Journal: Breast Cancer Research : BCR

Article Title: Nuclear PD-L1 drives IFN-γ-promoted lung metastasis of triple-negative breast cancer via POLR2A-mediated transcriptional activation of LY6E

doi: 10.1186/s13058-025-02193-5

Figure Lengend Snippet: LY6E is identified as a key downstream effector of nuclear PD-L1 in metastasis.​​ A ​​ Left: Venn diagram illustrating the strategy for identifying downstream targets of IFN-γ/nuclear PD-L1 from RNA-seq data of PD-L1 WT and KO 4T1 cells treated with or without 50 ng/mL IFN-γ for 24 h. Right: Heatmap of four candidate genes. ​​ B – C ​​ qPCR validation of Cldn1, Ly6e, Anxa8, and Gnb4 mRNA expression in the indicated treatment groups of 4T1 cells ( n = 4). ​​ D ​​ Western blot analysis of Claudin-1 and Ly6E protein expression in PD-L1 WT and KO 4T1 cells treated with or without 50 ng/mL IFN-γ for 24 h. ​​ E – F ​​ qPCR analysis of CLDN1 and LY6E mRNA expression in PD-L1 WT and PD-L1 KO MDA-MB-231 cells treated with or without 50 ng/mL IFN-γ for 48 h. ​​ G – H ​​ Western blot analysis of Claudin-1 and Ly6E protein expression in PD-L1 WT and KO MDA-MB-231 cells treated with or without 50 ng/mL IFN-γ for 48 h. I ​​ Analysis of LY6E and CD274 mRNA expression in TCGA TNBC tumor and adjacent normal tissue samples. ​​ J ​​ Correlation analysis of LY6E and CD274 mRNA expression in TCGA TNBC samples. ​​ K ​​ Representative images of lungs and quantification of metastatic nodules from LY6E overexpression cells. Data are presented as mean ± SD. Student’s t-test was used for two-group data analysis, while One Way ANOVA was used for multiple-group data analysis

Article Snippet: Proteins (20–30 μg) were separated by SDS-PAGE, transferred to nitrocellulose (NC) membranes, and probed with primary antibodies against PD-L1 (CST, #13684; #60475), POLR2A (CST, #2629), LY6E (Proteintech, 22144-1-AP), Claudin-1 (Proteintech, 13050-1-AP), STAT1 and phosphorylated STAT1 (CST, #14994; #9167), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: RNA Sequencing, Biomarker Discovery, Expressing, Western Blot, Over Expression

Journal: Breast Cancer Research : BCR

Article Title: Nuclear PD-L1 drives IFN-γ-promoted lung metastasis of triple-negative breast cancer via POLR2A-mediated transcriptional activation of LY6E

doi: 10.1186/s13058-025-02193-5

Figure Lengend Snippet: Nuclear PD-L1 forms a transcriptional complex with POLR2A to upregulate LY6E.​​ A ​​ Browser view of published ChIP-seq data showing lack of PD-L1 enrichment at the LY6E promoter region (highlighted in yellow) in MDA-MB-231 cells. ​​ B ​​ Transcription factors (TFs) predicted to bind the LY6E promoter in MDA-MB-231 cells, ranked by transcriptional potential score. ​​ C ​​ Venn diagram identifying POLR2A as the only TF overlapping between factors predicted to bind LY6E ( B ) and factors found to interact with PD-L1 by Co-IP/MS in MDA-MB-231 cells. ​​ D ​​ Browser view of ChIP-seq data showing POLR2A enrichment at the LY6E promoter region (highlighted in yellow) in MDA-MB-231 cells. ​​ E ​​ Predicted structural model of the PD-L1/POLR2A interaction generated by ZDOCK. Key interacting residues are labeled: Salt bridge (PD-L1 R140 - POLR2A E517), Electrostatic interactions (PD-L1 K136/K185 - POLR2A D452/D663). ​​ F ​​ Co-immunoprecipitation assay in MDA-MB-231 cells to detect protein interaction between POLR2A and PD-L1

Article Snippet: Proteins (20–30 μg) were separated by SDS-PAGE, transferred to nitrocellulose (NC) membranes, and probed with primary antibodies against PD-L1 (CST, #13684; #60475), POLR2A (CST, #2629), LY6E (Proteintech, 22144-1-AP), Claudin-1 (Proteintech, 13050-1-AP), STAT1 and phosphorylated STAT1 (CST, #14994; #9167), and GAPDH (Proteintech, 60004-1-Ig).

Techniques: ChIP-sequencing, Co-Immunoprecipitation Assay, Generated, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Human Wharton’s Jelly-Derived Mesenchymal Stem Cells Minimally Improve the Growth Kinetics and Cardiomyocyte Differentiation of Aged Murine Cardiac c-kit Cells in In Vitro without Rejuvenating Effect

doi: 10.3390/ijms20225519

Figure Lengend Snippet: List of antibodies for CC characterisation and differentiation.

