Journal: Medical Oncology (Northwood, London, England)

Article Title: Induction of anterior gradient 2 (AGR2) plays a key role in insulin-like growth factor-1 (IGF-1)-induced breast cancer cell proliferation and migration

doi: 10.1007/s12032-015-0577-z

Figure Lengend Snippet: Estrogen-independent activation of AGR2 by IGF-1 requires estrogen receptor and activation of ERK and AKT pathways. a , b and d , e MCF7 cells were incubated for 24 h in different treatments with IGF-1 and MEK, ER and AKT inhibitors. The concentration of IGF-1 is 10 nM in each group. MEK inhibitor U0126 (10 μM), ER inhibitor ICI 182, 780 (10 nM) and AKT inhibitor MK2206 (500 nM) and E2 (5 nM) were used. Level of AGR2 protein was detected by western blot with specific antibodies. β-Actin was detected as a loading control. c , f Luciferase construct with 1.9 kb of AGR2 promoter sequence and a pRL-TK control plasmid was co-transfected into MCF7 cells and treated with different inhibitors with or without the presence of 10 nM IGF-1 and 5 μM E2. The luciferase activity was detected with microplate reader and normalized by Renilla activity. g AGR2 expression induced by IGF-1 without E2 in the presence of four chemical inhibitors was detected by immunofluorescence in MCF7 cell by confocal microscopy. The inhibitor concentration was 10 μM U0126, 10 nM ICI 182,780, 500 nM MK2206 and 5 μM OSI-906. Cells were starved for 24 h before IGF-1 treatment. Nuclei were stained with DAPI ( blue ) as an internal reference, and AGR2 was stained with specific primary antibody ( red ). The IOD value of red fluorescence is quantified by Image-Pro, normalized to blue fluorescence and set in the right panel in the form of a histogram. The original magnification is ×200. Each experiment was repeated at least three times. * P < 0.05; ** P < 0.01

Article Snippet: In addition, chemical inhibitors included U0126-EtOH (MEK inhibitor), fulvestrant (ICI 182,780, estrogen receptor-α inhibitor) and MK2206 2HCl (AKT inhibitor), and SP00125 (JNK Inhibitor) and OSI-906 (IGF-1 receptor inhibitor), which were also purchased from Selleckchem, while monensin was from Beyotime.

Techniques: Activation Assay, Incubation, Concentration Assay, Western Blot, Control, Luciferase, Construct, Sequencing, Plasmid Preparation, Transfection, Activity Assay, Expressing, Immunofluorescence, Confocal Microscopy, Staining, Fluorescence

Journal: Cell Death & Disease

Article Title: Endometrial cancer-associated mutants of SPOP are defective in regulating estrogen receptor- α protein turnover

doi: 10.1038/cddis.2015.47

Figure Lengend Snippet: SPOP interacts with ER α in cells. ( a , b ) Ectopically expressed SPOP and ER α interact with each other. The 293T cells were co-transfected with FLAG-HA (FH)-SPOP and Myc-ER α constructs. After 24 h, cell lysates were prepared for co-IP with anti-FLAG antibody and WB analyzes. ( b ) co-IP assay was performed between ectopically expressed FH-ER α and Myc-SPOP. ( c ) Endogenous SPOP and ER α proteins interact with each other in Ishikawa cells. After being treated with 20 μ M MG132 for 4 h, cell lysates were prepared for co-IP with anti-ER α antibody and WB analyzes with indicated antibodies. ( d ) Schematic representation of SPOP deletion mutants. Binding capacity of SPOP to ER α is indicated with the symbol. ( e ) ER α binds to the MATH domain of SPOP. The 293T cells were co-transfected with FH-ER α and Myc-SPOP-WT or deletion mutants (ΔMATH, ΔBTB) constructs. After 24 h, cell lysates were prepared for co-IP assay with anti-FLAG antibody and WB analyzes. ( f ) Schematic representation of MATH domain deletion mutants of SPOP. Binding capacity of SPOP to ER α is indicated with the symbol. ( g ) The integrity MATH domain of SPOP is crtical for ER α binding. The 293T cells were co-transfected with FH-ER α and Myc-SPOP-WT or deletion mutants (D1–D8) constructs. After 24 h, cell lysates were prepared for co-IP assay with anti-Myc antibody and WB analyzes

Article Snippet: The following antibodies were used: SPOP (ab137537; Abcam, Cambridge, UK), GREB1 (ab72999; Abcam), Cyclin D1 (ab134175; Abcam), ER α (8644; Cell Signaling, Beverly, MA, USA), ubiquitin (6652-1; Epitomics, Burlingame, CA, USA), Myc (9E10; Sigma Aldrich, St. Louis, MO, USA), FLAG (M2; Sigma), actin (AC-74; Sigma) and HA (MM5-101R; Millipore, Darmstadt, Germany).

