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: 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: Journal of translational medicine

Article Title: ESRRG-PKM2 axis reprograms metabolism to suppress esophageal squamous carcinoma progression and enhance anti-PD-1 therapy efficacy.

doi: 10.1186/s12967-023-04347-5

Figure Lengend Snippet: Fig. 4 ESRRG inhibits the expression of key glycolytic enzyme PKM2 in ESCC cells. A A schematic diagram illustrates the regulation of glycolysis pathway. B Heatmap illustrates glycolysis related genes in transcript levels between TE1 cells with ESRRG overexpression and control. C The correlation between the relative levels of ESRRG and LDHA, PFKM, GPI, GAPDH, ENO1, PKM, TPI1, ALDOA mRNA transcripts in 82 ESCC tissues of TCGA database. D mRNA levels of 8 glycolysis-related genes in TE1 with ESRRG overexpression and ECa109 cells with ESRRG knockdown compared to relative control cells. E The correlation between the relative levels of ESRRG and PKM, determined by RT-PCR in 94 ESCC tissues. F Schematics of mutation strategies in the PKM promoter (− 2000 bp to + 50 bp). G Luciferase reporter assays exhibited that ESRRG bound to the PKM promoter at site 2 to induce its expression. H Effects of ESRRG overexpression and knockdown on PKM2 expression in ESCC cells. Immunohistochemistry (I) and Western blotting (J) for PKM2 protein level in xenograft tumor of mice after overexpression or knockout of ESRRG. Values are presented as mean ± SD (n = 3). *P < 0.05 or **P < 0.01 indicates significant differences from the vehicle group as assessed by a one-way ANOVA with a post hoc Dunnett’s test

Article Snippet: ESRRG (Proteintech, Cat No. 14017- 1-AP), PKM2 (Cell Signaling Technology, Cat No. 4053), β-actin (Cell Signaling Technology, Cat No. 4970), CD8 (Cell Signaling Technology, Cat No. 85336), and GAPDH (Santa Cruz Biotechnology, Cat No. sc47724) were used as primary antibodies.

Techniques: Expressing, Over Expression, Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Luciferase, Immunohistochemistry, Western Blot, Knock-Out

Journal: Journal of translational medicine

Article Title: ESRRG-PKM2 axis reprograms metabolism to suppress esophageal squamous carcinoma progression and enhance anti-PD-1 therapy efficacy.

doi: 10.1186/s12967-023-04347-5

Figure Lengend Snippet: Fig. 5 PKM2 is essential for ESRRG to inhibits tumor growth in ESCC cells. A–D Cell growth were determined in ESRRG knockdown ECa109 and KYSE510 cells with or without further knockdown of PKM employing CCK-8 assays(A, colony formation B and EDU assay (C, D). E Typical pictures of tumors isolated from nude mice with tumor xenografts derived from the indicated groups. F Tumour volumes were monitored at indicated time points. G The weight of tumours was measured at time of sacrificed. Values are presented as mean ± SD (n = 3–5). *P < 0.05 or **P < 0.01 indicates significant differences from the vehicle group as assessed by a one-way ANOVA with a post hoc Dunnett’s test

Article Snippet: ESRRG (Proteintech, Cat No. 14017- 1-AP), PKM2 (Cell Signaling Technology, Cat No. 4053), β-actin (Cell Signaling Technology, Cat No. 4970), CD8 (Cell Signaling Technology, Cat No. 85336), and GAPDH (Santa Cruz Biotechnology, Cat No. sc47724) were used as primary antibodies.

Techniques: Knockdown, CCK-8 Assay, EdU Assay, Isolation, Derivative Assay

Journal: Journal of translational medicine

Article Title: ESRRG-PKM2 axis reprograms metabolism to suppress esophageal squamous carcinoma progression and enhance anti-PD-1 therapy efficacy.

doi: 10.1186/s12967-023-04347-5

Figure Lengend Snippet: Fig. 8 Correlation of ESRRG and PKM2 in ESCC. A IHC analysis for ESRRG and PKM2 expression in clinical HCC samples. Scale bars = 100 μm. B Correlation between ESRRG expression and PKM2 expression (Chi-squared test). C Prognostic value of combining ESRRG and PKM2 levels was analyzed by Kaplan–Meier analysis in 94 ESCC samples. D Model illustrating tumor suppression mechanism regulated by ESRRG in ESCC progression

Article Snippet: ESRRG (Proteintech, Cat No. 14017- 1-AP), PKM2 (Cell Signaling Technology, Cat No. 4053), β-actin (Cell Signaling Technology, Cat No. 4970), CD8 (Cell Signaling Technology, Cat No. 85336), and GAPDH (Santa Cruz Biotechnology, Cat No. sc47724) were used as primary antibodies.

Techniques: Expressing