C2C12 myoblasts were obtained originally from H. Blau, Stanford University and a sub-clone A2 was derived in-house (Sachidanandan et al, 2002) and used for all experiments. Myoblasts were maintained in growth medium (GM; DMEM + 20% Fetal Bovine Serum (FBS) and antibiotics), ensuring that cells do not exceed 70-80% confluence before passaging. Differentiation was induced in low mitogen medium (DM: DMEM + 2% horse serum), for 5 days to form mature myotubes (MT). Synchronization in quiescence (G0) was induced by suspension culture of myoblasts for 48 hours in 1.3% methylcellulose medium prepared with DMEM containing 20% FBS, 10 mM HEPES, and antibiotics, as described (20). G0 cells were reactivated into the cell cycle by re-plating at a sub-confluent density in GM and harvested at defined times (30 minutes - 24hr) after activation.
Immunofluorescence analysis and microscopy
Cells that were either plated on coverslips, or harvested from suspension cultures were simultaneously fixed and permeabilized in 2% PFA, 0.2% Triton X-100 in modified cytoskeletal buffer (CSK) (52) at 4°C for 15 min, to ensure detection of stable microtubules of the ciliary axoneme and depolymerization of dynamic cytoplasmic microtubules. Cells were incubated in blocking buffer (either 10% FCS, 0.2% Triton X-100 in CSK buffer or 5% BSA, 0.2% Triton X-100 in CSK buffer) for 1 hour to reduce non-specific labeling. Primary antibodies were diluted in blocking buffer. Specific antibody labeling was detected using fluorescence tagged secondary antibodies from Invitrogen. Details of primary antibodies used are as follows: Acetylated tubulin (SIGMA, T7451, 1:5000), Gamma tubulin (SIGMA, T6557, 1:1000), Acetylated tubulin (Abcam, ab125356, 1:1000), Ki67 (Abcam, ab1667, 1:200), Myogenin (Santa Cruz, sc-576, 1:100), Pax7 (AVIVA, ARP32742_P050, 1:1000), Pax7 (DSHB, 1:20), Pericentrin (Santa Cruz, sc-28147, 1:100). All washes were done in CSK buffer at RT. For suspension culture samples, immunostaining was performed as described earlier (13). Samples were mounted in aqueous mounting agents with DAPI. Images were obtained using either the Leica TCS.SP5-II AOBS, Leica TCS SP8 or Zeiss LSM 700 confocal microscopes. Minimum global changes in brightness or contrast were made, and composites were assembled using Fiji (ImageJ).
Single muscle fiber analysis
Animal work was conducted in the NCBS/inStem Animal Care and Resource Center. All procedures were approved by the inStem Institutional Animal Ethics Committees following norms specified by the Committee for the Purpose of Control and Supervision of Experiments on Animals, Govt. of India.
Single skeletal muscle fibers were isolated using a technique modified from (53,54). Briefly, the Extensor Digitorum Longus (EDL) muscle of 6-week old male C57 BL/7 mice was dissected out and treated with Collagenase Type 1 (Cat# LS4196 Worthington 400U/ml final concentration) for 1 hour at 37°C. The resultant dissociated muscle fibers were transferred into fresh DMEM and triturated to release individual muscle fibers, which were then washed through transfer to fresh medium, and then fixed with 4% paraformaldehyde for 15 min at RT. For immunostaining, single fibers were washed with PBS twice and mounted on charged slides (Cat#12-550-15, Fisher Scientific). The fibers were permeabilized with 0.5% Tween 20 in PBS for 1 hour at RT followed by blocking with 2 mg/ml BSA in PBS, for 1 hour at RT. Primary antibody incubations were performed overnight at 4°C. Secondary antibody incubations were for 1 hour at RT using fluorescently tagged antibodies from Invitrogen. All antibody dilutions were made in a solution of 1 mg/ml BSA in 0.25% Tween 20 in PBS. Antibody incubations were followed by three washes with blocking solution. 4', 6-Diamidino-2-Phenylindole (DAPI) (Cat# 32670 Sigma) was used to stain the DNA. Images were acquired using Zeiss LSM 510 Meta confocal microscope.