Techniques: Transfection, Construct, Co-Immunoprecipitation Assay, Binding Assay

Journal: Cell Death & Disease

Article Title: Endometrial cancer-associated mutants of SPOP are defective in regulating estrogen receptor- α protein turnover

doi: 10.1038/cddis.2015.47

Figure Lengend Snippet: The SPOP-CUL3-RBX1 ubiquitin ligase complex targets ER α for ubiquitination and degradation. ( a ) SPOP regulates ER α protein levels through the proteasome pathway. The 293T cells were transfected with FH-ER α in combination with or without the Myc-SPOP constructs. After 24 h, cells were treated with MG132 (20 μ M), Bortezomib (200 nM), chloroquine (100 mM), or DMSO for 4 h before cell lysates were prepared for WB analyzes. Actin, a loading control. ( b ) The BTB and MATH domains in SPOP are essential for SPOP-mediated degradation of ER α . FH-ER α and different amounts of Myc-SPOP-WT or deletion mutants (ΔMATH, ΔBTB) constructs were transfected into 293T cells. After 24 h, cell lysates were prepared for WB analyzes. ( c ) SPOP regulates endogenous ER α protein levels. Ishikawa cells were transfected with Myc-SPOP-WT, or deletion mutants (ΔMATH, ΔBTB) constructs. After 24 h, cell lysates were prepared for WB analyzes. ( d ) Knockdown of SPOP increases endogenous ER α protein levels. Ishikawa cells were transfected with control or two SPOP-specific siRNAs. After 48 h, cell lysates were prepared for WB analyzes. ( e ) Quantitative RT-PCR measurement of the mRNA levels of SPOP and ESR1 in SPOP-knockdown Ishikawa cells. The mRNA level of GAPDH was used for normalization. The mean values (S.D.) of three independent experiments are shown. ( f , g ) Knockdown of SPOP prolongs ER α protein half-life. Ishikawa cells were transfected with control or SPOP-specific siRNA. After 48 h, cells were chased with 30 μ M cycloheximide (CHX). At the indicated time points, cell lysates were prepared for WB analyzes. ( f ) At each time point, the intensity of ER α was first normalized to the intensity of Actin (loading control) and then to the value of the 0-h time point ( g ). The mean values (S.D.) of three independent experiments are shown. ( h ) Knockdown of RBX1 or CUL3 increases endogenous ER α protein levels. Ishikawa cells were transfected with control siRNA or siRNAs for RBX1 or CUL3. After 48 h, cell lysates were prepared for WB analyzes. ( i ) SPOP promotes ER α polyubiquitination in vivo . FH-ER α , HA-Ub, and Myc-SPOP-WT or ΔBTB mutant constructs were co-transfected into 293T cells. After 24 h, cells were treated with 20 μ M MG132 for 4 h. ER α proteins were immunoprecipitated with anti-FLAG antibody and resolved by SDS/PAGE. The ubiquitinated forms of ER α were analyzed by WB with anti-Ub antibody. ( j ) Knockdown of SPOP decreases ubiquitination of endogenous ER α . Ishiwaka cells were transfected control or SPOP-specific siRNA. After 48 h, cells were treated with 20 μ M MG132 for 4 h and then the same procedure was performed as i . ( k ) Knockdown of SPOP promotes Ishikawa cells growth. Ishikawa cells were transfected with control or two SPOP-specific siRNAs. After 48 h, the cell growth was measured by CCK-8 assay at indicated days. The mean values (S.D.) of three independent experiments are shown

Article Snippet: The following antibodies were used: SPOP (ab137537; Abcam, Cambridge, UK), GREB1 (ab72999; Abcam), Cyclin D1 (ab134175; Abcam), ER α (8644; Cell Signaling, Beverly, MA, USA), ubiquitin (6652-1; Epitomics, Burlingame, CA, USA), Myc (9E10; Sigma Aldrich, St. Louis, MO, USA), FLAG (M2; Sigma), actin (AC-74; Sigma) and HA (MM5-101R; Millipore, Darmstadt, Germany).

Techniques: Ubiquitin Proteomics, Transfection, Construct, Control, Knockdown, Quantitative RT-PCR, In Vivo, Mutagenesis, Immunoprecipitation, SDS Page, CCK-8 Assay

Journal: Cell Death & Disease

Article Title: Endometrial cancer-associated mutants of SPOP are defective in regulating estrogen receptor- α protein turnover

