N sim

A 3D-SIM microscopy system (N-SIM, Nikon) was employed to carry out the 3D super-resolution imaging of U2OS cells. The wavelengths of 405 and 561 nm were used to excite DAPI and KR-TRF1 in the cell nucleus, respectively. An oil immersion objective (Nikon Apo TIRF 100x, NA = 1.49) was used for all 3D-SIM imaging. The lateral resolution of the 3D-SIM image is ∼100 nm and the axial resolution is ∼240 nm. The cells grown on a glass-bottomed petri dish were fixed with 3.7% (v/v) formaldehyde for 15 min at RT, followed by three washes with phosphate buffered saline (PBS) and then freshly made gelvatol was used as a mounting medium to cover the cells.

To obtain high-quality images of the bacterial flagella, super-resolution fluorescent imaging was performed using the Structured Illumination Microscopy (Nikon N-SIM). The microscope is equipped with an EMCCD camera (Andor iXon DU-897), a 100 × 1.49 NA TIRF objective (Nikon CFI Apo TIRF), a 488 nm excitation laser (Coherent Sapphire 488 LP), and a bandpass emission filter (500–550 nm, Chroma). Image acquisition and reconstruction were directed by the NIS-Elements Viewer (Nikon) software.

Spermatocyte spreads were prepared as we previously described^{65 (link),66 (link)}. In brief, the seminiferous tubules were treated with hypotonic extraction buffer for 30 min, and then germ cells were squeezed out in one drop of 100 mM sucrose solution and spread on slides with 1% PFA containing 0.15% Triton X-100. The slides were placed in a humidified chamber for at least 2 h and air-dried. Oocyte spreads were prepared from ovaries of 16.5–18.5 dpc female mice as previously reported^{9 (link),67} . Immunofluorescence staining was carried out as we previously described^{52 (link)}. The primary and secondary antibodies used and their dilutions are shown in Supplementary Table S3. Conventional fluorescence images were captured with an Olympus BX53 microscope (Olympus, Tokyo, Japan) with a scientific complementary metal-oxide-semiconductor camera (Prime BSI, Teledyne Photometrics Inc., USA) and processed with the Olympus cellSens software. Super-resolution images were captured using structured illumination microscopy (Nikon, N-SIM) equipped with a 100× oil-immersion objective lens (SR Apo TIRF 100×, NA 1.49) and a CCD camera (Andor, DU-897, X-11459), and images were processed using the NIS-Elements software.

Cells were fixed with 4% (vol/vol) formaldehyde and permeabilized with 0.5% Triton X-100. After blocking in 10% goat serum, HA-tagged E4-ORF3 from Ad3, 4, 5, 9, and 12, and endogenous TIF-1γ were detected with anti-HA and anti-TIF-1γ antibodies, respectively. Cell images were acquired on an Axiovert 200M digital deconvolution microscope (Zeiss) and analyzed using the AxioVision software. Subcellular localization of TIF-1γ wild-type and SUMO-deficient 4KR, E1B-55K, and endogenous Ubc9 was determined by structured illumination microscopy (N-SIM; Nikon), and images were reconstructed and analyzed using the NIS-Elements software (Nikon).

Cells overexpressing mtDsRed and TFAM-EYFP were seeded on coverslips and cultured for 24 h. Then, cells were fixed and mounted in slides and imaged by N-SIM (Nikon, Japan). The images were taken with a dual-color (laser 488 nm and laser 561 nm) SIM mode, using a 100 × oil (NA 1.49) objective with autofocus maintained by the Nikon Perfect Focus system. All images were reconstructed to maximum projections using NIS-Elements AR software (Nikon, Japan).