cd44  (R&D Systems)

Journal: Oncogenesis

Article Title: P4HB maintains Wnt-dependent stemness in glioblastoma stem cells as a precision therapeutic target and serum marker

doi: 10.1038/s41389-024-00541-2

Figure Lengend Snippet: A – C Kaplan–Meier survival curves for patients with glioma from the TCGA, CGGA, and REMBRANDT datasets, stratified by high and low expression of P4HB . P -values were calculated using the log-rank test and Wilcoxon test. D UMAP plot showing the clustering of different cell types within the GBM patients’ tissues, and the expression levels of P4HB across different cell clusters. E – G Relative mRNA expression levels of P4HB in X01, 448, and 83 GSCs, respectively, were measured by qRT-PCR after shRNA-mediated knockdown of P4HB . Data are presented as means ± SD, n = 3, *** P < 0.001, t-test. H Western blot analysis of P4HB, NESTIN, and SOX2 protein levels in X01 GSCs after shRNA-mediated knockdown of P4HB , α-tubulin was used as a loading control. I Limit-dilution assay for sphere-forming capacity in X01 GSCs following shRNA-mediated knockdown of P4HB . Log fraction without spheres is plotted against the number of initial cells per well. ** P < 0.01, *** P < 0.001, t-test. J Western blot analysis of P4HB, NESTIN, and SOX2 protein levels in 448 GSCs after shRNA-mediated knockdown of P4HB , α-tubulin was used as a loading control. K Limiting dilution assay for sphere-forming capacity in 448 GSCs following shRNA-mediated knockdown of P4HB . ** P < 0.01, *** P < 0.001, t-test. L Western blot analysis of P4HB, NESTIN, and CD44 protein levels in 83 GSCs after shRNA-mediated knockdown of P4HB , α-tubulin was used as a loading control. M Limiting dilution assay for sphere-forming capacity in 83 GSCs following shRNA-mediated knockdown of P4HB . ** P < 0.01, *** P < 0.001, t-test.

Article Snippet: Western blots were incubated with primary antibodies targeting P4HB (Proteintech, #D154013), α-Tubulin (Proteintech, #11224-1-AP), GAPDH (HUABIO, #EM1101), Vinculin (Proteintech, #26520-1-AP), Caspase-3 and Cleaved Caspase-3 (CC3, Cell Signaling Technology, #14220, #9664), PARP and Cleaved PARP (C-PARP, Cell Signaling Technology, #9542, #5625), BCL-2 (1:1000, Abcam, #ERP17509), β-catenin (Beyotime, #AC106), LRP6 (Cell Signaling Technology, #2560T), P-LRP6 (ZENBIO, R30284), Cyclin D1 (Proteintech, #60186-1-Ig), NESTIN (Thermo Fisher Scientific, #PA5-11887), SOX2 (R&D Systems, #AF2018-SP), CD44 (R&D Systems, #BBA10), and Lamin B1 (Proteintech, #12987-1-AP) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, shRNA, Knockdown, Western Blot, Control, Dilution Assay, Limiting Dilution Assay

Journal: Genes

Article Title: Enhancing Transcriptional Reprogramming of Mesenchymal Glioblastoma with Grainyhead-like 2 and HDAC Inhibitors Leads to Apoptosis and Cell-Cycle Dysregulation.

doi: 10.3390/genes14091787

Figure Lengend Snippet: Figure 1. GRHL2 represses mesenchymal proteins in GBM cells. Western blots for GRHL2, Slug, CD44, MMP2, and ZEB1 in LN229 cells with or without doxycycline (200 ng/mL) or 2.5 µM vorinostat (VOR). β-Actin was used as a loading control for protein expression. Graphs depict significant changes in protein expression levels from A. Graphs depict means +/−SEM for n = 3. * p < 0.05 t-test; **p < 0.01 t-test.

Article Snippet: The cells were incubated overnight with 10 μg/mL anti-CD44 antibody (R&D systems, BBA10) diluted in 1% BSA in PBS at 4 ◦C.

Techniques: Western Blot, Control, Expressing