Journal: Blood Cancer Journal

Article Title: Identification of the estrogen receptor beta as a possible new tamoxifen-sensitive target in diffuse large B-cell lymphoma

doi: 10.1038/s41408-022-00631-7

Figure Lengend Snippet: A Cell viability assay for DLBCL cell lines treated with tamoxifen (TAM) for 48 h in estrogen-containing culture medium. B Cell viability assay for cell lines treated with tamoxifen for 48 h in estrogen-free culture medium. C IC 50 values for DLBCL cell lines treated with tamoxifen for 48 h on normal culture medium and ES-free culture medium. D Western blot of U2932 treated with 10 (T10), 15 (T15), and 20 (T20) µM of tamoxifen in RPMI or ES-free RPMI. The effect of Tamoxifen treatment is shown on apoptosis with PARP and DNA damage with γH2AX. E Cell viability assay for cell lines treated with endoxifen for 48 h. F Cell viability assay for cell lines treated with PHTPP for 48 h. G Cell viability assay for cell lines treated with DPN for 48 h. H Competition assay for U2932 treated with tamoxifen and DPN for 48 h. Data were normalized to the control and plotted as the mean ± standard deviation (SD) of n = 3 in all panels. * P ≤ 0.05.

Article Snippet: Cells were incubated with increasing concentrations of tamoxifen (S1238, Selleckchem, Houston, TX, USA), Endoxifen (Selleckchem), PHTPP (Abcam) and diarylpropionitrile (DPN; Sigma-Aldrich) for 48 h. Cells were incubated with increasing concentrations of CHOP chemotherapy in combination with tamoxifen for 72 hours.

Techniques: Viability Assay, Western Blot, Competitive Binding Assay, Control, Standard Deviation

Journal: Blood Cancer Journal

Article Title: Identification of the estrogen receptor beta as a possible new tamoxifen-sensitive target in diffuse large B-cell lymphoma

doi: 10.1038/s41408-022-00631-7

Figure Lengend Snippet: A Western blot for ERβ in WT and KO cell line to confirm knockout. B Cell viability assay for WT and KO U2932 cell line treated with tamoxifen for 48 h. C Cell viability assay for WT and KO U2932 cell line treated with PHTPP for 48 h. D Cell viability assay for WT and KO U2932 cell line treated with endoxifen for 48 h. E Western blot for PARP1/cleaved PARP1 and γH2AX for U2932 and U2932 ERβO treated with 20 µM tamoxifen for 24 h. F Apoptosis induction with flow cytometry for AnV + (early) and AnV/PI + (late) apoptosis after treatment with tamoxifen (20 µM), endoxifen (15 µM), PHTPP (40 µM) and CHOP (1 µg/ml) in WT and ERβ KO U2932 cells. Average of three experiments. G Apoptosis for untreated WT and ERβ KO cells, flow cytometry for AnV and PI, and the average of four experiments.

Article Snippet: Cells were incubated with increasing concentrations of tamoxifen (S1238, Selleckchem, Houston, TX, USA), Endoxifen (Selleckchem), PHTPP (Abcam) and diarylpropionitrile (DPN; Sigma-Aldrich) for 48 h. Cells were incubated with increasing concentrations of CHOP chemotherapy in combination with tamoxifen for 72 hours.

Techniques: Western Blot, Knock-Out, Viability Assay, Flow Cytometry

Journal: PLoS ONE

Article Title: Male Sex is Associated with a Reduced Alveolar Epithelial Sodium Transport

doi: 10.1371/journal.pone.0136178

Figure Lengend Snippet: Primer sequences.

Article Snippet: The ER-β inhibitor 4-[2-Phenyl-5,7- bis (trifluoromethyl)pyrazolo[1,5- a ]pyrimidin-3-yl]phenol (PHTPP, # Cay16025-5, Biomol, Hamburg, Germany) was diluted in dimethyl sulfoxide (DMSO, # 472301, Sigma-Aldrich) and FDLE cells treated with 20 μM PHTPP [ , ] for 24 h prior to Ussing chamber analyses.

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Estrogen receptor β inhibits breast cancer cells migration and invasion through CLDN6-mediated autophagy

