Journal: RNA biology

Article Title: Estrogen receptor alpha (ERα) regulates PARN-mediated nuclear deadenylation and gene expression in breast cancer cells.

doi: 10.1080/15476286.2024.2413821

Figure Lengend Snippet: Figure 1. Estrogen receptor alpha (ERα) is an activator of PARN-mediated nuclear deadenylation in MCF7 (ERα+) cells. (A) nuclear extracts (NEs) for cells treated with different concentrations of 17β-estradiol (E2) for the indicated times were used in in vitro deadenylation assays with radiolabeled capped L3(A30) RNA substrate. Purified RNA was analysed by denaturing PAGE. Left panel: representative deadenylation reactions from three independent biological assays are shown. Positions of the polyadenylated RNA L3(A30) and the L3 deadenylated product are indicated. Right panel: bar graph of relative deadenylation (RD) is shown. (B–C) in vitro deadenylation assays using NEs from cells treated with (B) control (CTRL) or ERα siRNA for 24 h or (C) with increasing concentrations of fulvestrant for 2 h (FVT) were performed and analysed as in (A). (D) MCF7 cells were treated with either CTRL or PARN siRNA and subsequently treated with vehicle or E2. NEs were used for in vitro deadenylation as performed and analysed in (A). E) Cell-free deadenylation assays were performed in the presence of radiolabeled capped L3(A30) RNA substrates, limiting amount of his-PARN deadenylase and his-ERα and increasing amounts of GST-p53. Conditions for deadenylation assays were performed as in (A). F) NEs from untreated cells were used in endogenous reciprocal co-immunoprecipitation (e-ip) assays with polyclonal ERα, PARN, or p53 antibodies. NEs were treated with RNase A. Equivalent amounts of pellets (IP) and supernatants (SN) were resolved by SDS-PAGE, and proteins were detected by Western blot. Topo II was used as loading and IP specificity control. Ten percent of the NEs used in the e-ip assays are shown as input. All figures show representative deadenylation reactions and Western blot analyses from at least three independent biological assays analysed by triplicate (n = 3). Experiments with two groups were analysed using two-tailed unpaired Student’s t-test. The p-values are indicated as *(<0.01), **(<0.001) and ***(<0.0001).

Article Snippet: NEs were IPed with antibodies against ERα polyclonal (Cell Signaling, cat# 8644S), PARN polyclonal (Bethyl Laboratories, cat# A303-562A), or p53 polyclonal (Santa Cruz Biotechnology, cat# FL-393) using protein A-magnetic beads (Millipore, PureProteome cat# LSKMAGA10) per manufacturer’s instructions.

Techniques: In Vitro, Purification, Control, Immunoprecipitation, SDS Page, Western Blot, Two Tailed Test

Journal: Breast Cancer Research : BCR

Article Title: FGF1 supports glycolytic metabolism through the estrogen receptor in endocrine-resistant and obesity-associated breast cancer

doi: 10.1186/s13058-023-01699-0

Figure Lengend Snippet: FGF1 can simulate ER phosphorylation in endocrine-resistant breast cancer cells. A – C Immunoblot analysis and quantification of pER-S118 ( B ), pER-S167 ( C ), and total ER in MCF7 cells after 15 min of treatment with vehicle (Veh), E2 (10 nM), FGF1 (5 ng/mL), or E2 + FGF1. D – F Immunoblot analysis and quantification of pER-S118 ( E ), pER-S167 ( F ), and total ER in MCF7 TAMR cells after 15 min of treatment with vehicle (Veh), E2 (10 nM), FGF1 (5 ng/mL), or E2 + FGF1. G – I Immunoblot analysis and quantification of pER-S118 ( H ), pER-S167 ( I ), and total ER in MCF7 cells after 15 min of treatment with vehicle (Veh), E2 (10 nM), FGF1 (5 ng/mL), or E2 + FGF1. All experiments were performed independently at least three times. Data were analyzed with unpaired t -tests, comparing each treatment to vehicle. J Full representative capillary immunoblot image of MCF7 TAMR cell lysates analyzed for vinculin (loading control), pER-S118, pER-S167, total ER, pMAPK, or total MAPK as indicated on the right, in cells treated with vehicle (Con) or BGJ398 (100 nM) overnight prior to stimulation with vehicle, E2, FGF1, or E2 + FGF1. K – L Quantification of immunoblot data in ( J ) from two independent experiments. Data are expressed as pER-S118/total ER ( K ) or pER-S167/total ER ( L ) and plotted as fold change of treatment versus vehicle. Data were analyzed by comparing the BGJ398 group to the control group within each treatment (vehicle, E2, FGF1, E2 + FGF1) using unpaired t -tests

