Fv1000 confocal microscopy system

Cells were transfected with KR-TRF1 17 hr after passaging and 24 hr prior to imaging with the Olympus FV/1000 confocal microscopy system (Olympus) and FV/1000 software. Half-cells were selected and irradiated with a 100% power 559 laser (20 scan, 5 mW/scan) to activate the KR-TRF1 marked telomeres in the half-cell, or in the whole nucleus to induce damage. Then, telomere movement was tracked in the cell within the 1:8 zoom in 256 × 256 vision with a 2 μs/pixel scan speed and ZDC on. The time interval of the image acquisition with the above confocal setting is 0.429 s, and the total numbers of frames are 120. All of the experiments follow the same confocal setting as described. Cells were incubated at 37 °C on a thermos plate in normal media during imaging. The movement trend is exactly the same in the whole cell with/without activation of KR-TRF1 as in the half cell. System drift was analyzed using fixed KR-TRF1 or a four-color fluorescent bead (0.1 μm diameter, blue/green/orange/dark red fluorescence, Cat: T7279, Thermo Fisher Scientific).

HTC75 cells stably expressing GFP-actin cultured in DMEM with 10% FBS were transfected with siRNA for control, CAPN10 or MAP1B using the RNAiMAX transfection reagent (Invitrogen) and cultured for 48–72 hr before analysis. For FRAP (fluorescence recovery after photobleaching), siRNA-treated cells were incubated in HEPES-buffered DMEM and placed on a microscope stage that had been pre-warmed at 37 °C. The cells were observed and bleached using a FV1000 confocal microscopy system (Olympus). To visualize FRAP of GFP-actin, images were acquired sequentially every 2 sec. All values are indicated as mean ± standard deviation (SD). The unpaired student-t test was used for statistical analysis. Statistical analysis was performed with JMP® version 11 software (SAS Institute, Inc., Cary, NC).

Confocal images were acquired in the photon counting mode of a FV1000 confocal microscopy system with FV10-ASW software (Olympus). To calculate the population of OPG-expressing cells, we selected the region of M cells by setting a threshold of signal intensities for OPG and/or Spi-B and then calculated mean fluorescence intensities within these regions by using ImageJ software. Obtained values were plotted by Microsoft Excel software or saved as converted from a comma-separated values (csv) format to a flow cytometry standard (fcs) format using a CsvToFcs module of GenePattern software [https://gp.indiana.edu/gp/pages/index.jsf]. The fcs files were opened and analyzed by FlowJo software (TreeStar Inc., Ashland, OR, USA).

The Olympus FV1000 confocal microscopy system (Cat. F10PRDMYR-1, Olympus) and FV1000 software were used for the acquisition of images. Cells were cultured in 35 mm glass-bottom dishes (MatTek, P35GC-1.5-14-C) before observation. The damage was induced with a 405 nm laser. The laser passed through a PLAON 60X oil lens. Cells transfected with GFP-tagged proteins were incubated at 37 °C on a thermos plate in normal media during observation. For the evaluation of accumulation and kinetics, the mean intensity of each accumulated point or line was obtained after subtraction and quantified by ImageJ.

The Olympus FV1000 confocal microscopy system (Cat#: F10PRDMYR-1, Olympus; Waltham, MA, USA) and FV1000 software were used for acquisition of images. Cells were cultured in 35-mm glass-bottom dishes (P35GC-1.5-14-C, MatTek; Ashland, MA, USA) before observation. Activation of KR in bulky cells was completed by exposing them to a 15-W Sylvania (Wilmington, MA, USA) cool white fluorescent bulb for 25 min in a UVP stage. The intensity was measured by ImageJ 1.50i software. P values were calculated by the Student’s t test.

Mock or rgRSV-infected cells were fixed in acetone and methanol at the ratio of 1:1 for 30 min, and blocked with 10% goat serum. Slides were stained with all primary antibodies (1:100), washed three times with PBST, and stained with conjugated Alexa Fluor secondary antibodies Alexa Fluor 488/594 (1:300, Thermo-Fisher), and mounted with SlowFade™ Diamond Antifade Mountant with DAPI (Thermo-Fisher). Imaging was performed on an Olympus FV 1000 confocal microscopy system at The Ohio State University Campus Microscopy & Imaging Facility.

Cells were transfected with the WBP2 localization mutant plasmids, and seeded on μ-Slide 8 Well (ibidi) and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) diluted in PBS. Samples were then imaged using the FV1000 confocal microscopy system (Olympus). Image analysis was performed using ImageJ. The average pixel intensity in the nucleus and cytoplasm were recorded. The localization of WBP2 (L) was calculated as followed: $\mathit{L}=\mathit{Lo}{\mathit{g}}_2\left(\frac{{\overline{\mathit{P}}}_{\mathit{N}}}{{\overline{\mathit{P}}}_{\mathit{C}}}\right),$ where ${\overline{\mathit{P}}}_{\mathit{N}}$ is the average pixel intensity in the nucleus, and ${\overline{\mathit{P}}}_{\mathit{C}}\mathit{ }$ is the average pixel intensity in the cytoplasm. As $\mathit{L}$ is in a logarithmic scale, thus $\mathit{L}=0$ when the ${\overline{\mathit{P}}}_{\mathit{N}}={\overline{\mathit{P}}}_{\mathit{C}}$ , while $\mathit{L}>0$ and $\mathit{L}<0$ corresponds to ${\overline{\mathit{P}}}_{\mathit{N}}>{\overline{\mathit{P}}}_{\mathit{C}}$ and ${\overline{\mathit{P}}}_{\mathit{N}}<{\overline{\mathit{P}}}_{\mathit{C}}$ , respectively.