Journal: Cells

Article Title: A Heme-Binding Transcription Factor BACH1 Regulates Lactate Catabolism Suggesting a Combined Therapy for Triple-Negative Breast Cancer.

doi: 10.3390/cells11071177

Figure Lengend Snippet: Figure 2. BACH1-depleted TNBC cells are more sensitive to the MCT1 inhibitors for cell viability than control cells. (A) Diagram showing lactate catabolic pathways with small inhibitory molecules. AZD3965 and SR13800 inhibit MCT1, GSK2837808A inhibits LDHB, and UK5099 inhibits MPCs. (B,C) Colony formation of BM1-shBACH1 and control cells treated with SR13800 (50 µM) or AZD3965 (100 µM). After 48 h of drug treatment, cells were further incubated in DMEM (25 mM glucose, 2 mM glutamine) media for 10 days and stained using crystal violet. Quantified values of stained cells in the three replicates are shown. (D,E) Cell viability (%) of BM1-shBACH1 or re-expressed BACH1 in shBACH1 cells relative to the shControl cells that were treated with SR13800 (50 µM) or AZD3965 (100 µM) for 48 h and Calcein AM staining. Mean (n = 3/cells) ± s.e.m., p-values by two-tailed t-test. (F) Representative protein blots of BACH1 and beta-Actin as a loading control of cells used for (D,E).

Article Snippet: After 24 h of plating, inhibitors (as indicated) were added in the culture media (2 mM glutamine, 4 mM lactate), containing either low (1.25 mM) or high (25 mM) glucose, for 48–72 h. Viable cells were stained using 2 nM of Calcein AM dye (R&D systems, #5119) in 1x phosphate buffered saline (PBS) for 1 h at 37 ◦C to measure absorbance with excitation at 420 nm and emission at 520 nm using a Fisher Victor3 Spectrophotometer (Genomic Core, GWU).

Techniques: Control, Incubation, Staining, Two Tailed Test

Journal: Cells

Article Title: A Heme-Binding Transcription Factor BACH1 Regulates Lactate Catabolism Suggesting a Combined Therapy for Triple-Negative Breast Cancer.

doi: 10.3390/cells11071177

Figure Lengend Snippet: Figure 4. Combination treatment of hemin and the MCT1 inhibitors is effective to suppress TNBC growth. (A) Viability (%) of BM1-wt BACH1 or mut Bach1-expressing cells treated with hemin, SR13800 (50 µM), hemin+SR13800, or AZD3965 (100 µM), hemin+AZD3965. After incubation of cells for 48 h, viable cells were stained using Calcein AM; mean (n = 6/cells) ± s.e.m., p-values by two-tailed t-test. (B) Tumor volumes from athymic nude mice orthotopically injected with control and BM1-shBACH1 cells (shCont n = 4, shBACH1 n = 6, shCont+ AZD3965 n = 6, shBACH1+AZD3965 n = 3) and treated with AZD3965 (100 mg/kg body weight) by oral gavage for 17 days. (C) Tumor weights from athymic nude mice orthotopically injected with BM1 cells and treated with hemin (50 mg/kg body weight) daily by intraperitoneal injection and/or SR13800 for 10 days (vehicle n = 9, hemin n = 8, SR13800 n = 9, hemin + SR13800 n = 8). Representative tumor images are shown (below). (D) Schematic diagram indicating lactate catabolic pathways regulated by BACH1 in TNBC cells.

Article Snippet: After 24 h of plating, inhibitors (as indicated) were added in the culture media (2 mM glutamine, 4 mM lactate), containing either low (1.25 mM) or high (25 mM) glucose, for 48–72 h. Viable cells were stained using 2 nM of Calcein AM dye (R&D systems, #5119) in 1x phosphate buffered saline (PBS) for 1 h at 37 ◦C to measure absorbance with excitation at 420 nm and emission at 520 nm using a Fisher Victor3 Spectrophotometer (Genomic Core, GWU).

Techniques: Expressing, Incubation, Staining, Two Tailed Test, Injection, Control

Journal: Stem Cell Research & Therapy

Article Title: Interleukin-1β induces CXCR3-mediated chemotaxis to promote umbilical cord mesenchymal stem cell transendothelial migration

doi: 10.1186/s13287-018-1032-9

Figure Lengend Snippet: Effects of IL-1β in morphology and interaction between HUVECs and MSCs. Labeled MSCs with CellTracker™ Orange were seeded on HUVECs stained with Calcein AM and co-cultivated for 30 to 240 min. After a period of 60 min, MSCs attached to HUVECs and the morphology were still spherical but developed form of cytoplasmic offshoot. a After 60 min, MSC became flattened and adhered to HUVEC monolayer. IL-1β promoted adhesion (left, 60 min) and transendothelial migration abilities (right, 240 min) of MSCs. b After 180 and 240 min, MSCs extended long plasmic filopodia and integrated into the HUVEC monolayer. Orthogonal projections illustrate that MSCs inserted into HUVEC monolayer (left, 180 min) and formation of filopodia caused transendothelial migration (right, 240 min). Arrows indicate MSC migration through the HUVEC. Horizontal bar: XZ plane of confocal image stack; vertical bar: YZ plane of confocal image stack. Scale bar = 10 μm

Article Snippet: HUVEC monolayers were stained with 8 μM Calcein AM (Tocris, UK) in serum-free medium for 30 min at 37 °C, then gently washed three times with PBS.

Techniques: Labeling, Staining, Migration