Journal: Microorganisms

Article Title: Drebrin Is Involved in the Life Cycle of Pseudorabies Virus by Regulating the Actin Cytoskeleton

doi: 10.3390/microorganisms13091969

Figure Lengend Snippet: Effects of Drebrin on the different stages of PRV infection. ( A ) Schematic timeline for the time-of-addition assays. ( B ) BTP−2 (600 nM) was co-incubated with PRV-QXX (MOI = 10) at 4 °C for 2 h to simulate virus adsorption under low-temperature conditions in PK−15 cells. Unbound viral particles were removed, viral genomic DNA was extracted, and the copy numbers of the viral genome were evaluated by qRT-PCR. ( C ) PK−15 cells were firstly incubated with PRV-QXX (MOI = 10) at 4 °C for 1 h to allow virus adsorption. Subsequently, unbound virus was removed, and medium containing 600 nM BTP−2 was added. Cells were treated at 37 °C for 1 h, and viral genome copy number was evaluated by qRT-PCR. ( D ) PK−15 cells were incubated with PRV−QXX (MOI = 1) at 4 °C for 1 h, and the inoculum was then removed and replaced with maintenance medium containing 2% FBS/DMEM. Cells were incubated at 37 °C for 12 h to permit viral entry and early replication events. Subsequently, BTP2 was added, and cells were incubated at 37 °C for another 12 h. Then, the virus was collected by being frozen and thawed three times, and the TCID 50 assay was performed to evaluate the virus titer. ( E ) PK−15 cells were treated as in ( D ): the supernatant was collected and TCID 50 was performed to evaluate the extracellular viral yield. ( F ) The sgCtrl and sgDBN cells were incubated with PRV-QXX at 4 °C for 2 h, cells were rinsed 3 times with ice-cold PBS and incubated with maintenance solution at 37 °C for 30 min, and then the PRV genome copy number was determined by RT-qPCR. ( G ) Cells were treated as in ( D ): viruses were harvested with three freeze–thaw cycles, and then the viral titers were analyzed by TCID 50 assay. ( H , I ) The sgCtrl and sgDBN cells were incubated with PRV−QXX at 37 °C for 1 h, cells were rinsed three times with PBS, and the medium was replaced with 2% FBS/DMEM to maintain growth for 12 h and 24 h. Then, the cells and supernatant were collected to analyze the intracellular and extracellular viral yield by the TCID 50 assay, respectively. Significance levels relative to the corresponding control were denoted as non-significant * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: BTP−2 (HY-100831) and Puromycin (P8230) were obtained from MedChemExpress (South Brunswick Township, NJ, USA) and Solarbio (Beijing, China), respectively.

Techniques: Infection, Incubation, Virus, Adsorption, Quantitative RT-PCR, Control

Journal: APL Bioengineering

Article Title: Identification and regulation of EMT cells in vivo by laser stimulation

doi: 10.1063/5.0268350

Figure Lengend Snippet: EMT induced by photostimulation in vitro . (a) Schematic of the system setup for femtosecond laser stimulation of cells. The femtosecond laser (1030 nm, 220 fs, 1 MHz) is controlled by a shutter and galvanometer mirrors and focused by an objective (30 × , N.A. = 1.05) for scanning in a predefined arbitrary polygon region. (b)–(e) Representative images of Ca 2+ dynamics before and after photostimulation to cells (b); in the presence of 2-APB in Ca 2+ -free medium (c); in the presence of YM-58483 (d); and in the presence of 2-APB (e). Green fluorescence: Fluo-4/AM. Right: Kinetic plots of Ca 2+ levels following photostimulation at 10.5 mW [laser n = 43 cells, control n = 34 cells in (b); laser + 2-APB n = 42 cells, laser n = 61 in (c); laser + YM58483 n = 64 cells, laser n = 67 in (d); laser + 2-APB n = 58 cells, laser n = 53 in (e)]. (f) The fluorescence of H 2 DCFDA, the indicator of reactive oxygen species before and after laser stimulation (control and laser: n = 5 fields × 1 cell dishes per group). (g) The fluorescence of PI before and after the laser stimulation (control and laser: n = 5 fields × 1 cell dishes per group). (h) Experimental timeline. Cells were photostimulated on day 0, followed by immunofluorescence analysis on days 1, 2, 3, and 4. (i) Representative immunofluorescence images of Ki67 (green) in the cells on day 1. Blue: 4′,6-diamidino-2-phenylindole (DAPI). Right: Quantification of Ki67 levels (control and laser: n = 9 fields × 6 cell dishes per group). (j) Representative immunofluorescence images of CD90 (red) in the cells on day 1. Right: Quantification of CD90 levels (control and laser: n = 9 fields × 4 cell dishes per group). (k)–(m) Representative immunofluorescence images of E-cadherin (green), Vimentin (green), N-cadherin (red) in the cells on days 1, 2, 3, and 4, respectively. Blue: DAPI. Corresponding quantified expression levels are shown for E-cadherin and N-cadherin (n = 4 fields × 4 cell dishes per group for control and laser-treated cells) and for Vimentin (n = 4 fields × 3 cell dishes per group for control and laser-treated cells). Statistical significance was determined using a two-tailed t-test. Data represent mean ± SEM. * P < 0.05. ** P < 0.01. *** P < 0.001. **** P < 0.0001. Scale bar: 100 μ m.

Article Snippet: In , cells were treated with 2-APB (sigma, D9754) at 20 μ M and incubated at 37 °C for 1 h. Cells were treated with YM58483 (MCE, HY-100831) at 40 μ M and incubated at 37 °C for 1 h. Cells were stained with H2DCFDA (MCE, HY-D0940) at 5 μ M and incubated at 37 °C for 30 min.

Techniques: In Vitro, Fluorescence, Control, Immunofluorescence, Expressing, Two Tailed Test