Journal: International Journal of Biological Sciences

Article Title: The Interaction of CircESR1 and HNRNPAB Regulates Cell Cycle Transition of Breast Cancer Cell

doi: 10.7150/ijbs.126014

Figure Lengend Snippet: Estrogen promotes HNRNPAB expression via SP1. (A) Immunoblot assessment of HNRNPAB in 2 human normal breast epithelial cell lines and 11 human BC cell lines. (B) CPTAC database analyzed the relative HNRNPAB and ERα protein level in 105 BC patients. P value was determined by Pearson correlation analysis. (C) The relative expression of TFF1 and HNRNPAB mRNAs after stimulated with 10 nM E 2 in estrogen-deprived MCF-7 cells for 48 h analyzed by qRT-PCR. (D) Immunoblot assessment of HNRNPAB expression in MCF-7 with or without 10 nM E 2 in estrogen-deprived MCF-7 cells for 72 h, or in short-term oestrogen deprivation (STED) MCF-7 cells for 7 days and parental cells. (E) The schematic of HNRNPAB promoter region sequence selected from the transcription start site of -2 kb~+100 bp. (F) MCF-7 cells were transfected with pGL3 basic reporter vector containing HNRNPAB promoter, and pRL-TK reporter control vector containing luciferase activity, as well as control siRNA or different siRNA sequences of JUN , FOS , FOXA1 and SP1 . The relative fluorescence activity was measured. (G) CPTAC database analyzed the relative HNRNPAB and SP1 protein level in 68 ER+ BC patients. P value was determined by Pearson correlation analysis. (H) 293T cells were transfected with control or SP1 vector, and pRL-TK reporter control vector containing luciferase activity, as well as pGL3 basic reporter vector containing different regions of HNRNPAB promoter. The relative fluorescence activity was measured. (I) The schematic of three predicted conserved SP1 binding sites on the upstream -425 bp~+100 bp regions of the human HNRNPAB gene. Red boxes represent the predicted SP1 binding sites. (J) The DNA regions enriched by SP1 in ChIP assay were verified by PCR. (K) 293T cells were transfected with control or SP1 vector, and pRL-TK reporter control vector containing luciferase activity, as well as pGL3 basic reporter vector or containing “b” or “b” mutant regions. The relative fluorescence activity was measured. (L) The relative expression of SP1 and HNRNPAB mRNAs in MCF-7 cells bearing control or SP1 shRNAs analyzed by qRT-PCR. (M) Immunoblot assessment of SP1 and HNRNPAB expression in MCF-7 cells with or without SP1 shRNAs in the presence or absence of 10 nM E 2 for 48 h. Data was shown as mean ± S.D. from three independent experiments. Unpaired two-tailed Student's t test (C, H, K) and one-way ANOVA followed by Tukey's multiple comparisons test (F, L). ns, P >0.05; ***, P <0.001; ****, P <0.0001.

Article Snippet: For transcription factor mediated HNRNPAB expression, 0.2 μg pGL3 Basic luciferase reporter (RRID: Addgene_48743), 50 nmol JUN or FOS or FOXA1 or SP1 siRNAs and 0.02 μg pRL-TK plasmid (RRID: Addgene_11313) were transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Sequencing, Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay, Fluorescence, Binding Assay, Mutagenesis, Two Tailed Test

Journal: Communications Biology

Article Title: Endometrial stromal Menin supports endometrial receptivity by maintaining homeostasis of WNT signaling pathway through H3K4me3 during WOI

doi: 10.1038/s42003-025-08434-9

Figure Lengend Snippet: a qRT-PCR analysis of MEN1 , PRL , and IGFBP1 mRNA expression in human endometrial stromal cells (hESCs) treated with E2, MPA, and 8Br-cAMP for 0, 2, and 4 days ( n = 3). b ELISA analysis of the secreted levels of PRL in spent medium at D0 and D4 of decidualization in the shCtrl and sh MEN1 groups ( n = 3). c Western blotting assay of Menin and IGFBP1 protein expression in the shCtrl and sh MEN1 groups treated with E2, MPA, and 8Br-cAMP for 4 days ( n = 3). d Immunofluorescence (IF) staining of F-actin in primary hESCs transfected with shCtrl or sh MEN1 following 96 h of EPC treatment ( n = 3). e Volcano plot of differentially expressed genes detected by RNA sequencing (RNA-seq) in hESCs decidualized for 4 days with shCtrl and sh MEN1 . f Heatmap of decidualization-related genes in the shCtrl and sh MEN1 groups treated with E2, MPA, and 8Br-cAMP for 4 days ( n = 3) as determined by RNA-seq. g qRT-PCR analysis of HAND2 , FST , CEBPB , and EGR1 expression in the shCtrl and sh MEN1 groups treated with E2, MPA, and 8Br-cAMP for 4 days ( n = 3). h IF staining for EdU in the shCtrl and sh MEN1 groups and EdU-positive proportion (%) of the shCtrl and sh MEN1 groups ( n = 3); scale bar = 200 µm. i CCK-8 assay of the shCtrl and sh MEN1 groups treated with E2, MPA, and 8Br-cAMP for 0 and 2 days ( n = 4). Data are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, Student’s t test, one-way ANOVA and two-way ANOVA.

Article Snippet: Commercial ELISA kits (E-EL-H0141, Elabscience) were used to detect the PRL level.

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Staining, Transfection, RNA Sequencing, CCK-8 Assay