doi: 10.1038/cddis.2015.47

Figure Lengend Snippet: The S/T-rich motifs in ER α are degrons recognized by SPOP. ( a ) Schematic representation of wild-type ER α protein with the upper contiguous Ser/Thr residues indicating the S/T-rich motifs in its amino-acid sequence. The ER α point mutants (M1, M2, M3, and M4) were constructed starting from the FH-ER α -WT vector are schematically reported below the wild-type protein. On the right of each schematic protein is summarized its SPOP-binding capacity, sensitivity to SPOP-induced degradation or ubiquitination. ( b ) The S/T-rich motifs in ER α are required for its binding to SPOP. The 293T cells were transfected with the indicated constructs. After 24 h, cell lysates were prepared for co-IP assay with anti-FLAG antibody and WB analyzes. ( c ) The S/T-rich motifs in ER α are required for SPOP-mediated ER α degradation. The 293T cells were transfected with the indicated constructs. After 24 h, cell lysates were prepared for WB analyzes. SE, short exposure; LE, long exposure. ( d , e ) Mutation of the S/T-rich motifs prolongs the half-life of ER α . ER α -WT or M4 mutant was transfected into 293T cells. After 24 h, cells were treated with 30 μ M CHX. At the indicated time points, cell lysates were prepared for WB analyzes ( d ). At each time point, the intensity of ER α was first normalized to the intensity of Actin and then to the value of the 0-h time point ( e ). ( f )The S/T-rich motifs are required for SPOP-mediated ER α polyubiquitination. The 293T cells were transfected with the indicated constructs. After 24 h, cells were treated with 20 μ M MG132 for 4 h and the cell lysates were prepared for co-IP assay with anti-FLAG antibody and WB analyzes. The mean values (S.D.) of three independent experiments are shown. ( g ) Ser118 of ER α is not required for SPOP-mediated ER α degradation. The 293T cells were transfected with FH-ER α or S118A mutant in combination with or without Myc-SPOP constructs. After 24 h, cell lysates were prepared for WB analyzes

Article Snippet: The following antibodies were used: SPOP (ab137537; Abcam, Cambridge, UK), GREB1 (ab72999; Abcam), Cyclin D1 (ab134175; Abcam), ER α (8644; Cell Signaling, Beverly, MA, USA), ubiquitin (6652-1; Epitomics, Burlingame, CA, USA), Myc (9E10; Sigma Aldrich, St. Louis, MO, USA), FLAG (M2; Sigma), actin (AC-74; Sigma) and HA (MM5-101R; Millipore, Darmstadt, Germany).

Techniques: Sequencing, Construct, Plasmid Preparation, Binding Assay, Ubiquitin Proteomics, Transfection, Co-Immunoprecipitation Assay, Mutagenesis

Journal: Cell Death & Disease

Article Title: Endometrial cancer-associated mutants of SPOP are defective in regulating estrogen receptor- α protein turnover

doi: 10.1038/cddis.2015.47

Figure Lengend Snippet: Endometrial cancer-associated mutants of SPOP are defective in promoting ER α degradation and ubiquitination. ( a ) Distribution of the point mutations on the SPOP gene found in endometrial cancer samples. These mutations are exclusively located in the N-terminal MATH domain of SPOP. ( b ) Endometrial cancer-associated mutants of SPOP are defective in promoting ER α degradation. 293T cells were transfected with FH-ER α and wild-type or mutated SPOP constructs as indicated. After 24 h, cell lysates were prepared for WB analyzes. ( c ) Endometrial cancer-associated mutants of SPOP are defective in interacting with ER α . The 293T cells were transfected with the indicated constructs. After 24 h, cell lysates were prepared for co-IP assay with anti-FLAG antibody and WB analyzes. ( d ) Endometrial cancer-associated mutants of SPOP are defective in promoting ER α ubiquitination. The 293T cells were transfected with the indicated constructs. After 24 h, cells were treated with 20 μ M MG132 for 4 h and cell lysates were prepared for immunoprecipitation and WB analyzes

Article Snippet: The following antibodies were used: SPOP (ab137537; Abcam, Cambridge, UK), GREB1 (ab72999; Abcam), Cyclin D1 (ab134175; Abcam), ER α (8644; Cell Signaling, Beverly, MA, USA), ubiquitin (6652-1; Epitomics, Burlingame, CA, USA), Myc (9E10; Sigma Aldrich, St. Louis, MO, USA), FLAG (M2; Sigma), actin (AC-74; Sigma) and HA (MM5-101R; Millipore, Darmstadt, Germany).

Techniques: Ubiquitin Proteomics, Transfection, Construct, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: Endometrial cancer-associated mutants of SPOP are defective in regulating estrogen receptor- α protein turnover

doi: 10.1038/cddis.2015.47

Figure Lengend Snippet: Estrogen potentiates SPOP-mediated degradation of ER α . ( a ) Estrogen enhances the SPOP-ER α interaction. FH-ER α and Myc-SPOP constructs were co-transfected into 293T cells. After 24 h, cells were treated with the vehicle ethanol (EtOH,−) or 10 nM 17 β -estradiol (E2) for 4 h before cell lysates were prepared for co-IP and WB analyzes. ( b ) Estrogen enhances SPOP-mediated ER α degradation. The 293T cells were transfected with the indicated constructs. A small amount of Myc-SPOP constructs was used in transfection. After 24 h, cells were treated with the vehicle ethanol (EtOH) or 10 nM 17 β -estradiol (E2) for 4 h before cells lysates were prepared for WB analyzes. The density of ER α was determined by normalizing to actin (loading control) first and then to the normalized value in mock-treated cells. ( c ) Knockdown of SPOP attenuates estrogen-induced degradation of ER α . Ishikawa cells were transfected with control or SPOP-specific siRNA. After 48 h, cells were then treated with the vehicle ethanol (EtOH,−) or 10 nM 17 β -estradiol (E2) for 4 h before cell lysates were prepared for WB analyzes. ( d ) Estrogen potentiates SPOP-induced polyubiquitination of ER α . The 293T cells were transfected with the indicated constructs. After 24 h, cells were treated with the vehicle ethanol (EtOH,−) or 10 nM 17 β -estradiol (E2). Cells were then treated with MG132 for 4 h before cell lysates were prepared for IP and WB analyzes. ( e ) Ishikawa cells lines that stably transfected with control, SPOP-WT or SPOP mutants constructs were treated with 10 nM 17 β -estradiol (E2) for 24 h. The mRNA level of ER α target gene GREB1 was measured by qRT-PCR. The mRNA level of GAPDH was used for normalization. The mean values (S.D.) of three independent experiments are shown. **indicates statistical significance (** P <0.01). ( f , g ) Differential effects of estrogen on the protein level of ER α -WT and the SPOP degradation-resistant mutant (ER α -M4). The 293T cells were transfected with FH- ER α -WT or M4 mutant construct. After 24 h, cells were treated with vehicle ethanol (EtOH,−), 10 nM 17 β -estradiol (E2), 10 nM Tamoxifen (Tam), and 10 nM Fulvestrant (Ful) for 4 h before cell lysates were prepared for WB analyzes

