genome browser tracks showing the mRNA abundance and m 6 A peaks of ELK3 transcripts in KOPTK1 cells with or without FTO depletion. ( B ) qPCR analysis of ELK3 expression in FTO knockdown T-ALL cells as indicated. ( C ) mRNA (top) and protein (bottom) levels of Elk3 in primary murine T-ALL cells from Fto fl/fl or Fto −/− BMT as shown in fig. S2G ( n = 4 per group). ( D ) qPCR (left) and immunoblot (right) analysis of ELK3 expression in FTO -depleted KOPTK1 cells rescued with WT or MUT FTO. ( E ) Gene-specific m 6 A-qPCR analysis of the ELK3 mRNA in T-ALL cells as indicated. ( F ) FTO-RIP qPCR validation of FTO interaction with the ELK3 mRNA in T-ALL cells. ( G ) The mRNA half-life ( t 1/2 ) of ELK3 in FTO knockdown KOPTK1 cells with enforced expression of WT or MUT FTO. ( H ) Relative luciferase activity showing the WT or MUT ELK3 exon4 firefly luciferase reporter in 293T cells transduced with Control or FTO shRNA. ( I ) YTHDF2-RIP qPCR showing the binding of YTHDF2 to ELK3 mRNA in FTO knockdown leukemia cells. ( J ) The ELK3 mRNA level in FTO -deficient T-ALL cells with or without YTHDF2 shRNA expression. ( K ) Scatterplot showing the correlation of FTO and ELK3 in 108 T-ALL samples from databases of National Omics Data Encyclopedia (NODE) (OEP002748). The expression levels were log 2 (TPM). Pearson correlation coefficient ( R ) and P value ( P ) are marked. Data are presented as means (±SD) of technical triplicates, and the experiments were independently repeated at least twice with the similar results [(B) and (D) to (J)]. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons [(B), (D), and (J)], two-way ANOVA with Bonferroni’s multiple comparisons (H), and unpaired two-tailed Student’s t test [(C), (E), (F), and (I)]. *** P < 0.001, n.s., nonsignificant.

Journal: Science Advances

Article Title: FTO regulates ELK3-mediated metabolic rewiring and represents a unique therapeutic target in T cell leukemia

doi: 10.1126/sciadv.adq3052

Figure Lengend Snippet: ( A ) Genome browser tracks showing the mRNA abundance and m 6 A peaks of ELK3 transcripts in KOPTK1 cells with or without FTO depletion. ( B ) qPCR analysis of ELK3 expression in FTO knockdown T-ALL cells as indicated. ( C ) mRNA (top) and protein (bottom) levels of Elk3 in primary murine T-ALL cells from Fto fl/fl or Fto −/− BMT as shown in fig. S2G ( n = 4 per group). ( D ) qPCR (left) and immunoblot (right) analysis of ELK3 expression in FTO -depleted KOPTK1 cells rescued with WT or MUT FTO. ( E ) Gene-specific m 6 A-qPCR analysis of the ELK3 mRNA in T-ALL cells as indicated. ( F ) FTO-RIP qPCR validation of FTO interaction with the ELK3 mRNA in T-ALL cells. ( G ) The mRNA half-life ( t 1/2 ) of ELK3 in FTO knockdown KOPTK1 cells with enforced expression of WT or MUT FTO. ( H ) Relative luciferase activity showing the WT or MUT ELK3 exon4 firefly luciferase reporter in 293T cells transduced with Control or FTO shRNA. ( I ) YTHDF2-RIP qPCR showing the binding of YTHDF2 to ELK3 mRNA in FTO knockdown leukemia cells. ( J ) The ELK3 mRNA level in FTO -deficient T-ALL cells with or without YTHDF2 shRNA expression. ( K ) Scatterplot showing the correlation of FTO and ELK3 in 108 T-ALL samples from databases of National Omics Data Encyclopedia (NODE) (OEP002748). The expression levels were log 2 (TPM). Pearson correlation coefficient ( R ) and P value ( P ) are marked. Data are presented as means (±SD) of technical triplicates, and the experiments were independently repeated at least twice with the similar results [(B) and (D) to (J)]. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons [(B), (D), and (J)], two-way ANOVA with Bonferroni’s multiple comparisons (H), and unpaired two-tailed Student’s t test [(C), (E), (F), and (I)]. *** P < 0.001, n.s., nonsignificant.

