Journal: Radiotherapy and Oncology

Article Title: Regulation of O 2 consumption by the PI3K and mTOR pathways contributes to tumor hypoxia

doi: 10.1016/j.radonc.2014.02.007

Figure Lengend Snippet: Hypoxia is reduced in a 3D spheroid model and is predicted to decline in vivo . (A) EMT6 and FaDu spheroids were treated for 24 h with BEZ235 or BKM120, prior to fixation and sectioning. Sections were DAB-stained for pAKT (Ser473) expression. Representative images are shown for BEZ235 (50 nM) and BKM120 (5 μM). (B) The percentage of DAB-positive pixels in the viable region of the spheroid was reduced with all treatments. (C) Spheroids were treated with 50 nM BEZ235 or 5 μM BKM120 for 24 h, 48 h or 24 h followed by 24 h incubation in drug-free medium (24 h + wash). Hypoxia was assessed by staining central spheroid sections for EF5 (red) with Hoechst as a counterstain (blue). Data shown are representative of two independent experiments, n = 10 spheroids. (D) Mean EF5 fluorescence in spheroids treated for 24 h was reduced in both cell lines tested. (E) To assess whether hypoxia was recoverable, mean EF5 fluorescence in spheroids treated for 24 h was compared with that in spheroids treated for either 48 h or 24 h followed by the 24 h wash in drug-free medium. In cells treated with BEZ235, the 24 h wash period led to a return of the hypoxia signal. (F and G) Simulated distribution of oxygen partial pressure (mmHg) in a representative 1497 μm × 1482 μm vascular network derived from ex vivo samples where oxygen consumption was either 100% (F) or 55% (G) of base level. Highly oxygenated regions are in red, scaling down to poorly oxygenated regions in blue. (H) Average hypoxic fraction (defined as <10 mmHg) calculated across 10 example networks as in F as a function of varying oxygen consumption. All data points represent mean ± 95% Confidence Interval. One-way ANOVAs were performed with Bonferroni-corrected post t -tests. ∗ P < 0.01.

Article Snippet: To assess signaling inhibition in spheroids, sections were stained with anti-pAKT antibody using ImmPRESS™ reagent kit (MP-7401, VectorLabs) and DAB Peroxidase substrate kit (SK-4100, VectorLabs).

Techniques: In Vivo, Staining, Expressing, Incubation, Fluorescence, Derivative Assay, Ex Vivo

Journal: Oncotarget

Article Title: A Pyrazolo[3,4- d ]pyrimidine compound inhibits Fyn phosphorylation and induces apoptosis in natural killer cell leukemia

doi: 10.18632/oncotarget.11496

Figure Lengend Snippet: On 4c treated or not primary NK cells sorted from PBMCs of 3 NK-CLPD patients (UPN: 6–8) ( A ) Trypan blue count, ( B ) PKH67 proliferation test, ( C ) Apoptosis and ( D ) Caspase 3/7 activity assay, ( E ) Cell cycle analysis were performed. Proliferation, apoptosis and caspase 3/7 level were analyzed on CD56 + /CD16 + and CD56 + /CD16 − NK cell populations. P -values are indicated by asterisk: * p < 0.05, ** p < 0.01. (MFI= mean fluorescent intensity).

Article Snippet: NK primary cells were stained with PKH67 (PKH67 Green Fluorescent Cell Linker Kit for General Cell Membrane Labeling, Sigma Aldrich) as manufacturer's protocol, treated with 4c compound or with DMSO vehicle control for 24 hours and analyzed by FACSCalibur cytometer (BD).

Techniques: Activity Assay, Cell Cycle Assay

Journal: Cell

Article Title: The 7q11.23 Protein DNAJC30 is an Auxiliary Component of ATP Synthase and Links Mitochondria to Brain Development

doi: 10.1016/j.cell.2018.09.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Avidin-Biotin blocker , Vector Laboratories , Cat#SP-2001.