Article Snippet: The following antibodies were used: SPOP (ab137537; Abcam, Cambridge, UK), GREB1 (ab72999; Abcam), Cyclin D1 (ab134175; Abcam), ER α (8644; Cell Signaling, Beverly, MA, USA), ubiquitin (6652-1; Epitomics, Burlingame, CA, USA), Myc (9E10; Sigma Aldrich, St. Louis, MO, USA), FLAG (M2; Sigma), actin (AC-74; Sigma) and HA (MM5-101R; Millipore, Darmstadt, Germany).

Techniques: Construct, Transfection, Co-Immunoprecipitation Assay, Control, Knockdown, Stable Transfection, Quantitative RT-PCR, Mutagenesis

Journal: Cell Death & Disease

Article Title: Endometrial cancer-associated mutants of SPOP are defective in regulating estrogen receptor- α protein turnover

doi: 10.1038/cddis.2015.47

Figure Lengend Snippet: Models depicting SPOP-mediated ER α degradation in physiological and pathological conditions in endometrial cancer. ( a ) Unmutated SPOP promotes degradation of wild-type ER α . ( b ) Endometrial cancer-associated mutants of SPOP are defective in promoting ER α ubiquitination and degradation. ( c ) Estrogen potentiates SPOP-mediated ER α degradation

Article Snippet: The following antibodies were used: SPOP (ab137537; Abcam, Cambridge, UK), GREB1 (ab72999; Abcam), Cyclin D1 (ab134175; Abcam), ER α (8644; Cell Signaling, Beverly, MA, USA), ubiquitin (6652-1; Epitomics, Burlingame, CA, USA), Myc (9E10; Sigma Aldrich, St. Louis, MO, USA), FLAG (M2; Sigma), actin (AC-74; Sigma) and HA (MM5-101R; Millipore, Darmstadt, Germany).

Techniques: Ubiquitin Proteomics

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: PDZK1 is a novel factor in breast cancer that is indirectly regulated by estrogen through IGF-1R and promotes estrogen-mediated growth.

doi: 10.2119/molmed.2011.00001

Figure Lengend Snippet: Figure 2. PDZK1 is not an immediate early ER-α–dependent gene product but is required for E2- dependent growth of MCF-7 cells. (A) MCF-7 cells were treated with 1 nmol/L E2 for different time intervals or left untreated. Total RNA was subjected to RT-PCR analysis with primers specific to PDZK1 or β-actin (upper two panels). Protein extracts were subjected to immunoblot analysis with antibodies against PDZK1 or GAPDH (lower two panels). (B) MCF-7 cells were treated with 1 nmol/L E2 for 48 h or left untreated. Cells were subjected to immunofluorescence with anti- bodies against PDZK1. Note the partial nuclear localization of PDZK1 in E2-treated MCF-7 cells (C). (D) MCF-7 cells were treated with 1 nmol/L E2 in the presence or absence of the ER-α an- tagonist ICI 182,780, the selective estrogen modulator tamoxifen or the EGFR kinase inhibitor AG1478 for 48 h, after which protein extracts were subjected to immunoblot analysis with anti- bodies against PDZK1 or GAPDH. (E) MCF-7 cells were transiently transfected with siRNA target- ing PDZK1 (siPDZK1-1) or control siRNA and treated with 1 nmol/L E2 or left untreated for 48 h. Protein extracts were prepared and subjected to immunoblot analysis with antibodies against PDZK1 or GAPDH (inset). MCF-7 cells were subjected to PDZK1 knockdown with siPDZK1-1 (se- quence 1) or PDZK1-2 (sequence 2), after which cell proliferation was assessed by using the MTT assay. *Difference from respective untreated controls; #difference from E2-treated cells; p < 0.05. (F) MCF-7 cells were subjected to PDZK1 knockdown with the siPDZK1, after which cells in the S phase were assessed by fluorescence-activated cell sorter analysis. *Difference from un- treated control; #difference from E2-treated cells; p < 0.05. (G) MCF-7 cells were subjected to PDZK1 knockdown with the siPDZK1-1 sequence and treated with 1 nmol/L E2 for 48 h. Protein extracts were subjected to immunoblot analysis with antibodies against c-Myc or GAPDH. (H) MCF-7 cells were subjected to ER-α knockdown with siRNA and treated with 1 nmol/L E2 for 48 h. Protein extracts were subjected to immunoblot analysis with antibodies against c-Myc, PDZK1 or GAPDH. Note that the immunoblot for ER-α was run on a different gel and its GAPDH loading control is depicted in Supplementary Figure S4; the latter figure also depicts im- munoblot for ER-α in protein extracts from cells treated with control siRNA.