Article Snippet: Antibodies used in the experiments include FTO (Proteintech, 27226-1-AP), ELK3 (OriGene, TA503603; NOVUS, NBP2-01264), ALKBH5 (Abcam, ab195377), and β-ACTIN (Abclonal, AC026).

Techniques: Expressing, Knockdown, Western Blot, Biomarker Discovery, Luciferase, Activity Assay, Transduction, Control, shRNA, Binding Assay, Two Tailed Test

( A ) Growth curves of ELK3 -depleted T-ALL cell lines (CUTLL1 and KOPTK1) and primary T-ALL cells. Data are presented as the mean ± SD from two biological replicates, each with three technical replicates. ( B ) KEGG pathway analysis of down-regulated genes in ELK3 -depleted KOPTK1 cells. ( C ) GSEA plot showing enrichment of gene sets of hallmark glycolysis in ELK3 knockdown CUTLL1 cells. ( D ) Glycolytic proton efflux rate (glycoPER) analyzed in ELK3 -depleted CUTLL1 cells using a Seahorse extracellular flux analyzer. ( E ) Heatmap presentation of glycolysis-related metabolites from metabolomic analysis in KOPTK1 cells ( n = 3 per group). ( F ) qPCR analysis of glycolytic gene expression in ELK3 -depleted KOPTK1 cells. ( G ) Density plot of ELK3 (red) and H3K27ac (blue) ( GSE76783 ) occupancy around the transcription start site (TSS) of genes significantly down-regulated in ELK3-depleted KOPTK1 cells. ChIP-seq signal normalized with input. ( H ) Genome browser tracks of ELK3 and H3K27ac ChIP-seq signals at the promoters of PGK1 (top) and PFKFB4 (bottom). ( I ) ChIP-qPCR validation of ELK3 and H3K27ac binding to PGK1 and PFKFB4 promoters in KOPTK1 cells. ( J ) qPCR analysis of glycolysis-related genes in FTO -depleted KOPTK1 cells with or without the overexpression of ELK3. ( K ) Glycolytic proton efflux rate (glycoPER) analyzed in FTO -depleted CUTLL1 cells with or without the overexpression of ELK3 . Data are presented as means (±SD) of technical triplicates, and the experiments were independently repeated at least twice with the similar results [(D), (F), (I), (J), and (K)]. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons (J), two-way ANOVA with Bonferroni’s multiple comparisons (A), and unpaired two-tailed Student’s t test [(D), (F), and (I)]. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Journal: Science Advances

Article Title: FTO regulates ELK3-mediated metabolic rewiring and represents a unique therapeutic target in T cell leukemia