Article Snippet: FITC Rat Anti-mouse Sca-1 Antibody (Clone D7) , 1:10 , FC , Miltenyi Biotec, Germany (130-102-297).

Techniques:

Journal: eLife

Article Title: Epigenetic regulation of Wnt7b expression by the cis -acting long noncoding RNA Lnc-Rewind in muscle stem cells

doi: 10.7554/eLife.54782

Figure Lengend Snippet: ( A ) Table represents the values obtained by analysing the local sequence alignment between the human and murine Lnc-Rewind transcripts. Data were produced by using the implementation of the Smith–Waterman algorithm available at http://www.ebi.ac.uk/Tools/psa/emboss_water/ . ( B ) Semiquantitative RT-PCR (sqRT-PCR) quantification of the human hs_Lnc-Rewind transcript in proliferating (GM) myoblasts from a healthy donor. Mature Gapdh was used as endogenous control. ( C ) FACS plot showing MuSC isolation strategy from WT mice. MuSCs are isolated, among the lineage (CD45/CD31/Ter119) negative cells, as α7integrin+/Sca1− cells (magenta box) (left and middle panels). The right plot shows the check purity of sorted MuSCs. ( D ) qRT-PCR quantification of Myog and Mck in C 2 C 12 and MuSC-derived myoblasts in growth (GM) and differentiated (DM) conditions. Data represent the mean ± SEM of three biological replicates and were normalized on Gapdh mRNA. ( E ) sqRT-PCR quantification of Lnc-Rewind, using different primers, in cytoplasmic (Cyt) and nuclear (Nuc) fractions from proliferating C 2 C 12 and MuSC-derived myoblasts. The quality of fractionation was tested with mature ( Gapdh ) and precursor ( pre-Gapdh ) RNAs. –rt represents the negative control ( F ) Representative 60X confocal images of MuSC-derived myoblasts cell cultures hybridized with probes set specific for Lnc-Rewind (upper panels) and for a human mRNA (dlc1), as negative control (neg ctrl) (bottom panels). Autofluorescence (gray) is shown with false colour to visualize the cell body. DAPI, 4’,6-diamidino-2-phenylindole (blue); scale bar: 25 μm. ( G ) Representative 60× confocal images of C 2 C 12 cell cultures hybridized with probes set specific for Lnc-Rewind (upper panels) and for a human mRNA (dlc1), as negative control (neg ctrl) (bottom panels). Autofluorescence (grey) is shown with false colour to visualize the cell body. DAPI, 4’,6-diamidino-2-phenylindole (blue); scale bar: 25 μm. Data information: *p<0.05, unpaired Student’s t-test. Figure 1—figure supplement 1—source data 1. Source data for .

Article Snippet: Cells were incubated with primary antibodies CD31-PE (MiltenyBiotec, 130111540; RRID: AB_2657296 ), CD45-PE (MiltenyBiotec, 139110797; RRID: AB_2658218 ), Ter119-PE (MiltenyBiotec, 130112909; RRID: AB_2654115 ) 1:25; Sca1-FITC (MiltenyBiotec, 130116490; RRID: AB_2751322 ) 1:50; α7Integrin-APCVio770 (MiltenyBiotec, 130095212; Custom) 1:20 for 45 min on ice diluted in HBSS containing 0.2% BSA, 1% penicillin-streptomycin, and 1% DNAse I.

Techniques: Sequencing, Produced, Reverse Transcription Polymerase Chain Reaction, Control, Isolation, Quantitative RT-PCR, Derivative Assay, Fractionation, Negative Control

Journal: eLife

Article Title: Epigenetic regulation of Wnt7b expression by the cis -acting long noncoding RNA Lnc-Rewind in muscle stem cells

doi: 10.7554/eLife.54782

Figure Lengend Snippet:

Article Snippet: Cells were incubated with primary antibodies CD31-PE (MiltenyBiotec, 130111540; RRID: AB_2657296 ), CD45-PE (MiltenyBiotec, 139110797; RRID: AB_2658218 ), Ter119-PE (MiltenyBiotec, 130112909; RRID: AB_2654115 ) 1:25; Sca1-FITC (MiltenyBiotec, 130116490; RRID: AB_2751322 ) 1:50; α7Integrin-APCVio770 (MiltenyBiotec, 130095212; Custom) 1:20 for 45 min on ice diluted in HBSS containing 0.2% BSA, 1% penicillin-streptomycin, and 1% DNAse I.

Techniques: Sequencing, Control, Clone Assay, SYBR Green Assay, Magnetic Beads, Recombinant, Software