Sp8 dls

Cells were fixed on coverslips using growth medium containing 3.7% PFA for 15 min at room temperature. After PBS washes, cells were permeabilized using 0.1% Triton X-100 in PBS for 5 min and washed with PBS again. Coverslips were incubated with primary antibodies (Supplementary Table 3) for 1 h and washed three times with PBS before incubation with secondary antibodies (AlexaFluor (Invitrogen)) for 45 min in the dark. Upon incubation, cells were washed with PBS, stained with 4,6-diamidino-2-phenylindole, washed again with PBS and mounted using ProLong Gold (Invitrogen). Images were acquired using a Leica SP8-DLS laser-scanning confocal microscope equipped with an ×100 oil HC PL APO CS2 objective (numerical aperture (NA) 1.4) or Leica DMI 6000 B wide-field fluorescence microscope equipped with an ×100 oil HCX PL APO CS 23°–37 °C objective (NA 1.46).

HeLa cells transfected with indicated constructs were plated on 13 mm glass coverslips in 24 well dishes. Cells were washed with room temperature PBS and fixed with 3.7% PFA in PBS at room temperature for 20 min. Cells were permeabilized in PBS containing 0.2% Triton X‐100. Samples were blocked in PBS containing 0.1% BSA for 30 min before incubating overnight with primary IF‐antibody reconstituted in blocking solution with dilutions indicated above. The secondary IF‐antibody (Invitrogen, Alexa Flour) was reconstituted in blocking buffer and incubated for 1 h, before washing the samples and mounting the coverslips onto microscopy slides with Prolong Gold Antifade Mountant (Invitrogen, Cat# P10144). Image acquisition was performed with a Leica SP8‐DLS laser‐scanning confocal microscope using a 100× oil objective (Leica HC PL APO 100×/1.4 OIL CS2; Cat# 11506372).

U2OS cells (WT, HPF1KO, PARGKO) were cultured on glass coverslips, treated as indicated, and fixed with ice-cold methanol for 20 min at −20 °C. The cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min, then blocked with 3% normal goat serum (Invitrogen) for 5 min. The coverslips were incubated with primary antibody (AbD43647 IgG, 1:500 dilution) for 1 h at room temperature, followed by 1 h incubation at room temperature with Alexa Fluor anti-mouse secondary antibodies (Anti-Mouse Alexa-Fluor 594-conjugated goat secondary Invitrogen Cat# A11005) at 1:500 dilution and with DAPI at 1:1000 dilution, then mounted with Prolong Diamond Antifade (ThermoScientific). Cells were imaged using a Leica SP8-DLS inverted laser-scanning confocal microscope using 63X objective. Image analysis and quantification was performed using the Fiji Software. Nuclei were identified based on DAPI, and used as a mask to measure the pixel intensity of other image channels.

Cells were fixed using 4% PFA in 0.1 m PBS for 2 h, after which they were washed three times with PBS. The cells were permeabilized by incubation overnight in 2% Triton X-100 in PBS (PBST) at room temperature. The cells were then kept in the refrigerator overnight in blocking buffer consisting of 1% Triton X-100 and 10% normal goat serum (catalog #G9023, Sigma-Aldrich). Primary antibody incubation was performed for 2 d in 4°C with antibodies diluted at the fractions indicated in the subsection Chemicals and culture components, in 1% normal goat serum and 0.2% Triton X-100. Next, cells were washed three times with 0.1% Triton X-100 in PBS and left overnight at 4°C. Secondary antibodies in 1% normal goat serum and 0.2% Triton X-100 were applied and left at 4°C for 1 d. Samples were once again washed three times with blocking buffer and left at 4°C overnight. Samples were stained with DAPI (1.5 μg/ml) for an hour and then cleared with RapiClear 1.49 (SUNJin Lab) by incubation at least overnight at 4°C. Samples were mounted on slides and were sealed with nail polish and kept at 4°C until further use. Confocal images were taken using a microscope (model SP8 DLS, Leica) and captured with a 20× oil-immersion objective.

Jejunal sections were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned. Slides were deparaffinized and antigen retrieval was performed by boiling with pH 6 citrate buffer. Primary antibodies used were: p62/SQSTM (Abcam, 56416; 1:100 dilution), pH3 (CST, 4370; 1:100 dilution), lysozyme (Thermo Fisher Scientific, PA5-16668; 1:300 dilution) and Man2B1 (St John’s Laboratory, 640-850; 1:100 dilution). Primary antibodies were detected using Alexa Flour 488-, Alexa Flour 594- and Alexa Flour 633-conjugated anti-rabbit or anti-mouse secondary antibodies (Thermo Fisher Scientific; 1:500 dilution). Sections were mounted in DAPI-containing mounting medium (Vectashield H1200) and imaged using a confocal Leica SP8-DLS or SP8-X microscope, with Leica Application Suite X software (v3.x, Leica Microsystems).

Bright-field images were acquired using the Evos FL Auto 2 microscope. Quantification of the AlizarinrRed S staining was done in ImageJ software; measurement of intensity was done after making RGB stacks for each image and applying the same thresholds to all samples from each experiment. Immunofluorescent images were acquired using confocal SP8-X and SP8-DLS microscopes (Leica). All immunofluorescent images were processed identically in ImageJ; in particular, images are shown after background subtraction (rolling ball radius:50) and noise despeckle.

Prepupae treated with dsmalE or dsTcLRPPRC were collected and the first and second abdominal segments were cut using the micro scissors. The fat body and alimentary canal were removed using forceps, and the integument was cut along sides to separate ventral and dorsal parts. Then the integuments were incubated in Ex-cell 420 medium (Sigma-Aldrich, St. Louis, MO) containing MitoView™ 650 Mitochondria staining Dye (Biotium, Fremont, CA) for 15 min. The tissues were then mounted on a slide and examined under Leica SP8 DLS using 638 nm excitation filter.