Two mouse ES cell lines were used during our work: USP1  and CGR8 . USP1 ES cells were maintained in an undifferentiated state on mouse embryonic fibroblast (MEF) feeder layer, mitotic inactivated with mitomycin C (Sigma-Aldrich), in DMEM/F12 with 15% (v/v) knockout serum replacement, 2 mM L-glutamine, 1% (v/v) nonessential amino acids, 0.1 mM β-mercaptoethanol (Invitrogen), 40 μg/mL gentamicin sulfate (Scheing-Plough) and 1000 U/mL of leukemia inhibitory factor (LIF, Invitrogen). CGR8 murine ES cells were cultured on 0,2% (w/v) gelatin (Sigma) coated tissue culture plates in Glasgow's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, 2 mM L-glutamine, 1% (v/v) nonessential amino acids, 0.1 mM β-mercaptoethanol, 1 mM sodium pyruvate, 50 U/mL penicillin, 50 mg/mL streptomycin (Invitrogen). 1000 U/mL of LIF were added prior to use. All cultures were incubated at 37°C in a humidified 5% CO2/95% air.
Formation and treatment of embryoid bodies (EBs)
A standard protocol for EB formation was used. Briefly, USP1 ES cells and MEF cell monolayers were dissociated with TrypLE™ Express (Invitrogen) and were plated on gelatin-treated dishes for 30 min to remove the feeder cells. This procedure was repeated twice. CGR8 ES cells were used directly. For EBs formation, ES cells (4 × 105/mL) were passed to a 6-well plate covered with 2% (w/v) gelatin (Sigma) and cultured for 48 h (d0-d2) in ES medium as described above. Colonies were then treated with TrypLE™ Express for 5 minutes at 37°C, transferred to non-adherent plate dishes (2.5 × 105/mL) with EB medium (DMEM/F12 medium with 15% (v/v) fetal bovine serum (Invitrogen), 2 mM glutamine, 1% (v/v) nonessential amino acids, 0.1 mM β-mercaptoethanol, 50 U/mL penicillin, 50 mg/mL streptomycin without LIF). ES cells were plated as hanging drops on a lid of 10-cm non-adherent Petri dish. The lid was inverted over the bottom of the dish filled with culture medium. Spherical cell aggregates (EBs) were formed during 4 days (d3-d6). The resulting EBs were transferred into Petri dishes (10-15 EBs per 6-cm dish) in 4 mL of the EB medium, and treated with all-trans-retinoic acid (RA, Sigma-Aldrich) during additional 4 days (from d7 to d10) to induce neural differentiation. At d7, EBs were divided in 2 groups: a) 0.02% (v/v) vehicle (DMSO) and b) 2 μM RA. The medium was changed every two days.
For studying the neuronal differentiation, EBs were plated onto 1 μg/mL laminin (Invitrogen) and 1 μg/mL fibronectin (Invitrogen) coated dishes at day 10 and cultured in DMEM/F12, with 1% (v/v) N2 supplement and 20 ng/mL of FGF-2 (Invitrogen) for additional 4 days.
Transduction of mouse ES cells by lentivirus
Mouse ES cells were cultured described above. The shRNA lentiviral particles transduction was performed according to the manufacturer's protocol (Santa Cruz). Synemin shRNA lentiviral particles (sc-60526-V, Santa Cruz) and control shRNA lentiviral particles (sc-108080, Santa Cruz) were used for this study. Synemin shRNA (m) Lentiviral Particles (sc-60526, Santa Cruz) is a pool of 3 different shRNA plasmids. Corresponding siRNA sequences are following:
A: Sense GCAAGACUAUGUUUGGAAAtt/Antisense UUUCCAAACAUAGUCUUGCtt
B: Sense GCAAGGCAUUGCCUAUGAAtt/Antisense UUCAUAGGCAAUGCCUUGCtt
C: Sense CAGUCACUCUGGAGUCAAAtt/Antisense UUUGACUCCAGAGUGACUGtt
The transduced mouse ES cell clumps were selected via Puromycin dihydrochloride selection (2 μg/ml), and then analyzed by qRT-PCR, Western blot and immunocytochemistry.
Different quantities (1 × 104, 2 × 104, 4 × 104 and 8 × 104 cells) of three selected synemin knockdown and control clones of mouse ES cells were cultured in 96-well flat-bottomed plates in a triplicate pattern. After eighteen or Forty-two hours, 20 μl 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/ml) was added to each well and incubated for 3.5 h at 37°C. Then media were carefully removed, 200 μl of DMSO was added to each well and the plate was agitated for 10 min at 37°C. Finally, Absorbance of the converted dye of each well is measured at a wavelength of 570 nm.
Total RNA from ES cells and mouse embryos was isolated with Trizol Reagent (Invitrogen) and then treated with RNase-free DNase (Qiagen). Aliquots (1 mg) of total RNA were reverse-transcribed into cDNA with first-strand DNA synthesis kit (Roche Diagnostic) following the manufacturer's instructions, and RT-PCR products were amplified as previously described . For quantitative RT-PCR, the PCR analysis was performed with SYBR green PCR technology (Light Cycler 480 system, Roche Diagnostics). Primers were selected with the Primer3 program http://biotools.umassmed.edu. Relative quantification was achieved with the following equation: R = 2ΔCttarget(control-sample)-ΔCtref(control-sample), Ct = cycle threshold http://www.gene-quantification.de. HPRT was used as the reference transcript. Results from 3 independent RT-PCR analyses were expressed as the ratio between mutant and control samples.