Article Snippet: Protein extracts were subjected to immunoblot analysis with antibodies to phospho-MEK, MEK, phospho(T202/Y204)-ERK1/2, ERK1/2, c-Src (32G6) and EGFR (all purchased from Cell Signaling Technology); ER-α, c-Myc or GADPH (all from Santa Cruz Biotechnology); or PDZK1(EPR3751) (Novus Biologicals).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Transfection, Control, Knockdown, Sequencing, MTT Assay, Fluorescence

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: PDZK1 is a novel factor in breast cancer that is indirectly regulated by estrogen through IGF-1R and promotes estrogen-mediated growth.

doi: 10.2119/molmed.2011.00001

Figure Lengend Snippet: Figure 4. Expression of IGF-1R is critical for PDZK1 expression upon E2 stimulation in MCF-7 cells. (A) MCF-7 cells were treated with 1 nmol/L E2 for 6 h, after which cell lysates were prepared and incubated at 4°C overnight with a membrane containing fixed antibodies against the indicated growth factor. Bound growth factors were detected by enhanced chemiluminescence according to the manufacturer’s instructions. The relative signal inten- sity was quantified by using the VersaDoc imaging system. (B) MCF-7 cells were treated with 1 nmol/L E2 for different time intervals or left untreated. Total RNA was subjected to RT- PCR analysis with primers specific to human IGF-1R or β-actin. (C) MCF-7 cells were tran- siently transfected with siRNA targeting ER-α or control siRNA (Supplementary Figure S4). Cells were treated with 1 nmol/L E2 or left untreated for 48 h (left panel), after which pro- tein extracts were prepared and subjected to immunoblot analysis with antibodies against IGF-1R or GAPDH. (D) MCF-7 cells were treated with 1 nmol/L E2 in the presence or ab- sence of different doses of the IGF-1R antagonist AG1024 for 48 h or with human IGF-1 (10 ng/mL) for the indicated time intervals (right panel), after which protein extracts were prepared and subjected to immunoblot analysis with antibodies against PDZK1 or GAPDH.

Article Snippet: Protein extracts were subjected to immunoblot analysis with antibodies to phospho-MEK, MEK, phospho(T202/Y204)-ERK1/2, ERK1/2, c-Src (32G6) and EGFR (all purchased from Cell Signaling Technology); ER-α, c-Myc or GADPH (all from Santa Cruz Biotechnology); or PDZK1(EPR3751) (Novus Biologicals).

Techniques: Expressing, Incubation, Membrane, Imaging, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Western Blot

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: PDZK1 is a novel factor in breast cancer that is indirectly regulated by estrogen through IGF-1R and promotes estrogen-mediated growth.

doi: 10.2119/molmed.2011.00001

Figure Lengend Snippet: Figure 7. PDZK1 interacts with the Src/ER-α/EGFR complex and enhances EGF-mediated sig- nal transduction. (A) MCF-7 cells were treated with 1 nmol/L E2 for 48 h or left untreated, after which protein extracts were subjected to immunoprecipitation with antibodies against c-Src. The immunoprecipitates, along with 10% input, were subjected to immunoblot analysis with antibodies against c-Src, ER-α, PDZK1, EGFR or GAPDH. The same protein extracts were subjected to immunoprecipitation with control IgG followed by immunoblot analysis with antibodies against c-Src, PDZK1 or GAPDH (Supplementary Figure S8). (B) The same protein extracts were subjected to immunoprecipitation with antibodies against IGF-1R followed by immunoblot analysis with antibodies against PDZK1, IGF-1R or GAPDH. (C) MCF-7 cells were transiently transfected with siRNA targeting PDZK1 and were treated with 1 nmol/L E2 or left untreated for 48 h. In the absence of E2, cells were treated with 20 ng/mL EGF for different time intervals. Cells were collected and protein extracts were prepared and subjected to immunoblot analysis with antibodies against phospho-ERK1/2 (pERK), ERK, PDZK1 or GAPDH. Immunoblots for pERK were quantified by using Adobe Photoshop CS, and data are ex- pressed as the fold-change from control. *Difference from untreated WT control; #difference from similarly E2-treated nontransfected cells; p < 0.05.

Article Snippet: Protein extracts were subjected to immunoblot analysis with antibodies to phospho-MEK, MEK, phospho(T202/Y204)-ERK1/2, ERK1/2, c-Src (32G6) and EGFR (all purchased from Cell Signaling Technology); ER-α, c-Myc or GADPH (all from Santa Cruz Biotechnology); or PDZK1(EPR3751) (Novus Biologicals).