Article Snippet: We evaluated the total protein and phosphorylated proteins in cell lysates by the Simple Western system that uses an automated capillary electrophoresis to perform protein separation (Protein Simple, San Jose, CA, SM-W004-1, PS-ST01, PN-009-050), immobilized the separated protein onto the capillary wall, immuno-probe for the target protein using a primary antibodies total ERα (Thermo Scientific RM9101-50), ERα S118 (Abcam Cat. No. 32396 diluted 1:50), ERα S167 (Cell Signaling Technologies, Boston, MA, Cat. No. 64508 s), p44/42 MAPK (Erk1/2)(Thr202/Thr204) (Cell Signaling Technologies, Boston, MA, Cat. No. 4370S diluted 1:200), p44/42 MAPK(Erk1/2) (Cell Signaling Technologies, Boston, MA, Cat. No. 9102 diluted 1:200) and vinculin was used as loading control (Cell Signaling Technologies, Boston, MA, 1390, diluted 1:1000) and secondary anti-rabbit HRP antibody conjugate (1X) (Protein Simple, San Jose, CA, DM-001).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Breast Cancer Research : BCR

Article Title: FGF1 supports glycolytic metabolism through the estrogen receptor in endocrine-resistant and obesity-associated breast cancer

doi: 10.1186/s13058-023-01699-0

Figure Lengend Snippet: FGFR levels across cell lines and FGFR1 overexpression in MCF7 cells. A – D Expression levels of A FGFR1, B FGFR2, C FGFR3, and D FGFR4 in MCF7, MCF7 TAMR, and UCD12 cells measured by RNA sequencing. E Immunoblot analysis of FGFR1 and actin protein in control or FGFR1-overexpressing MCF7 cells treated with vehicle, E2, or FGF1 for 15 min. F – G Immunoblot analysis of pER-S118 and pER-S167 relative to total ER in control or FGFR1-overexpressing MCF7 cells treated with vehicle, E2, or FGF1 for 15 min. Representative immunoblots are shown in ( G ) with vinculin loading control, pMAPK, or total MAPK

Article Snippet: We evaluated the total protein and phosphorylated proteins in cell lysates by the Simple Western system that uses an automated capillary electrophoresis to perform protein separation (Protein Simple, San Jose, CA, SM-W004-1, PS-ST01, PN-009-050), immobilized the separated protein onto the capillary wall, immuno-probe for the target protein using a primary antibodies total ERα (Thermo Scientific RM9101-50), ERα S118 (Abcam Cat. No. 32396 diluted 1:50), ERα S167 (Cell Signaling Technologies, Boston, MA, Cat. No. 64508 s), p44/42 MAPK (Erk1/2)(Thr202/Thr204) (Cell Signaling Technologies, Boston, MA, Cat. No. 4370S diluted 1:200), p44/42 MAPK(Erk1/2) (Cell Signaling Technologies, Boston, MA, Cat. No. 9102 diluted 1:200) and vinculin was used as loading control (Cell Signaling Technologies, Boston, MA, 1390, diluted 1:1000) and secondary anti-rabbit HRP antibody conjugate (1X) (Protein Simple, San Jose, CA, DM-001).

Techniques: Over Expression, Expressing, RNA Sequencing, Western Blot, Control

Journal: Cell reports

Article Title: Challenges and Reinterpretation of Antibody-Based Research on Phosphorylation of Tyr 307 on PP2Ac

doi: 10.1016/j.celrep.2020.02.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: P-SRC Y419 , Cell Signaling , Cat#2101; Lot#4; RRID:AB_331697.

Techniques: Recombinant, Protease Inhibitor, Cloning, Mutagenesis, Isolation, Bicinchoninic Acid Protein Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Vitamin D Compounds PRI-2191 and PRI-2205 Enhance Anastrozole Activity in Human Breast Cancer Models

doi: 10.3390/ijms22052781

Figure Lengend Snippet: Biomol GmbH Human Estrogen Signaling Primer Library, Cat. No: HESR-1—a set of commercial primers used to analyze the expression of the examined genes in MCF-7 breast cancer cells.

Article Snippet: Human Estrogen Signaling Primer Library (HESR-I, Biomol GMBH, Hamburg, Germany) was used for this purpose.

Techniques: Expressing, Binding Assay, Virus