doi: 10.1126/sciadv.adq3052

Figure Lengend Snippet: ( A ) Growth curves of ELK3 -depleted T-ALL cell lines (CUTLL1 and KOPTK1) and primary T-ALL cells. Data are presented as the mean ± SD from two biological replicates, each with three technical replicates. ( B ) KEGG pathway analysis of down-regulated genes in ELK3 -depleted KOPTK1 cells. ( C ) GSEA plot showing enrichment of gene sets of hallmark glycolysis in ELK3 knockdown CUTLL1 cells. ( D ) Glycolytic proton efflux rate (glycoPER) analyzed in ELK3 -depleted CUTLL1 cells using a Seahorse extracellular flux analyzer. ( E ) Heatmap presentation of glycolysis-related metabolites from metabolomic analysis in KOPTK1 cells ( n = 3 per group). ( F ) qPCR analysis of glycolytic gene expression in ELK3 -depleted KOPTK1 cells. ( G ) Density plot of ELK3 (red) and H3K27ac (blue) ( GSE76783 ) occupancy around the transcription start site (TSS) of genes significantly down-regulated in ELK3-depleted KOPTK1 cells. ChIP-seq signal normalized with input. ( H ) Genome browser tracks of ELK3 and H3K27ac ChIP-seq signals at the promoters of PGK1 (top) and PFKFB4 (bottom). ( I ) ChIP-qPCR validation of ELK3 and H3K27ac binding to PGK1 and PFKFB4 promoters in KOPTK1 cells. ( J ) qPCR analysis of glycolysis-related genes in FTO -depleted KOPTK1 cells with or without the overexpression of ELK3. ( K ) Glycolytic proton efflux rate (glycoPER) analyzed in FTO -depleted CUTLL1 cells with or without the overexpression of ELK3 . Data are presented as means (±SD) of technical triplicates, and the experiments were independently repeated at least twice with the similar results [(D), (F), (I), (J), and (K)]. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons (J), two-way ANOVA with Bonferroni’s multiple comparisons (A), and unpaired two-tailed Student’s t test [(D), (F), and (I)]. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Antibodies used in the experiments include FTO (Proteintech, 27226-1-AP), ELK3 (OriGene, TA503603; NOVUS, NBP2-01264), ALKBH5 (Abcam, ab195377), and β-ACTIN (Abclonal, AC026).

Techniques: Knockdown, Gene Expression, ChIP-sequencing, ChIP-qPCR, Biomarker Discovery, Binding Assay, Over Expression, Two Tailed Test

( A ) Growth curves of indicated T-ALL cells treated with varying doses of Dac51. ( B ) CCK8 viability assessment of various T-ALL cells and normal BM cells (BM #1 and #2) after Dac51 treatment for 24 hours. ( C ) qPCR (left) and immunoblots (right) showing the ELK3 mRNA and protein levels upon Dac51 (150 or 300 nM) treatment for 36 hours. ( D ) qPCR analysis of glycolysis-related genes in CUTLL1 cells following Dac51 treatment (300 nM, 36 hours). ( E ) Assessment of glycolytic proton efflux rate (glycoPER) in KOPTK1 cells treated with 300 nM Dac51 for 36 hours. ( F ) Growth curves of 300 nM Dac51 treated CUTLL1 cells with or without ectopic ELK3 expression. ( G ) Schematic of Dac51 treatment in vivo. Primary human or murine T-ALL cells were transplanted into NPG or WT C57BL/6J mice, respectively, followed by Dac51 (4 mg/kg) treatment. ( H ) Assessment of leukemia cells in the PB from T-ALL mice shown in (G). Percentages of hCD45 + leukemia cells were analyzed at day 20 post-transplantation (patient #1 and #2), and Kras G12D /Notch1 L1601P GFP + murine leukemia cells were analyzed at day 14. ( I ) H&E and immunohistochemistry (PCNA) staining of spleens from T-ALL mice shown in (H) at 28, 20, and 14 days post-transplantation, respectively. Scale bar, 20 μm. ( J ) Kaplan-Meier survival curves of T-ALL mice shown in (H). Data are presented as the mean (±SD) from two biological replicates, each with three technical replicates [(A) and (F)], and from technical triplicates with experiments repeated at least twice [(B) to (E)]. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons (C), two-way ANOVA with Bonferroni’s multiple comparisons [(A) and (F)], unpaired two-tailed Student’s t test [(D) and (H)] and log-rank test (J). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Journal: Science Advances

Article Title: FTO regulates ELK3-mediated metabolic rewiring and represents a unique therapeutic target in T cell leukemia