Western blot analysis
Lysates of adherent cultures or suspension cells were obtained after harvesting PBS washed cells by centrifugation, and resuspended in Laemmli sample buffer (2% SDS, 5% β-mercaptoethanol, 50 mM Tris-Cl pH 6.8) supplemented with Protease Inhibitor Cocktail and PhosStop (Roche) for isolation of total cellular protein. Protein amount in lysates was estimated using Amido Black staining and quantification of absorbance at 630 nm. Proteins were resolved on 8-12% Acrylamide gels, and transferred to PVDF membranes. The membrane was washed in 1X TBS with 0.1% Tween20 (TBST) and blocked in 5% blocking reagent (5% w/v nonfat milk in TBST) for 1 hour at room temperature, followed by incubation with primary antibody overnight at 4°C, then washed in 1X TBS + 0.1% Tween for 10 mins each followed by incubation with secondary antibody conjugated with HRP (Horse radish peroxidase) for 1 hour. After a brief wash with TBS-T for 10 minutes, ECL western blotting detection reagent for HRP was used for chemiluminescent detection using the ChemiCapt (Vilber Lourmat) gel documentation system. Details of antibodies used in this study are as follows: Cyclin A2 (Abcam, ab7956, 1:500), Cyclin B1 (Abcam, ab52187, 1:500), Cyclin D1 (Abcam, ab40754, 1:200), Cyclin E1 (Abcam, ab3927, 1:200), IFT88 (Proteintech, 13967-1-AP, 1:1000), p130 (Santa Cruz, SC-317, 1:200), p27 (BD, 610242, 1:2000), p21 (BD, 556430, 1:2000), MyoD (DAKO, M3512, 1:500), GAPDH (Abcam, ab9484, 1:2000), Myogenin (Santa Cruz, SC-12732, 1:1000), Active-Beta Catenin (Millipore, 05-665, 1:2000), Total-Beta Catenin (BD, 610153, 1:2000), PDGFR-alpha (Santa Cruz, SC-338, 1:500), Hes1 (Abcam, ab71559, 1:500), Phospho - mTOR (CST, 2971, 1:1000), Total - mTOR (CST, 2972, 1:2000), Phospho - rpS6 (CST, 4858, 1:1000), Total - rpS6 (CST, 2217, 1:2000), Phospho-4E-BP1 (Thr37/46) (CST, 2855, 1:1000), Total 4E-BP1 (53H11) (CST, 9644, 1:1000), Phospho-IGF-I Receptor β (Tyr1135) (CST, 3918p, 1:1000), IGF-I Receptor β (CST, 9750p, 1:1000), Arl13b (Proteintech, , 17711-1-AP, 1:1000).
Cell cycle analysis
Adherent cells were trypsinized, washed in PBS and pelleted by centrifugation. Suspension-arrested cells were recovered from methylcellulose by dilution with PBS followed by centrifugation as described earlier. Cell pellets were dispersed in 0.75 ml of PBS, and fixed by drop wise addition into 80% ice-cold ethanol with gentle stirring, following which they were briefly washed with PBS and resuspended in PBS with 40 μM of the DNA dye DRAQ5TM (Cat. No DR50050, Biostatus) per 106cells. Cell cycle analysis was performed on a FACS Caliber® Cytometer (Becton Dickenson) using CelQuest® software and analyzed using FlowJo® software. At least 10,000 cells were acquired for each sample. Forward scatter and side scatter were used to gate cell populations and doublets were removed from analysis.
Analysis of p27 levels
A stable C2C12 line expressing a p27 sensor (p27-mVenus, a fusion protein consisting of mVenus and a defective mutant of p27-CDKI, (p27K(-)) (55) was generated, transfected with either control or IFT88 siRNAs, and placed in suspension arrest. After recovery from methyl cellulose medium, these cells were assayed for proportion of mVenus positive cells by flow cytometry using a FACS Caliber cytometer (Becton Dickenson). CelQuest® software was used for acquisition and FlowJo® software was used to analyze the data.