doi: 10.1186/s13046-019-1359-9

Figure Lengend Snippet: E2 regulates CLDN6 expression via ERβ. a Western blot analysis of ERα and ERβ expression in MCF-7 cells treated with E2. Actin served as a loading control. b Western blot analysis of ERβ in MDA-MB-231 cells treated with E2. c MDA-MB-231 cells were transfected with either negative control (sh-NC) or three different ERβ shRNAs for 48 h and were then subjected to western blot analysis to detect the protein abundance of ERβ. Actin was used as the loading control. d ERβ knockdown abolished the CLDN6 expression induced by E2. e MDA-MB-231 cells were incubated with DPN for 24 h at the indicated concentration. CLDN6 gene and protein expression levels were detected by using semiquantitative RT-PCR and western blot. f Immunofluorescence of CLDN6 (red) was prominent along the edges of the MDA-MB-231 cells upon DPN treatment. Nuclei were stained with 4, 6-diamino-2-phenylindole (DAPI) (blue) (scale bar, 20 μm). g Tight junctions (white arrowheads) between cells were prominent in MDA-MB-231 cells after DPN treatment as observed by TEM. h The migration (scale bar, 200 μm) and invasion (scale bar, 50 μm) abilities of MDA-MB-231 cells treated with DPN were decreased. i CLDN6 knockdown rescued the migration and invasion abilities of MDA-MB-231 cells after DPN treatment. The ERβ antagonist PHTPP (10 μM, 24 h) ( j ) and ERβ knockdown ( k ) abolished the DPN-induced CLDN6 expression. Overexpression of ERβ induced CLDN6 upregulation in SR-BR-3 ( l ) and MDA-MB-231 ( m ) cells after treatment with DPN. Data are presented as mean ± SD. The data shown are representative results of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The 17β-estradiol (E2) (10006315) and PHTPP (16025) were purchased from Cayman Chemical (Denver, USA).

Techniques: Expressing, Western Blot, Control, Transfection, Negative Control, Quantitative Proteomics, Knockdown, Incubation, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Migration, Over Expression

Journal: bioRxiv

Article Title: A gatekeeping role of ESR2 to maintain the primordial follicle reserve

doi: 10.1101/2020.02.06.937953

Figure Lengend Snippet: Gonadotropin stimulation resulted in an increased number of antral follicles in Esr2-/- ovaries. Wildtype (WT) and Esr2-/- rats were treated with exogenous gonadotropins on PND28. 48h after PMSG (30IU) injection, rats were administered with hCG (30IU). 4h after the hCG injection, ovaries were collected and processed for histological examination or collection of COCs. Histological examination demonstrated the presence of ovarian follicles at different stages of development in WT rats ( A ). In contrast, Esr2-/- rat ovaries showed an increased number of antral follicles ( B ). Cumulus cells were detached from the oocytes by mechanical pipetting before counting under microscope. Oocyte yield was about three-fold higher in Esr2-/- rat ovaries ( C ). Serially sectioned whole ovaries were stained with H&E, and follicles at different stages were counted in WT and Esr2-/- rats ( D ). Follicle counting demonstrated a decreased number of primordial follicles and an increased number of activated follicles in Esr2-/- rats ( E-G ). Data shown as mean ± SE, n≥3. * P ≤ 0.05.

Article Snippet: A selective ESR2 antagonist PHTPP ( ) or a selective ESR2 agonist DPN ( ) (Cayman Chemical, Ann Arbor, MI) was dissolved in DMSO (5µg/µl).

Techniques: Injection, Microscopy, Staining

Journal: bioRxiv

Article Title: A gatekeeping role of ESR2 to maintain the primordial follicle reserve

doi: 10.1101/2020.02.06.937953

Figure Lengend Snippet: Increased activation of primordial follicles in Esr2-/- rat ovaries. Follicle counting in PND28 ( A-C, J, M ), PND16 ( D-F, K, N ), and PND8 ( G-I, L, O ) wildtype (WT) and Esr2-/- rats showed increased activation of primordial follicles in Esr2-/- ovaries. Primordial follicle activation was near two-fold starting on PND8 ( G-I, L, O ). A greater number of atretic follicles were present within the pool of activated follicles at PND28 ( A, B ) PND16 ( D, E ) and PND8 ( G, H ) in Esr2-/- ovaries. Data shown as mean ± SE, n≥3. * P ≤ 0.05. Pd, Primordial follicle; Py, Primary follicle.

Article Snippet: A selective ESR2 antagonist PHTPP ( ) or a selective ESR2 agonist DPN ( ) (Cayman Chemical, Ann Arbor, MI) was dissolved in DMSO (5µg/µl).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: A gatekeeping role of ESR2 to maintain the primordial follicle reserve

doi: 10.1101/2020.02.06.937953

Figure Lengend Snippet: Premature ovarian senescence in Esr2-/- rats. The total number of follicles in PND8 ovaries were similar between wildtype (WT) and Esr2-/- rats; however, the number of primordial follicles were decreased in Esr2-/- ovaries ( A ). Follicle counts in 4wk, 12wk, and 24wk old WT and Esr2-/- rats revealed a sharp decline in the primordial follicle reserve in Esr2-/- ovaries ( B-H ). 24wk old Esr2-/- rats showed a significantly lower level of serum estradiol and AMH compared to WT ( I, J ). Data shown as mean± SE, n≥3 (follicle counting) and n≥6 (hormone assays), * P ≤ 0.05. Pd, Primordial follicle; Py, Primary follicle.

Article Snippet: A selective ESR2 antagonist PHTPP ( ) or a selective ESR2 agonist DPN ( ) (Cayman Chemical, Ann Arbor, MI) was dissolved in DMSO (5µg/µl).