Techniques: Transduction, Immunoprecipitation, Western Blot, Control, Transfection

Journal: Advanced Science

Article Title: ARL3 Enhances ERα Stability via USP10 Deubiquitination to Promote Endocrine Resistance and Drive Mitochondrial Metabolic Reprogramming in HR+ Breast Cancer

doi: 10.1002/advs.202509769

Figure Lengend Snippet: ARL3 correlates with the luminal subtype and predicts poor prognosis in breast cancer. A) Venn diagram of shared differentially expressed genes among tamoxifen‐resistant MCF7 ( GSE159968 and GSE148878 datasets), T47D ( GSE125738 dataset), and endocrine therapy‐relapsed patients ( GSE9893 dataset). B‐D) Comparative ARL3 mRNA (TCGA dataset) and protein (CPTAC dataset) expression profiles in breast cancer tissues. E,F) ARL3 mRNA (E) and protein expression levels (F) across breast cancer cell lines. G,H) Overall survival analysis (GEPIA database) (G) and Kaplan‐Meier survival curves based on ARL3 protein expression levels (KM Plot database) (H). I) Quantitative PCR analysis of ARL3 expression in 31 paired breast cancer tissues and adjacent normal tissues ( ** p < 0.01, paired two‐tailed Student's t ‐test). J) Representative immunohistochemical staining of ARL3 and ESR1 in human breast cancer tissue and matched adjacent normal tissue. Scare bars, 20 µm. K) Quantitative analysis of ARL3‐positive cells in luminal breast cancer ( n = 34, *** p < 0.001) and triple‐negative breast cancer (TNBC) ( n = 12, ** p < 0.01) tissues versus adjacent normal tissues (paired two‐tailed Student's t ‐test).

Article Snippet: Immunohistochemical analysis of ARL3, ESR1, and Ki67 was performed on 4 μm FFPE sections from breast cancer specimens (First Affiliated Hospital of CQMU, Ethics Approval K2023‐468) using validated rabbit monoclonal antibodies (ARL3, Proteintech 10961‐1‐AP, 1:200, ESR1, Proteintech 21244‐1‐AP, 1:100, Ki67, Servicebio GB111499 ‐100 1:500) with citrate‐based antigen retrieval and EnVision detection system.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test, Immunohistochemical staining, Staining

Journal: Advanced Science

Article Title: ARL3 Enhances ERα Stability via USP10 Deubiquitination to Promote Endocrine Resistance and Drive Mitochondrial Metabolic Reprogramming in HR+ Breast Cancer

doi: 10.1002/advs.202509769

Figure Lengend Snippet: ARL3 promotes tamoxifen resistance by increasing ESR1 expression. A) KEGG analysis of RNA‐seq (sgNC vs ARL3‐KO). B–D) GSEA plots (B,C) and GPSA (D) correlation diagrams generated from both the ESR1‐KD dataset obtained through the GPSA online platform and our corresponding RNA‐seq analysis. E,F) Quantitative PCR analysis of mRNA expression and Western blot detection of protein levels in the estrogen receptor (ER) downstream signaling pathway following ARL3 knockout (ARL3‐KO). G‐J) Functional assays assessing wound healing (G), cellular proliferation (CCK‐8, H), transwell assay(I), and tamoxifen sensitivity (J) following ESR1 reconstitution in ARL3‐knockout (ARL3‐KO) MCF7 cells. Mean ( n = 3) ± s.d. Two‐way ANOVA ( * p < 0.05, ** p < 0.01, *** p < 0.001, *** p < 0.0001). Scare bars, 250 µm (wound healing), 75 µm (transwell).

Article Snippet: Immunohistochemical analysis of ARL3, ESR1, and Ki67 was performed on 4 μm FFPE sections from breast cancer specimens (First Affiliated Hospital of CQMU, Ethics Approval K2023‐468) using validated rabbit monoclonal antibodies (ARL3, Proteintech 10961‐1‐AP, 1:200, ESR1, Proteintech 21244‐1‐AP, 1:100, Ki67, Servicebio GB111499 ‐100 1:500) with citrate‐based antigen retrieval and EnVision detection system.

Techniques: Expressing, RNA Sequencing, Generated, Real-time Polymerase Chain Reaction, Western Blot, Knock-Out, Functional Assay, CCK-8 Assay, Transwell Assay

Journal: Advanced Science

Article Title: ARL3 Enhances ERα Stability via USP10 Deubiquitination to Promote Endocrine Resistance and Drive Mitochondrial Metabolic Reprogramming in HR+ Breast Cancer