EdU incorporation Assay
Cells to be analyzed for DNA synthesis were pulsed with 10 μM EdU for 30 minutes, washed in PBS and fixed as described earlier. Samples were stored in PBS at 4°C till further processing. For staining, cells were permeabilized and blocked in PBS having 10% FBS and 0.5% TX-100 and labeling was detected using Click-iT® imaging kit (Invitrogen) as per manufacturer’s instructions. Samples were mounted in aqueous mounting agents with DAPI. Images were obtained using the Leica TCS SP8 confocal microscope. Minimum global changes in brightness or contrast were made, and composites were assembled using Fiji (ImageJ).
OPP incorporation Assay
Cells to be analyzed for protein synthesis were pulsed with 20μM OPP for 30 minutes, washed in PBS and fixed as described earlier. Samples were stored in PBS at 4°C till further processing. For staining, cells were permeabilized and blocked in PBS having 10% FBS and 0.5% TX-100 and labeling was detected using Click-iT® imaging kit (Invitrogen) as per manufacturer’s instructions. Samples were mounted in aqueous mounting agents with DAPI. Images were obtained using the Leica TCS SP8 confocal microscope. Image intensity was calculated using Fiji (ImageJ) software, and corrected mean intensity (CMI = total intensity of signal – (area of signal × mean background signal)) was determined.
Knockdown of gene expression using siRNA
C2C12 cells were cultured in growth medium until 80% confluent. The cells were then trypsinized and plated on to tissue culture dishes at appropriate cell density according to size of the dish. Approximately 16 hours post plating, the cells were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen) as per manufacturer's instructions. The cells were incubated with the RNA-Lipid complex for at least 18-24 hours following which they were used for further experimental analysis. Both single siRNA as well as SMARTpools of 4 siRNAs were used for knockdown experiments. At least one replicate of all experiments, barring the microarray assay, was performed using SMARTpools. Typically, siRNA-mediated knockdown was in the range of 50 - 90% at RNA level. Details of siRNA used in this study are as follows. For IFT88 knockdown: 5’-GCUGUGAACUCGGAUAGAU-3’ (Eurogentec) or siGENOME Mouse Ift88 (21821) siRNA-SMARTpool (Dharmacon, M-050417-00-0010); For Control: Scrambled siRNA (Eurogentec SR-NP001-001) or siGENOME Non- Targeting Pool #1 (Dharmacon, D-00126-13-20)
Isolation of RNA from cultured cells and Quantitative real-time RT-PCR
RNA was isolated from cells using Trizol® (Invitrogen) according to manufacturer’s instructions, dissolved in nuclease-free water, quality checked by agarose gel electrophoresis and quantitated by spectrophotometry using the NanoDrop ND-1000UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA was prepared from 1 μg total RNA using Superscript III (Invitrogen) and used in SYBR-Green (Applied Biosystems) based quantitative real time PCR analysis performed on an ABI 7900HT thermal cycler (Applied Biosystems) normalized to GAPDH levels. Fold change was calculated using normalized cycle threshold value differences2−ΔΔct. Primer sequences used in this study are as follows: GAPDH: forward 5’-AATGTGTCCGTCGTGGATCTGA -3’, reverse, 5’-GATGCCTGCTTCACCACCTTCT -3’; IFT88: forward, 5’-ATGTGGAGCTGGCCAACGACCT-3’, reverse, 5’-TGGTCGCAGCTGCACTCTTCACT-3’; FEZ1: forward, 5’-TGTACTTCGGTGCCAGGATG -3¢, reverse, 5’-GAGAGGGAAGGGTCCTCCAG-3’; PDGFRα: forward, 5’-GGTGGCCTGGACGAACAGAG-3’, reverse, 5’-GGAACCTGTCTCGATGGCACTC-3’; YPEL3: forward, 5’-TGCGGGCCAGCAGAAGAGCG-3’, reverse, 5’-GGAGCTAGGTCAGTCCCAGCCGT-3’; YPEL5: forward, 5’-GGCGCCACTGGTAGAGCATT-3’, reverse, 5’-CAGGATCACACGGCCTTCCT-3’; AURKA: forward, 5’-CGGTGCATGCTCCATCTTCC-3’, reverse, 5’-CTTCTCGTCATGCATCCGGC-3’; Cdc20: forward, 5’-CCGGCACATTCGCATTTGGA-3’, reverse, 5’-GTTCTGGGCAAAGCCGTGAC-3’; CenpF: forward, 5’-CAGCTGGTGGCAGCAGATCA-3’, reverse, 5’-GCTGGGAGTTCTTGGAAGGC-3’; Bub1: forward, 5’-TGCTCAGTAACAAGCCATGGAAC-3’, reverse, 5’-CCTTCAGGTTTCCAGACTCCTCC-3’; FoxM1: forward, 5’-ACTTTAAGCACATTGCCAAGCCA-3’, reverse, 5’-TGGCACTTGGGTGAATGGTCC-3’; Ki67: forward, 5’-TGGAAGAGCAGGTTAGCACTGT-3’, reverse, 5’-CAAACTTGGGCCTTGGCTGT-3’, Mcm3: forward, 5’-CCAGGACTCCCAGAAAGTGGA-3’, reverse, 5’-TGGAACACTTCTAAGAGGGCCG-3’; Mcm5: forward, 5’-TCAAGCGCCGTTTTGCCATT-3’, reverse, 5’-CTCACCCCTGCGTAGCATGA-3’; Plk4: forward,5’-GAAGGACTTGGCCACACAGC -3’, reverse, 5’-GAACCCACACAGCTCCGCTA -3’.
Global transcription profiling using microarray
1 mg of total RNA isolated from Control or IFT88 KD cells was converted to cDNA using One-cycle labeling kit and amplified using IVT labeling kit following manufacturer's instructions (Affymetrix). The normalized cRNA was fragmented, hybridized to mouse Affymetrix Gene-chips (430A 2.0), washed, stained and scanned as per Affymetrix protocols. The experiment was repeated with three different biological replicates and data analyzed using Affymetrix Gene Chip operating software (GCOS). All the CEL (cell intensity) files generated by Expression Console were then loaded into the R Bioconductor "affy" package for microarray analysis (56). Briefly, the CEL files were normalized using Loess Normalization before proceeding to further analysis of differential expression between Control and IFT88KD samples for each specific cell state. Genes showing >1.5-fold differential expression with P ≤ 0.05 were selected and a subset validated by real time Q-RT-PCR. The raw data is available on the GEO database (Series GSE110742).
Analysis of microarray data
STRING analysis: The lists of genes that were either up-regulated or down-regulated in IFT88KD myoblasts under G0 conditions were used as input to search for networks in the STRING database using default settings with only connected nodes being represented in the network diagram.
GSEA: Normalized expression values for Control (SCR) and IFT88KD were used as input dataset and the gene sets used were those from the C5 collection and those related to signaling, which are available on the Molecular Signatures Database (MSigDB), as well as a gene set identified from earlier published datasets (pSig) (36). The enrichment analysis was carried out as described in the GSEA User guide (http://software.broadinstitute.org/gsea/doc/GSEAUserGuideFrame.html).
Luciferase reporter assays for testing Wnt pathway activity
All assays were performed as described on stable clones expressing either the Super8X-TOP-flash (TCF site) construct (57), TFC-1, or the FOP-flash (mutated TCF site) construct (57), FFC-15. TFC-1 and FFC-15 clones were derived earlier (11,22).
Luciferase activity was measured in lysates of TFC-1 and FFC-15 cells that were transfected with either IFT88 or negative control siRNAs. Assays were performed using the Luciferase Reporter Gene Assay Kit (Roche) to obtain data as relative light units (RLU). TCF activity was finally expressed as an Arbitrary Unit (AU), which was obtained after normalizing RLU to total protein estimated using BCA kit (Pierce).
Unless otherwise mentioned, all data represented are values derived from at least 3 biological replicates and is represented as mean ± s.e.m, analyzed using Student’s t-test, where p<0.05 was taken as significant.