doi: 10.1126/sciadv.adq3052

Figure Lengend Snippet: ( A ) Growth curves of indicated T-ALL cells treated with varying doses of Dac51. ( B ) CCK8 viability assessment of various T-ALL cells and normal BM cells (BM #1 and #2) after Dac51 treatment for 24 hours. ( C ) qPCR (left) and immunoblots (right) showing the ELK3 mRNA and protein levels upon Dac51 (150 or 300 nM) treatment for 36 hours. ( D ) qPCR analysis of glycolysis-related genes in CUTLL1 cells following Dac51 treatment (300 nM, 36 hours). ( E ) Assessment of glycolytic proton efflux rate (glycoPER) in KOPTK1 cells treated with 300 nM Dac51 for 36 hours. ( F ) Growth curves of 300 nM Dac51 treated CUTLL1 cells with or without ectopic ELK3 expression. ( G ) Schematic of Dac51 treatment in vivo. Primary human or murine T-ALL cells were transplanted into NPG or WT C57BL/6J mice, respectively, followed by Dac51 (4 mg/kg) treatment. ( H ) Assessment of leukemia cells in the PB from T-ALL mice shown in (G). Percentages of hCD45 + leukemia cells were analyzed at day 20 post-transplantation (patient #1 and #2), and Kras G12D /Notch1 L1601P GFP + murine leukemia cells were analyzed at day 14. ( I ) H&E and immunohistochemistry (PCNA) staining of spleens from T-ALL mice shown in (H) at 28, 20, and 14 days post-transplantation, respectively. Scale bar, 20 μm. ( J ) Kaplan-Meier survival curves of T-ALL mice shown in (H). Data are presented as the mean (±SD) from two biological replicates, each with three technical replicates [(A) and (F)], and from technical triplicates with experiments repeated at least twice [(B) to (E)]. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons (C), two-way ANOVA with Bonferroni’s multiple comparisons [(A) and (F)], unpaired two-tailed Student’s t test [(D) and (H)] and log-rank test (J). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Antibodies used in the experiments include FTO (Proteintech, 27226-1-AP), ELK3 (OriGene, TA503603; NOVUS, NBP2-01264), ALKBH5 (Abcam, ab195377), and β-ACTIN (Abclonal, AC026).

Techniques: Western Blot, Expressing, In Vivo, Transplantation Assay, Immunohistochemistry, Staining, Two Tailed Test

Elevated FTO expression in T-ALL maintains low levels of m 6 A methylation on many transcripts including the ELK3 mRNA, which prevents the YTHDF2-mediated degradation. Augmented ELK3 expression in turn up-regulates glycolysis-related genes like PGK1 and PFKFB4 , thereby enhancing glycolysis. Selective FTO inhibitor Dac51 leads to increased m 6 A modification on ELK3 mRNA, resulting in decreased ELK3 expression, ultimately lowering glycolytic capacity and suppressing T-ALL progression. See more details in the text.

Journal: Science Advances

Article Title: FTO regulates ELK3-mediated metabolic rewiring and represents a unique therapeutic target in T cell leukemia

doi: 10.1126/sciadv.adq3052

Figure Lengend Snippet: Elevated FTO expression in T-ALL maintains low levels of m 6 A methylation on many transcripts including the ELK3 mRNA, which prevents the YTHDF2-mediated degradation. Augmented ELK3 expression in turn up-regulates glycolysis-related genes like PGK1 and PFKFB4 , thereby enhancing glycolysis. Selective FTO inhibitor Dac51 leads to increased m 6 A modification on ELK3 mRNA, resulting in decreased ELK3 expression, ultimately lowering glycolytic capacity and suppressing T-ALL progression. See more details in the text.

Article Snippet: Antibodies used in the experiments include FTO (Proteintech, 27226-1-AP), ELK3 (OriGene, TA503603; NOVUS, NBP2-01264), ALKBH5 (Abcam, ab195377), and β-ACTIN (Abclonal, AC026).