Primers for RT-PCR:
Synemin H or M: SynF (5'-AGTCAGGGAGCGTTTCTGTGGACG-3') and SynR (5'-ATCGCTTCTCGTGTCGCTCAAATCC-3');
Synemin L: SynLF (5'-AGAGTGATTGACAGCCTGGAGGAT-3') and SynLR (5'-ACTGTGTGCAATTCTCCAGCCACC-3');
Nestin: NesF (5'-AGGCTTCTCTTGGCTTTCCT-3') and NesR (5'-TGGATCATCAGGGAAGTGGT-3');
GFAP: gfapF (5'-TGGATTTGGAGAGAAAGGTTGAAT-3') and gfapR (5'-CGATAGTGGTTAGCTTCGTGTTTG-3').
Primers for qRT-PCR:
Synemin: F (5'-GGTGGCCTCAGATGAGAAGA-3') and R (5'-GGCTTGCATGTCGGTATTTT-3');
Keratin 8: K8F (5'-TCATCCTATGGGGGACTCAC-3') and K8R (5'-TCTTCACAACCACAGCCTTG-3');
Keratin 18: K18F (5'-CTTGCTGGAGGATGGAGAAG-3') and K18R (5'-GCCTCAGTGCCTCAGAACTC-3')
Oct4: Oct4F (5'-GGGGCTGTATCCTTTCCTCT-3') and Oct4R (5'-GCTGGTGCCTCAGTTTGAAT-3');
Nanog: NanogF (5'-GGACTTTCTGCAGCCTTACG-3') and NanogR (5'-TTTCACCTGGTGGAGTCACA-3');
SOX2: Sox2F (5'-TACCTCTTCCTCCCACTCCA-3') and Sox2R (5'-CTGGGCCATGTGCAGTCTAC-3').
Production of the antibody against synemin H
A cDNA fragment encoding the exon 4a (formerly named intron IV) of human synemin , a specific fragment for synemin H (amino acid residues 1151-1462) was cloned into a pQE32 plasmid (Qiagen). The antibody anti-synemin H was produced as described previously . The resulting rabbit antiserum was characterized by Western blotting analysis. This antibody recognizes both human and mouse synemin H isoforms.
ES cells were fixed with cold methanol during 5 minutes, washed with PBS, and then with PBS-Triton X-100 (0.1% v/v). Nonspecific sites were blocked with PBS + 5% (w/v) bovine serum albumin (BSA) during 45 minutes and incubated with primary antibodies for 90 minutes at room temperature (RT). The primary antibodies were polyclonal anti-synemins H/M (1:400), anti-synemin H (1:400) and anti-synemin L (1:100) produced in our laboratory , mouse monoclonal antibodies anti-Oct-3/4 (1:100, Santa Cruz), anti-stage-specific embryonic antigen-1 (SSEA-1, 1:100, Chemicon), anti-glial fibrillary acidic protein (GFAP, 1:1000, Sigma), anti-nestin (1:200, Chemicon), anti-β-III tubulin (1:500, Sigma) or rat anti-keratin 8 (TROMA 1, 1:100) . The secondary antibodies used were goat anti-mouse, anti-rat or anti-rabbit immunoglobulins, coupled to the fluorochromes Alex 488 (1:400, Invitrogen) or Cy3 (1:500, Jackson) for 40 minutes at RT. Controls were performed without the primary antibody.
One- and two-dimensional PAGE and Western blotting
Protein separation by 1D- or 2D-PAGE was carried out as described previously [49, 50]. For 2D-PAGE, total protein extract of ES cells was separated by using pH 4-6 gradients IPG strips and 8% SDS/PAGE gels. The proteins were transferred onto nitrocellulose Hybond-C+ or PVDF (GE Healthcare) membranes and nonspecific protein-binding sites were blocked with TBS-T (20 mM Tris-HCl, pH 7.5, 136.8 mM NaCl and 0.1% (v/v) Tween 20) containing 5% (w/v) non-fat milk. The blots were reacted with primary antibodies against synemins H/M (1:3000), synemin L (1:1000), nestin (1:1000), GFAP (1:5000), β-III tubulin (1:3000), Oct-3/4 (1:500), GAPDH (1:5000, Sigma) or α-tubulin (1:5000, Dako) in TBS-T containing 2% (w/v) non-fat milk overnight at 4°C followed by corresponding secondary antibodies anti-rabbit or mouse IgG HRP-conjugated (1:5000; GE Healthcare). The specific binding was revealed by an enhanced chemiluminescence (ECL) detection system (GE Healthcare). For synemin phosphorylation study, the anti-phosphoserine CDKs substrate antibody (1:1000, Cell Signaling) in TBS-T containing 5% (w/v) BSA were used. This antibody detects phosphosérine in a (K/R)(S*)PX(K/R) cyclin-dependent kinase (CDK) motif.