Sp8 falcon

In order to understand the subcellular distributions of cbx2, ISH and FISH were performed on gonadal sections and verified by hematoxylin and eosin staining. ISH was performed as previously described [24 (link)]. Briefly, the 552 nt of cbx2 cDNA and 794 nt of vasa cDNA were inserted into a pGEM-T vector, respectively. The recombinant plasmids were linearized with spel or sphl to transcribe DIG-labeled or FITC-labeled antisense or sense probes according to RNA Labeling kit instructions (Roche, Basel, Switzerland). The synthetic probe was incubated with DNaseI (Thermo Fisher, Carlsbad, CA, USA) and precipitated by LiCl at –80 °C. The ISH experiments of cbx2 and vasa as the marker gene were performed and stained with BCIP/NBT. FISH was carried out with a TSA™ Plus Fluorescence Systems kit according to the instructions (NEL756, PerkinElmer, Waltham, MA, USA). The nucleus was dyed with DAPI and the slide was fixed with Anti-Fade Mounting Medium (Invitrogen, Carlsbad, CA, USA).
Photography and observation of ISH were performed using an upright microscope (Leica DM500, Heerbrugg, Switzerland) and FISH images were observed and photographed using a Leica SP8 FALCON with the confocal image system (LAS X).

Samples were imaged using a Nikon Eclipse E800 or Leica DM6000B fluorescence microscope and Zeiss LSM 710 or LSM 510 and Leica SP8 Falcon confocal microscope. Figures were assembled in Adobe Photoshop CS4. Images presented in the figures are raw data images processed for orientation and cropped to size and pixel density with the following exception: In Figure 2 and Supplementary Figures 9, 11, the image brightness was increased to better visualize the data.

Two assays were used to evaluate the effects on cells’ mitochondrial membrane potential. Mitochondrial membrane potential (MMP) was analyzed using JC-1 (T4069, Sigma, MO, United States) in both flow cytometric and cell fluorescence assays. All cells were washed with ice-cold 1 × PBS and stained with 5 mg/mL JC-1 for 30 min in the dark. Then, each cell suspension was quantified by flow cytometry (BD FACS Accuri C6; BD Biosciences, MO, United States). Fluorescence images were acquired using a confocal microscope (Leica, SP8 FALCON, Germany).

The cells that climbed onto the carry sheet glass (20 mm) were cultured in an incubator. At the start of the experiment, the cells were washed three times with 1 × PBS. Then, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and then washed again with 1 × PBS. After fixation, cells were incubated in a 0.5% Triton X-100 solution for 5 min, and then washed again with 1 × PBS. The cytoskeleton was stained with TRITC-conjugated phalloidin (CA1610, Solarbio, Beijing, China) for 30 min, away from any light, at room temperature. Next, the cells were washed three times with 1 × PBS. Finally, DAPI (100 nM in PBS, Invitrogen, CA, United States) was used to counterstain the nuclei at room temperature for 40 s. Their fluorescence images were captured under a confocal microscope (Leica, SP8 FALCON, Germany).

NADH FLIM was performed on fibroblasts grown in glass-bottomed dishes (NEST, Cat# 801001) in fibroblast medium. Fibroblasts were imaged using a Leica SP8 FALCON with a DIVE laser scanning microscope. An oil-immersion objective of ×63/1.4NA was used for image acquisition. The NADH FLIM signal was collected using 750 nm 2-photon excitation (Spectra-Physics InSight X3 tunable laser, 0.8 mW average power) and 408–479 nm emission. Fluorescence was monitored using a multi-detector approach (Fast FLIM). Five images were obtained for each sample. Five to six fibroblasts with clear boundaries and the correct relative positions were selected from each image. The region of interest of each fibroblast was manually extracted to calculate free and protein-bound NADH. Proper fitting of the lifetime curve was evaluated using χ², and the mean lifetime (τmean) was calculated.