Techniques: Expressing, Methylation, Modification

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 – c Determination of ELK3 degradation pathways. The cell lysates extracted from HeLa cells treated with MG132 ( a ), chloroquine ( b ), or MLN4924 ( c ) were used to visualize ELK3 by WB. d ELK3 interaction with Cullin3. The cell lysates extracted from HEK293T cells transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the interaction between ELK3 and each of Cullins by IP and WB. e Cullin3 decreases ELK3 half-life. The cell lysates extracted from HEK293T cells transfected with indicated plasmids and treated with cycloheximide (CHX, 10 μg/ml) were used to evaluate the half-life of ELK3 by WB. f Knockdown of Cullin3 increases ELK3. The cell lysates extracted from HeLa cells stably expressing sh-mock or sh-CUL3 were used to evaluate the ELK3 protein levels by WB. g Cullin3 knockdown inhibits ELK3 destabilization. The cell lysates extracted from PC-3 cells stably expressing sh-mock or sh-Cul3 and treated with CHX (10 μg/ml) were used to evaluate the ELK3 half-life by WB. h Cullin3 induces ELK3 ubiquitination. The cell lysates extracted from HEK293T cells transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the ELK3 ubiquitination by IP and WB. Graphs in e and g The band intensity ELK3 bands measured using NIH image J computer program and normalized by β-actin intensity were plotted. Data, three independent experiments; Significance, * p < 0.05 obtained by Student t test.

Article Snippet: Subsequently, the tissues were incubated with primary antibodies against ELK3 (dilution 1:200; cat. #: NBP2-01264, Novus) or Brd4 (dilution 1:300; cat. #: ab128874, Abcam, Cambridge, UK) at 4 °C overnight.

Techniques: Transfection, Knockdown, Stable Transfection, Expressing, Ubiquitin Proteomics

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 , b ELK3 interacts with SPOP. The cell lysates extracted from HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to determine ELK3 interaction to E3 ligases by IP and WB. c Evaluation of endogenous interaction between ELK3 and SPOP. The cell lysates extracted from HeLa cells treated with MG132 (10 μM) for 8 h were used to evaluate the ELK3 and SPOP interaction by IP and WB. d Confirmation of ELK3 and SPOP interaction. The cell lysates extracted from HEK293T cells transiently transfected with His-ELK3 plasmid were used to confirm the ELK3 and partially purified bacterial His-SPOP by pulldown assay (PD) and WB. e SPOP induces ELK3 destabilization. The cell lysates extracted from HEK293T cells transiently transfected with His-ELK3 and dose-increasing Myc-SPOP plasmids were used to evaluate ELK3 protein level by WB. GFP was used as an internal control for equal transfection. f SPOP accelerates ELK3 destabilization. The cell lysates extracted from HEK293T cells transiently transfected with indicated plasmids and treated with CHX (10 μg/ml) were used to evaluate the ELK3 half-life by WB. g SPOP knockdown increases ELK3 protein levels. The cell lysates extracted from HeLa cells stably expressing sh-mock or sh-SPOP were used to evaluate the ELK3 protein levels by WB. h Depletion of SPOP did not affect ELK3 RNA expression. The levels of ELK3 mRNA extracted from HeLa cells stably expressing sh-mock or sh-SPOP were assessed by quantitative real-time RT-PCR using TaqMan RNA-to-CT 1-step kit. i SPOP knockdown prolongs ELK3 half-life. The cell lysates extracted from PC-3 cells stably expressing sh-mock or sh-SPOP and treated with CHX (10 μg/ml) were used to evaluate ELK3 half-life by WB. j , k SPOP induces ELK3 ubiquitination in cell and in vitro ubiquitination assay system. The cell lysates extracted from HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate ELK3 ubiquitination by IP and WB ( j ). In vitro ubiquitination assay was conducted using commercially available E1, E2, and ubiquitin (Ubi) proteins, along with His-tagged ELK3 purified from E. coli and Myc-tagged SPOP isolated from HEK293T cells overexpressing SPOP ( k ). Graphs in f and i The band intensity of ELK3 quantified by NIH image J computer program normalized by β-actin intensity was plotted. Data, three independent experiments; Significance, * p < 0.05 obtained by Student t test.