doi: 10.1002/advs.202509769

Figure Lengend Snippet: Knockout of ARL3 Promotes Degradation of ERα via Enhancing Polyubiquitination at K252 residue. A–C) Cycloheximide (CHX, 100 µg mL −1 ) chase assays in ARL3‐overexpressing and knockout cells. Quantified ERα decay curves with calculated half‐lives (C) (Mean ( n = 3) ± s.d. Student's t ‐test ( * p < 0.05). β‐Tubulin serves as loading control. D) Immunoblots showing MG132 (10 µ m , 6 h) rescues ERα levels in ARL3 knockout cells. β‐Tubulin serves as a loading control. E) Representative confocal images showing ARL3 (red) and ERα (green) co‐localization in MCF7 cells. Nuclei were counterstained with DAPI (blue). Scale bar, 20 µm. F) Co‐IP assays of ARL3 and ERα in MCF7 and T47D cells. Input was shown as an expression control. G,H) Schematic outlines of ARL3 and ESR1 structural features (G). The indicated constructs were transfected into HEK293T cells. After 24 h, cells were subjected to immunoprecipitation (IP) using anti‐FLAG or anti‐HA antibodies (H). I–K) MCF7 and T47D cells were co‐transfected with the designated plasmids and treated with MG132 prior to cell lysis. Immunoprecipitation (IP) was performed on cell lysates using an anti‐ERα antibody, followed by Western blotting (WB) with antibodies targeting ubiquitin (Ub) (I), Lys48 (K48)‐linked ubiquitin (J), Lys63 (K63)‐linked ubiquitin (K), and ERα. L) Ubiquitination mapping in ARL3‐deficient MCF7 cells, identifying characteristic ubiquitin signatures at K252 and K416 (arrows). M) Denaturing immunoprecipitation analysis of wild‐type (WT) ESR1 and lysine‐to‐arginine mutants (K252R, K416R) in ARL3 knockout (KO) cells.

Article Snippet: Immunohistochemical analysis of ARL3, ESR1, and Ki67 was performed on 4 μm FFPE sections from breast cancer specimens (First Affiliated Hospital of CQMU, Ethics Approval K2023‐468) using validated rabbit monoclonal antibodies (ARL3, Proteintech 10961‐1‐AP, 1:200, ESR1, Proteintech 21244‐1‐AP, 1:100, Ki67, Servicebio GB111499 ‐100 1:500) with citrate‐based antigen retrieval and EnVision detection system.

Techniques: Knock-Out, Residue, Control, Western Blot, Co-Immunoprecipitation Assay, Expressing, Construct, Transfection, Immunoprecipitation, Lysis, Ubiquitin Proteomics

Journal: Advanced Science

Article Title: ARL3 Enhances ERα Stability via USP10 Deubiquitination to Promote Endocrine Resistance and Drive Mitochondrial Metabolic Reprogramming in HR+ Breast Cancer

doi: 10.1002/advs.202509769

Figure Lengend Snippet: ARL3 promotes mitophagy through ERα upregulation. A) Gene Set Enrichment Analysis (GSEA) of RNA‐seq data (sgNC VS ARL3‐KO). B) GO term enrichment analysis of ARL3‐interacting proteins identified by immunoprecipitation‐mass spectrometry (IP/MS). C) Immunoblots of OXPHOS complex subunits (CI‐NDUFB8, CII‐SDHB, CIII‐UQCRC2, CIV‐MTCO1, CV‐ATP5A) in MCF7 and T47D cells transfected with ARL3 overexpression (OE) or knockout (KO) plasmids. β‐Tubulin serves as a loading control. D,E) Mitochondrial functional profiling in sgNC, ARL3‐KO cells, ATP production (D), and seahorse analysis (E). F,G) Autophagic flux analyzed by mRFP‐GFP‐LC3 reporter via confocal microscopy. Scare bars, 20 µm (F). Representative confocal images showing LC3B (red) and TOMM20 (green) co‐localization in sgNC, ARL3‐KO cells. Nuclei were counterstained with DAPI (blue). Scale bar, 50 µm (G). H) Representative immunoblots of p‐mTOR (Ser2448)/total mTOR, p‐AMPKα (Thr172)/total AMPKα, p62, LC3B‐I/II, and β‐tubulin (loading control) in sgNC, ARL3‐KO cells. β‐Tubulin serves as loading control. I) Cellular ATP levels in sgNC, ARL3‐KO, ARL3‐KO+ESR1‐OE MCF7, and T47D cells. Mean ( n = 3) ± s.d. Two‐way ANOVA ( *** p < 0.001, **** p < 0.0001). J) Immunoblots of OXPHOS complex subunits (CI‐NDUFB8, CII‐SDHB, CIII‐UQCRC2, CIV‐MTCO1, CV‐ATP5A) in sgNC, ARL3‐KO, ARL3‐KO+ESR1‐OE MCF7 and T47D cells. β‐Tubulin serves as a loading control. K) Autophagic flux analysis using mRFP‐GFP‐LC3 reporter. Yellow puncta (mRFP+GFP+) represent autophagosomes, red puncta (mRFP+GFP‐) indicate autolysosomes. Scale bar, 20 µm. L) Immunoblots of p‐mTOR, total mTOR, p‐AMPKα, total AMPKα, p62, and LC3B‐I/II in sgNC, ARL3‐KO, ARL3‐KO+ESR1‐OE cells. β‐Tubulin serves as a loading control. M) Seahorse analysis of sgNC, ARL3‐KO, ARL3‐KO+ESR1‐OE, and ARL3‐KO+ESR1‐OE +MHY1485 in MCF7 and T47D cells. N) Representative confocal images showing LC3B (red) and TOMM20 (green) co‐localization in sgNC, ARL3‐KO, ARL3‐KO+ESR1‐OE, and ARL3‐KO+ESR1‐OE +MHY1485 cells. Nuclei were counterstained with DAPI (blue). Scale bar, 20 µm.