Techniques:

Journal: bioRxiv

Article Title: A gatekeeping role of ESR2 to maintain the primordial follicle reserve

doi: 10.1101/2020.02.06.937953

Figure Lengend Snippet: Regulation of primordial follicle activation is ESR2-dependent. Deletion of the ESR2 DBD increased primordial follicle activation at PND28 ovaries ( A, B, G, I, J ), but this was not observed in ovaries from Esr1-/- rats ( C, G, K ). Administration of a selective ESR2 antagonist, PHTPP, into wildtype rats increased primordial follicle activation ( D, E, H, L, M ), whereas treatment with an ESR2 agonist, DPN, suppressed the activation ( F, H, N ). Data shown as mean ± SE, n ≥ 3. * P ≤ 0.05. Rel., Relative. Pd, Primordial follicle; Py, Primary follicle.

Article Snippet: A selective ESR2 antagonist PHTPP ( ) or a selective ESR2 agonist DPN ( ) (Cayman Chemical, Ann Arbor, MI) was dissolved in DMSO (5µg/µl).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: A gatekeeping role of ESR2 to maintain the primordial follicle reserve

doi: 10.1101/2020.02.06.937953

Figure Lengend Snippet: Activation of AKT, ERK, and mTOR signaling in Esr2-/- ovaries. Expression of ESR2 was detected in PND4, 6, and 8 rat ovaries using RT-qPCR ( A ) and western blotting ( B ). Primordial (Pd) ( C ), and primary (Py) ( D ) follicles were isolated from rat ovaries by digestion with liberase followed by size fractionation with strainers. RT-qPCR analysis demonstrated Esr2 expression in both Pd and Py follicles ( E ). Western blot analyses of PND8 ovaries and quantification of signal intensities demonstrated a significant increase in AKT ( F-I ) and ERK1/2 ( L-M ) activation. This was associated with increased activation of mTORC1 ( P-Q ) and its targets P70S6K ( R-S ) and RPS6 ( T-U ). But no difference was observed in PTEN ( J-K ) and pTSC2 ( N-O ) levels. Signal quantification data are presented as mean ± SEM. n ≥ 6. * P ≤ 0.05. Rel., Relative.

Article Snippet: A selective ESR2 antagonist PHTPP ( ) or a selective ESR2 agonist DPN ( ) (Cayman Chemical, Ann Arbor, MI) was dissolved in DMSO (5µg/µl).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Isolation, Fractionation

Journal: bioRxiv

Article Title: A gatekeeping role of ESR2 to maintain the primordial follicle reserve

doi: 10.1101/2020.02.06.937953

Figure Lengend Snippet: Transcript levels of known activators of the AKT and mTOR pathways. RT-qPCR was performed on PND8 wildtype (WT) and Esr2-/- rat ovaries to analyze the expression of genes involved in activation of the AKT and mTOR pathways and activation of primordial follicles. While the expression of Kitlg ( A ), Kit ( B ), Igf1 ( C ), Bmp15 ( H ) and Gdf9 ( I ) was moderately upregulated in Esr2-/- rat ovaries, Adcyap1r1 ( F ), Bmp4 ( G ), Gata4 ( N ), and Npm2 ( O ) were highly upregulated. But no significant differences in the expression of Fgf2 ( D ), Ntrk2 ( E ), Amh ( J ), Bdnf ( K ), Nobox ( L ) and Sohlh1 ( M ) were observed between the WT and Esr2-/- rat ovaries. RT-qPCR data represent the mean ± SEM. n ≥ 8. * P ≤ 0.05. Rel., Relative.

Article Snippet: A selective ESR2 antagonist PHTPP ( ) or a selective ESR2 agonist DPN ( ) (Cayman Chemical, Ann Arbor, MI) was dissolved in DMSO (5µg/µl).

Techniques: Quantitative RT-PCR, Expressing, Activation Assay

Journal: bioRxiv

Article Title: A gatekeeping role of ESR2 to maintain the primordial follicle reserve

doi: 10.1101/2020.02.06.937953

Figure Lengend Snippet: ESR2 signaling in the regulation of primordial follicle activation. Loss of ESR2 leads to upregulation of both granulosa cell and oocyte derived factors that can activate the AKT, ERK and mTOR pathways. Increased levels of KITLG, KIT and IGF1, as well as NPM2, and ADCYAP1R1 can activate the AKT pathway followed by the mTOR pathway. Moreover, upregulation of NPM2 and BMP4 can activate the ERK pathway. Activated AKT, ERK and mTOR pathways in association with the transcriptional regulator GATA4 can promote the transition of primordial follicles to primary follicles in Esr2-/- ovaries.

Article Snippet: A selective ESR2 antagonist PHTPP ( ) or a selective ESR2 agonist DPN ( ) (Cayman Chemical, Ann Arbor, MI) was dissolved in DMSO (5µg/µl).

Techniques: Activation Assay, Derivative Assay