Techniques: Transfection, Plasmid Preparation, Purification, Control, Knockdown, Stable Transfection, Expressing, RNA Expression, Quantitative RT-PCR, Ubiquitin Proteomics, In Vitro, Isolation

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 ELK3 interacted with MATH domain of SPOP. Upper panel Schematic diagram of SPOP domains. The numbers at the MATH domain indicates SPOP mutations observed in PCa patients. Bottom panels The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated MG132 (10 μM) for 4 h were used to determine the SPOP’s binding domain by IP and WB. b MATH domain deletion of SPOP suppressed ELK3 ubiquitination. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated MG132 (10 μM) for 4 h were used to evaluate the SPOP-dependent ELK3 ubiquitination by IP and WB. c The deletion of the MATH or BTB domain of SPOP abolishes ELK3 destabilization. The cell lysates of HEK293T cells transiently transfected with indicated plasmids were used to evaluate ELK3 protein levels by WB. GFP was used as an internal control for equal transfection. d MATH domain mutation of SPOP abrogates interaction with ELK3. The cell lysates of HEK293T cells transiently transfected with the indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate the interaction between SPOP and ELK3 by IP and WB. e MATH-F133V mutation of SPOP critically affects ELK3 stability. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with CHX (10 μg/ml) for indicated period were used to evaluate ELK3 protein levels by WB. The band intensity of ELK3 quantified by NIH image J computer program normalized by β-actin intensity was plotted. GFP was used an internal control for equal transfection. Data, three independent experiments; Significance, * p < 0.05 obtained by Student t test. f SPOP mutations at MATH domain inhibits endogenous ELK3 destabilization. The cell lysates of 22Rv1 cells stably expressing SPOP-wt or each of SPOP mutants (Y87C, W131G, and F133V) were used to evaluate ELK3 protein levels by WB. g SPOP mutations at MATH domain abolishes ELK3 ubiquitination. The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate ELK3 ubiquitination by IP and WB.

Techniques: Transfection, Binding Assay, Ubiquitin Proteomics, Control, Mutagenesis, Stable Transfection, Expressing

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 Identification of ELK3 degron motifs for SPOP. ELK3 amino acid sequences were aligned to search putative degron motifs for SPOP. b Determination of ELK3 degron motif interacting to SPOP. 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 ELK3 degron motif by IP and WB. c Involvement of ELK3 Deg1 in its stabilization. The cell lysates of HEK293T cells transiently transfected with indicated plasmids were used to determine the ELK3 degron motif by WB. GFP was used an internal control for equal transfection. d ELK3 Deg1 deletion abrogates SPOP-mediated destabilization. Left panels The cell lysates of HEK293T cells transiently transfected with indicated plasmids and treated with CHX (10 μg/ml) for an indicated time were used to evaluate the ELK3 protein levels by WB. GFP was used as an internal control for equal transfection. Graph The band intensity of ELK3 quantified by NIH image J computer program normalized by β-actin intensity was plotted. GFP was used an internal control for equal transfection. Data, three independent experiments; Significance, * p < 0.05 obtained by Student t test. e ELK3 Deg1 deletion abolishes SPOP-mediated ubiquitination. The cell lysate of HEK293T cells transiently transfected with indicated plasmids and treated with MG132 (10 μM) for 4 h were used to evaluate SPOP-mediated ELK3 ubiquitination by IP and WB. f , g Protein–protein docking of ELK3 and SPOP. The SPOP structure (PDB ID: 3HQI) and ELK3 built by amino acid similarly modeling using Discovery Studio were used to predict SPOP and ELK3 interaction interface. Magenta, ELK3; Green, SPOP. The amino acids and interaction types involved in interaction between ELK3 and SPOP are provided in Supplementary Fig. .

Techniques: Transfection, Control, Ubiquitin Proteomics

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.

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: 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 , b SPOP inhibits ELK3-mediated c-fos promoter activity. HEK293T cells transiently transfected with indicated plasmids were used to evaluate c-fos promoter activity by luciferase assay. c SPOP knockdown increases c-fos promoter activity. HEK293T cells stably expressing sh-SPOP were transfected with c-fos -luc reporter plasmid and evaluated c-fos promoter activity by luciferase assay. d Confirmation of increased c-Fos protein by SPOP knockdown. The cell lysates of 22Rv1 cells stably expressing sh-SPOP were used to evaluate the c-Fos protein level by WB. e Establishment of ELK3 and SPOP double knockdown cells. The cell lysates of 22Rv1 cells stably expressing sh-SPOP and/or sh-ELK3 as indicated were used to evaluate the SPOP and c-Fos protein levels by WB. f Increased cell proliferation by SPOP knockdown suppresses cell proliferation by ELK3. The 22Rv1 cells in e were used to evaluate the effects of cell proliferation by CCK-8 assay. g Increased cell invasion by SPOP knockdown abrogated by ELK3 knockdown. The 22Rv1 cells in e were used to evaluate the effects of cell invasion by cell invasion assay using Matrigel Invasion Chamber. a – c and f , g Data, three independent experiments; Significance, * p < 0.05 obtained by Student t -test.