Techniques: Recombinant, Protease Inhibitor, Modification, Avidin-Biotin Assay, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Knock-Out, Sequencing, Clone Assay, Software, Microscopy, Digital PCR, Fluorescence, Transmission Assay

Journal: Cell Proliferation

Article Title: Haematopoietic lineage‐committed bone marrow cells, but not cloned cultured mesenchymal stem cells, contribute to regeneration of renal tubular epithelium after HgCl 2 ‐induced acute tubular injury

doi: 10.1111/j.1365-2184.2008.00545.x

Figure Lengend Snippet: Examples of engraftment of haematopoietic lineage marrow cells (HLMCs) and mesenchymal stem cells at 3 days after HgCl2. (a) GFP‐positive control shows proximal tubular cells (PHA‐L, blue colour) co‐stained for GFP (brown colour). (b) GFP‐negative control (wild type) showing no detection of GFP. (c) BMT mouse treated with HgCl2 demonstrating GFP‐positive proximal tubular epithelial cells. Dashed black arrows indicate PHA‐L stained donor HLMC‐derived tubular cells. (d) Male positive control; Y chromosomes were detected by indirect FISH, black arrows point to Y chromosomes (brown dot) in proximal tubular cells histochemically stained with PHA‐L (red colour). (e) Female control showing the lack of Y chromosome detection within proximal tubular cells histochemically stained with PHA‐L. (f) Scattered Y chromosomes (brown dot) in renal interstitial area, not in proximal tubular cells. (g) High magnification of yellow box in (f); a–f, ×400; g, ×600. BMT, bone marrow transplantation; FISH, fluorescence in situ hybridization; PHA‐L, Phaseolus vulgaris leucoagglutinin.

Article Snippet: After GFP staining, tissue sections were microwaved in 10 m m tri‐sodium citrate (pH 6.0) for 10 min and biotin was blocked using a biotin blocking kit (DAKO), and then incubated for 45 min with biotinylated Phaseolus vulgaris leucoagglutinin (PHA‐L) 1/100 (B‐1115, Vector Laboratories, Burlingame, CA, USA) to detect proximal convoluted tubules, or biotinylated peanut agglutinin (PNA) 1/800 (B‐1075, Vector Laboratories) to detect distal convoluted tubules.

Techniques: Positive Control, Staining, Negative Control, Derivative Assay, Transplantation Assay, Fluorescence, In Situ Hybridization

Journal: Cell Proliferation

Article Title: Haematopoietic lineage‐committed bone marrow cells, but not cloned cultured mesenchymal stem cells, contribute to regeneration of renal tubular epithelium after HgCl 2 ‐induced acute tubular injury

doi: 10.1111/j.1365-2184.2008.00545.x

Figure Lengend Snippet: (a) Changes in the abundance of GFP‐positive cells within the PHA‐L stained cell population in control mice and mice treated with HgCl2 (n = 5 per treatment time point). *P < 0.05 versus the same group at day 0; +P < 0.05 versus group CON at the corresponding time point. The percentages of GFP‐positive cells were adjusted based on a correction factor derived from the actual cell count of 41% of PHA‐L stained cells being GFP‐positive in GFP donor mice. (b) Changes in the 3H‐LI of PHA‐L stained proximal tubular cells of: combined indigenous and donor HLMC (left panel); indigenous origin (central panel); donor HLMC origin (right panel). n = 5 per group. *P < 0.05 versus the same group at day 0; +P < 0.05 versus group CON at the corresponding time point. (c–g) Examples of chimerism and proliferation (3H‐thymidine labelling) of GFP+ HLMC‐derived PHA‐L‐stained cells at 3 days after HgCl2. (c) Black arrowheads indicate PHA‐L stained (blue colour in tubular cell apical membrane) donor GFP+ HLMC‐derived tubular cells (brown colour in cytoplasm) under bright field, and black arrows indicate 3H‐thymidine labelling of PHA‐L‐stained HLMC‐derived tubular cells, ×500. (d) The same field under dark field, white arrowheads point to silver grains (3H‐thymidine labelling) and white arrows indicate 3H‐thymidine labelling of PHA‐L‐stained HLMC‐derived tubular cells (×500). (e) Bright field and (f) dark field are the higher magnification of yellow box area in (c). (g) The images in (e) and (f) were combined to help to show silver grains over cells that are donor GFP+ HLMC‐derived PHA‐L‐stained tubular cells. GFP, green fluorescent protein; HLMC, haematopoietic lineage marrow cell; PHA‐L, Phaseolus vulgaris leucoagglutinin.

Article Snippet: After GFP staining, tissue sections were microwaved in 10 m m tri‐sodium citrate (pH 6.0) for 10 min and biotin was blocked using a biotin blocking kit (DAKO), and then incubated for 45 min with biotinylated Phaseolus vulgaris leucoagglutinin (PHA‐L) 1/100 (B‐1115, Vector Laboratories, Burlingame, CA, USA) to detect proximal convoluted tubules, or biotinylated peanut agglutinin (PNA) 1/800 (B‐1075, Vector Laboratories) to detect distal convoluted tubules.

Techniques: Staining, Derivative Assay, Cell Counting