Journal: Circulation Research

Article Title: Jmjd3 Controls Mesodermal and Cardiovascular Differentiation of Embryonic Stem Cells

doi: 10.1161/circresaha.113.302035

Figure Lengend Snippet: Figure 1. Aberrant differentiation of Jmjd3-/- ESCs. (A) qPCR analysis of Jmjd3 in WT and Jmjd3-/- ESCs. (B) Western blot analysis of Jmjd3 and Histone marks in WT and Jmjd3-/- ESCs. Histone H3 is used as a loading control. Quantification is shown in the right panel (N=3-5). (C) Upper panel: Morphology of WT and Jmjd3-/- ESCs on feeder cells. Lower panel: Alkaline phosphatase staining of undifferentiated WT and Jmjd3-/- ESCs. (D) Western blot analysis of Jmjd3 and Oct4 in WT ESCs during differentiation. Į-Tubulin is used as a loading control. (E) Bright field image of EBs at day 5. Scale bar indicates 200μm. (F) Gene ontology analysis for more than 2-fold repressed genes in Jmjd3-/- ESCs compared to WT ESCs 4 days after differentiation. The most highly represented categories are presented with ontology terms on the y-axis and p-values for the significance of enrichment are shown on the x-axis. (G) Gene expression changes of pluripotency and lineage-specific markers in WT and Jmjd3-/- ESCs after spontaneous differentiation by LIF withdrawal. N=4.

Article Snippet: Immunofluorescence was performed as previously described 4 using antibody against Oct4 (2750 Cell Signaling), Nanog (ab80892 abcam), H3K27me3 (CS200603 Millipore), αMHC (sc-20641 Santa Cruz), Pecam-1 (550274 BD Biosciences) and Phalloidin (MFP-A1379 Mo Bi Tec).

Techniques: Western Blot, Control, Staining, Gene Expression

Journal: BMC Medicine

Article Title: ISG15-driven immune modulation and tumor progression in breast cancer metastasis: insights from single-cell and spatial transcriptomics

doi: 10.1186/s12916-025-04614-w

Figure Lengend Snippet: Impact of ISG15 on self-renewal and migration invasion of CSCs. Note: A Expression levels of the Isg15 gene in different cell types; B illustration of immunomagnetic bead separation of CSCs; C immunofluorescence staining of ISG15 and CD133 expression in primary and lymph node metastatic tumors; D western blot analysis of ISG15 protein levels in bulk tumor cells, CD133 + , and CD133 − subpopulations; E application of immunofluorescence technique to detect stem cell markers CD133, Nestin, CD44, and OCT4 in control group cells and isolated CSC cells, where blue represents DAPI nuclear staining and red represents cells positive for CD133, Nestin, CD44, and OCT4 (scale bar = 25 μm). OCT4 staining was performed post-fixation/permeabilization and served as additional validation for stemness marker expression but was not used for functional experiments; F overexpression of ISG15 detected by western blotting in 4T1-S cells; G knockdown of ISG15 detected by western blotting in 4T1-S cells; H protein expression levels of stemness markers NESTIN, OCT4, and SOX2 in different groups of 4T1-S cells determined by western blotting; I spheroid formation assay showing the sphere-forming ability of different groups of 4T1-S cells (scale bar = 100 μm); J clonogenic assay demonstrating the clonogenic ability of different groups of 4T1-S cells; K CCK-8 assay measuring the proliferation ability of different groups of 4T1-S cells; L Transwell assay assessing the migration and invasion ability of different groups of 4T1-S cells (scale bar = 50 μm). * indicates a significant difference between groups ( P < 0.05). All cell experiments were performed in triplicate

Article Snippet: The membrane was blocked with 5% skim milk at room temperature (RT) for 1 h, then incubated overnight at 4 °C with primary antibodies against ISG15 (1:1000, ab308219), NESTIN (1:1000, ab221660), OCT4 (1:10,000, ab200834), SOX2 (1:1500, ab92494), STAT3 (1:1500, ab68153), p-JAK2 (1:1500, ab32101), JAK2 (1:5000, ab108596), PD-L1 (1:1000, ab213480), Arg (1:1000, ab203490), CD206 (1:1200, ab64693), CD163 (1:1000, ab182422), CD86 (1:1200, ab220188), and GAPDH (1:2500, ab9485) as the internal reference (all from Abcam, Cambridge, UK), as well as p-STAT3 (1:1000, NB100-82213, Novus Biologicals).

Techniques: Migration, Expressing, Immunofluorescence, Staining, Western Blot, Control, Isolation, Biomarker Discovery, Marker, Functional Assay, Over Expression, Knockdown, Tube Formation Assay, Clonogenic Assay, CCK-8 Assay, Transwell Assay