Techniques: Activity Assay, Transfection, Luciferase, Knockdown, Stable Transfection, Expressing, Plasmid Preparation, CCK-8 Assay, Invasion Assay

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 Inverse correlation of ELK3 and phospho-CHK1/2 protein levels under docetaxel treatment. The cell lysates of 22Rv1 cells treated with docetaxel (300 nM) for an indicated time were used to evaluate the indicated protein levels by WB. b Rescue of docetaxel-mediated decreased ELK3 protein level by MG132 treatment. The 22Rv1 cell lysates treated with docetaxel (300 nM) in the presence or absence of MG132 (10 μM) were used to evaluate ELK3 protein levels by WB. c Docetaxel-mediated decreased ELK3 protein is rescued by SPOP knockdown. The cell lysates of 22Rv1 cells stably expressing sh-SPOP and treated with docetaxel (300 nM) were used to evaluate ELK3 protein level by WB. d Docetaxel induces SPOP and ELK3 interaction. The cell lysates of HEK293T cells transfected with indicated plasmids and treated with docetaxel (300 nM) were used to evaluate the SPOP and ELK3 interaction by IP and WB. e Docetaxel induces ELK3 ubiquitination. The cell lysates of HEK293T cells transfected with indicated plasmids and treated with docetaxel (300 nM) were used to evaluate ELK3 ubiquitination by IP and WB. f Diminished interaction between SPOP and ELK3 increases PCa cell survivability. 22Rv1 cells stably expressing SPOP-WT or each of SPOP mutants treated with docetaxel (1 μM) for 48 h were used to cell survivability by CCK-8 assay. g SPOP and ELK3 double knockdown inhibits cell survivability by docetaxel in SPOP knockdown cells. 22Rv1 cells stably expressing sh-SPOP and/or sh-ELK3 treated with docetaxel (1 μM) for 48 h were used to evaluate cell survivability by CCK-8 assay. h SPOP and ELK3 double knockdown enhances apoptosis signaling by docetaxel. The cell lysates of 22Rv1 cells stably expressing sh-SPOP and/or sh-ELK3 treated with docetaxel (300 nM) were used to evaluate the protein levels of apoptosis-related signaling molecules by WB. i , j The SPOP-ELK3 axis plays a critical role in docetaxel-induced cell death. The cell lysates of 22Rv1 cells stably expressing sh-SPOP and/or SPOP were used to evaluate the protein levels of ELK3 and SPOP by WB ( i ), and to assess cell viability using the CCK-8 assay ( j ). k Increased ELK3 protein levels in PCa cancer tissues. IHC images of ELK3 in prostate cancer tissues ( n = 117) stained by DAB staining were analyzed by TissueFAXS system. Data, DAB intensity in normal vs. cancer tissues; Significance was obtained by Anova t test. l Increased ELK3 protein levels in PCa patient harboring SPOP-F133V mutation. Representative IHC image of ELK3 protein levels by DAB staining in prostate cancer tissues harboring SPOP-WT or SPOP-F133V. f , g Data, three independent experiments; Significance, * p < 0.05 obtained by Student t test.

Techniques: Knockdown, Stable Transfection, Expressing, Transfection, Ubiquitin Proteomics, CCK-8 Assay, Staining, Mutagenesis

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 schematic diagram illustrates the working model in which SPOP mutants enhance ELK3 stabilization, leading to increased resistance to docetaxel-induced cell death. Consequently, the SPOP-ELK3 axis plays a pivotal role in the progression of prostate cancer (PCa).

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