Article Snippet: Immunohistochemical analysis of ARL3, ESR1, and Ki67 was performed on 4 μm FFPE sections from breast cancer specimens (First Affiliated Hospital of CQMU, Ethics Approval K2023‐468) using validated rabbit monoclonal antibodies (ARL3, Proteintech 10961‐1‐AP, 1:200, ESR1, Proteintech 21244‐1‐AP, 1:100, Ki67, Servicebio GB111499 ‐100 1:500) with citrate‐based antigen retrieval and EnVision detection system.

Techniques: RNA Sequencing, Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Western Blot, Transfection, Over Expression, Knock-Out, Control, Functional Assay, Confocal Microscopy

Journal: Cell Death & Disease

Article Title: BRCA1 negatively regulates IGF-1 expression through an estrogen-responsive element-like site

doi: 10.1038/cddis.2012.78

Figure Lengend Snippet: Binding of endogenous BRCA1 and ER α to the promoter regions of human IGF-1. ( a ) Schematic diagram of the human IGF-1 promoter showing the location and sequence of the ERE-like (EREL) sequence. ( b ) Cells pretreated with siRNA (control versus BRCA1) were used for ChIP assay. Endogenous promoter regions associated with BRCA1 and/or ER α were immunoprecipitated with anti-BRCA1 or anti-ER α antibody, respectively. The relative amounts of IGF-1 promoter-specific DNA containing the EREL site in immunoprecipitated complexes were then determined by semiquantitative PCR as described in Materials and Methods. The non-ERE region in IGF-1 promoter was used as negative control. ( c ) Using the same DNA samples obtained in ( b ) qRT-PCR was performed. ( b and c ) Representative data from two independent experiments performed in duplicate are shown as mean±S.E.M. *** P <0.001

Article Snippet: The sheared chromatin was immunoprecipitated with an ER- α antibody (HC-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), BRCA1 antibody (Ab1+Ab4; Calbiochem, Gibbstown, NJ, USA), or normal IgG.

Techniques: Binding Assay, Sequencing, Control, Immunoprecipitation, Negative Control, Quantitative RT-PCR

Journal: Mediators of Inflammation

Article Title: Anti-Inflammatory and Antimicrobial Effects of Estradiol in Bovine Mammary Epithelial Cells during Staphylococcus aureus Internalization

doi: 10.1155/2016/6120509

Figure Lengend Snippet: Oligonucleotides used in this study.

Article Snippet: The anti-phospho-ER α (2511S, Ser118) was obtained from Cell Signaling and the anti-ER β (517700) was acquired from Life Technologies.

Techniques: Sequencing

Journal: Mediators of Inflammation

Article Title: Anti-Inflammatory and Antimicrobial Effects of Estradiol in Bovine Mammary Epithelial Cells during Staphylococcus aureus Internalization

doi: 10.1155/2016/6120509

Figure Lengend Snippet: Participation of ERs in E2-treated bMECs. (a) The relative fluorescence intensities of ER α activation in bMECs treated with E2 and infected with S. aureus are shown. Fluorescence intensity was estimated from 10,000 events. An inserted histogram plot that shows ER α staining data in vehicle-treated bMECs (black line), cells that were challenged with S. aureus (blue line), cells that were stimulated with E2 (red line), and E2-treated bMECs infected with S. aureus (green line). The bMEC nuclei were fixed and stained with an anti-pER α antibody overnight and analyzed with flow cytometry. (b) The ER α mRNA expression was analyzed by RT-qPCR and GAPDH was used as endogenous gene in all conditions. Fold-change values greater than 2 or less than 0.5 were considered as significant differentially expressed mRNAs. Each bar shows the mean of triplicates ± SD of three independent experiments. (c) The relative fluorescence intensities of ER β activation in bMECs treated with E2 and infected with S. aureus are shown. Fluorescence intensity was estimated from 10,000 events. An inserted histogram plot that shows ER β staining data in vehicle-treated bMECs (black line), cells that were challenged with S. aureus (blue line), cells that were stimulated with E2 (red line), and E2-treated bMECs infected with S. aureus (green line). The bMEC nuclei were fixed and stained with an anti-ER β antibody overnight and analyzed with flow cytometry. (d) The ER β mRNA expression was analyzed by RT-qPCR and GAPDH was used as endogenous gene in all conditions. Fold-change values greater than 2 or less than 0.5 were considered as significant differentially expressed mRNAs. Each bar shows the mean of triplicates ± SD of three independent experiments. Different letters indicate significant changes among treatments ( P < 0.05). Vehicle: 1% ethanol.

Article Snippet: The anti-phospho-ER α (2511S, Ser118) was obtained from Cell Signaling and the anti-ER β (517700) was acquired from Life Technologies.

Techniques: Fluorescence, Activation Assay, Infection, Staining, Flow Cytometry, Expressing, Quantitative RT-PCR