Journal: bioRxiv

Article Title: Role of mitotic diffusion barriers in regulating the asymmetric division of activated CD8 T cells

doi: 10.1101/2021.09.10.458880

Figure Lengend Snippet: (A) Experimental outline. Murine naïve CD8 T cells, sorted from adult mouse spleen and lymph nodes, were cultured under homeostatic proliferation condition (IL7 and IL15) and subjected to lentiviral transduction on RetroNectin coated cell culture plates, followed by activation or phenotypic analysis. (B) Representative images of CD8 T cells expressing Sec61α-GFP (MemER-GFP) that stained with for CD8. (C and D) Anaphase to telophase transition time of activated CD8 T cells in the imaging medium compared to thermo-reversible hydrogel. Differential interference contrast (DIC) microscopy of dividing CD8 T cells (C) and quantification of anaphase to telophase transitions of dividing CD8 T cells in hydrogel (blue, n=15) or imaging medium (purple, n=18) (unpaired Welch’s t-test; mean ± SEM) (D) . (E) FACS analysis of phenotypic markers (CD62L, CD127, CD44, CD25 and CD69) of lentivirally transduced (purple) or non-transduced CD8 T cells (black) prior to activation (blue), or 48 hrs after TCR-mediated activation (pink). Data are representative of three independent experiments. Unpaired Welch’s t-test; mean ± SEM. Scale bars, 10 μm.

Article Snippet: Such isolated naïve CD8 T cells were cultured for 3d prior transducton with 3rd generation lentiviral vectors on RetroNectin (Takara) coated cell culture plates (according to the manufacturer’s protocol) for 2d without washing.

Techniques: Cell Culture, Transduction, Activation Assay, Expressing, Staining, Imaging, Microscopy

Journal: bioRxiv

Article Title: Impaired oxidative phosphorylation drives primary tumor escape and metastasis

doi: 10.1101/2025.01.08.631936

Figure Lengend Snippet: (A) Western blot analysis of the yeast complex I protein NDI1 stable expression in wild-type RENCA cells. Western blot analysis of ECAD protein expression in 40 µg of protein lysed from EV and NDI1 expressing RENCA cells. (B) Abdominal MRI demonstrating a peri-renal lymph node metastasis in a 70-year-old male HLRCC patient. Mitochondria imaged using Transmission Electron Microscopy (TEM) in (C) normal adjacent kidney and (D) tumor from the HLRCC patient in (B). Scale bars are 0.5 µm. (E) Tumor cell culture from patient in (B), designated as the continuous tumor cell line UOK365. (F) Seahorse flux analyses of FH -/- UOK262 and UOK365 tumor cells and FH +/+ control cells. (G) GSEA Hallmark analysis of bulk-RNA sequencing of 17 primary HLRCC tumors, 12 HLRCC metastatic tumors, 6 HLRCC normal adjacent kidneys, and 12 normal kidneys from donors. While (H) oxidative phosphorylation was enriched in normal kidney compared with HLRCC tumor, (I) glycolysis and (J) EMT were enriched in tumors compared with normal kidneys.

Article Snippet: NDUFA4L2 and NDI1 3 rd -generation lentiviral expression vectors were designed with a puromycin resistance cassette and purchased from Vectorbuilder Inc. HEK293T cells were used to package the vectors into lentiviruses.

Techniques: Western Blot, Expressing, Transmission Assay, Electron Microscopy, Cell Culture, Control, RNA Sequencing Assay