Apotome slider module

All the antibodies are listed in the “Supplementary Materials and Methods” file.
Immunohistochemistry was performed on a glioma tumor array (US Biomax, Inc, Rockville, MD, USA), using a specific anti-SETD8 antibody.
To perform immunofluorescence, cells were spotted on cover glass and treated with DMSO or UNC0379 or with siRNAs for 48 h. After treatment, cells were processed as described [20 (link)]. For details, see also the “Supplementary Materials and Methods” file. DNA was stained by DAPI (Sigma-Aldrich). Samples were observed and photographed using an Axiovert 200 M inverted microscope equipped with the Apotome slider module with 40× and 63× objectives (Zeiss, Oberkochen, Germany).
Immunoblot analyses of glioblastoma cells were performed upon chemical and genetic inhibition of SETD8. After 48 h of treatment, cells were collected and lysed. Lysis buffer was 0.1% NP40-PBS, plus phosSTOP and Complete Protease inhibitor (Roche Life Science, Penzberg, Germany). Cell lysates were put on ice for 10 min and centrifuged twice at 14,000 × g for 10 min. Supernatants were collected and protein lysates were separated on SDS/PAGE and blotted. Western blots were repeated three times. Full and uncropped western blots are uploaded in the “Supplementary Figures” file.

FISH was performed to confirm the occurrence of prokaryotes on 13 nematodes (Table 2). In the field or quickly after sampling, some nematodes were fixed (see Nematode sorting section). Later, in the laboratory, they were hybridized with universal probes (Eurogentec, Liège, Belgium) (Table 3). Nematodes were rinsed in a 30% formamide buffer^{53 (link)} and incubated in a final volume of 30 µl hybridization buffer containing 30% formamide and 3 µl of each probe (8 µM) for 3.5 h at 46 °C. After that, the nematodes were rinsed in a washing buffer for 45 min at 48 °C. This step was ended by a final wash in milliQ water at room temperature for 10 min. After a quick drying period, the labelled organisms were mounted on a slide in an anti-fade mounting medium (SlowFade® Gold anti-fade reagent, Invitrogen) containing DAPI (4′6-diamidino-2-phenylindole, dilactate), a DNA intercalary agent. Observations were performed using the Imager.Z2 microscope (Zeiss, Oberkochen, Germany) equipped with an Apo-Tome slider module (Zeiss) and Colibri light technology (Zeiss) and using an AxioCam MRm (Zeiss) camera. Micrographs were analysed using the Zen (Zeiss) software.

Anti-phospho-serine Cdk Substrates (P-S2-100; recognizing K/HpSP), anti phosphorylated MAPK/CDK substrates (recognizing PXpSP or pSPXK/R) and anti phospho-ENSA/ARPP19 (pS67/pS62), anti phosphoT320-PP1cα and Cleaved Caspase-3 antibodies were purchased from Cell Signaling Technology (Danvers, MA); anti-MASTL antibodies from Bethyl Laboratories (Montgomery, TX) and NOVUS (Littleton, CO); anti-Fcp1 antibodies from Bethyl Laboratories and Santa Cruz Biotechnology (Dallas, TX). Other antibodies were from Santa Cruz Biotechnology. Immunoprecipitations and immunoblots were performed as previously described (Visconti et al., 2012 (link)). For immunofluorescence, cells were grown or spun on microscopy slides, washed in PBS, fixed with 4% formaldehyde in PBS for 10 min and permeabilized with 0.2% Triton X-100 in PBS for further 10 min. After blocking with 3% BSA in PBS for 1 hr, samples were incubated with primary antibodies in PBS + 1% BSA for 3 hr. After 3 PBS washes, samples were incubated with secondary antibodies (Jackson ImmunoResearch Laboratories Inc., Westgrove, PA) in PBS + 1% BSA for 1 hr at room temperature. DNA was stained by incubation with Hoechst 33258 (10 µg/ml; Santa Cruz Biotechnology) in PBS. Samples were observed and photographed using an Axiovert 200M inverted microscope equipped with the Apotome slider module with 63X or 40X objectives (Zeiss, Germany).