Cell lines
The human CRC cell lines, including RKO, SW480, HCT116, and LOVO were obtained from the Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in a minimum Eagle’s medium (MEM, Gibco, USA), Mccoy’s 5A medium (Gibco, USA) and Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, USA) with 10% fetal bovine serum (Gibco, USA). Cells were then cultured and incubated at 37˚C with 5% CO2.
Immunofluorescence
Localization of endogenous transgelin in RKO, SW480, HCT116 and LOVO cell lines and the expression of PARP1 in RKO cells were determined by immunofluorescence. The primary antibody (anti-transgelin, 1:500, Abcam, USA; anti-PARP1, 1:500, Cell signaling technology, USA), secondary antibody (Alexa Flour 594 goat anti-rabbit IgG, Alexa Flour 488 goat anti-rabbit IgG, 1:500, Invitrogen, USA), and the VECTASHIELD mounting medium (Vector Laboratories, USA)) with 4', 6-diamidino-2-phenylindole (DAPI) were used. The immunofluorescence images were taken and preserved under the laser scanning confocal microscope using a 63× oil-immersion objective lens (Carl Zeiss, USA).
Transfection
The SW480 and RKO cells were cultured in 12-well plates and transfected with pcDNA6/myc-His B-TAGLN-flag and pcDNA6/myc-His B-flag plasmids (Takara, Japan). The RKO cells were transfected with pENTER-TAGLN-Flag and pENTER-Flag control plasmids (Vigene Biosciences, USA) in the co-immunoprecipitation experiment. Transfection was conducted using Lipofectamine 2000/ Lipofectamine 3000 (Thermo Fisher Scientific, USA). Cells were then harvested at 48 hours after transfection for further analysis.
RNA isolation, reverse transcription and polymerase chain reaction (RT-PCR)
Extraction of total RNA was performed using Trizol (Invitrogen) followed by reverse transcription (RT). Real-time polymerase chain reaction (PCR) was carried out using a Light Cycler 480 SYBR Green I Master mix (Roche, USA) on a Light Cycler 480 System (Roche, USA) according to the manufacturer’s instructions. The PCR conditions were as follows: 95C for 30 s, 35 cycles at 95C for 5 s, then 60C for 30 s. PCR primers are listed in Supplementary Table 2, Additional file 4.
Immunoblotting
The nuclear and plasma proteins from HCT116, SW480, LOVO and RKO cell lines were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, USA). The protein concentration of the extracted cytoplasmic and nuclear proteins was determined. Immunoblotting was performed with the primary antibody anti-transgelin (1:500, Abcam, USA, or 1:500, R&D, USA), anti-GADPH (1:400, Abcam, USA or 1:500, Cell signaling technology, USA), anti-PARP1 (1:500, Cell signaling technology, USA), anti-Lamin B1(1:1000, Cell signaling technology, USA), anti-flag (1:500, Cell signaling technology, USA) and the secondary antibody (horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG, 1:30000, Sigma-Aldrich, USA) or IgG Detector (IgG Detector Solution v2, HRP labeled, 1:1000, Takara, Japan). Antibody detection was performed using a chemiluminescence substrate and the protein bands were visualized with Syngene G: BOX Chemi XT4 fluorescence and chemiluminescence gel imaging system (Cambridge, UK).
Immunoprecipitation
The RKO cells were cultured conventionally and transfected with pcDNA6/ myc-His B-TAGLN-flag and pcDNA6/ myc-His B-flag plasmids. The RKO cells were transfected with pENTER-TAGLN-Flag and pENTER-Flag control plasmids in the validation experiment. Cells were then harvested at 48 hours after transfection for further analysis. Antibody immobilization, cell lysis, pretreatment of cell lysate with control agarose resin, immunoprecipitation, immunoprecipitation elution, and immunoblotting analysis were performed in sequence according to the protocol of the Pierce Crosslink Immunoprecipitation Kit (Thermo Fisher Scientific, USA). Anti-flag antibody (10ug, Sigma-Aldrich, USA, for the subsequent mass spectrometry; 1:50, Cell signaling technology, USA, for the validation experiment) and the control rabbit IgG (1:50, Cell signaling technology, USA) were used.
Mass spectrometry
A fraction of the protein samples after immunoprecipitation was analyzed using SDS-PAGE and silver staining (Invitrogen, USA). Another fraction of the samples was used for high-performance liquid chromatography assay (EASY-nLC™, Thermo Fisher Scientific, USA) after filter-aided sample preparation (FASP) and enzymatic hydrolysis. The samples were then analyzed with a Q-Exactive Mass Spectrometer (Thermo Finnigan, USA). The mass/charge ratios of peptides and fragments of peptides were collected. Maxquant 1.3.0.5 software was used to retrieve the Uniprot database by using the raw file as source. The search in the database was set up with specific parameters (Enzyme, trpsin; De-Isotopic, True; Max Missed Cleavages, 2; Fixed modifications, Carbamidomethyl (C); Variable modifications, Oxidation (M); First search ppm, 20ppm; Main search ppm, 6ppm; Decoy database pattern, reverse; Min. Reporter PIF, 0.75; Peptides false discovery rate (FDR) ≤0.01; Protein FDR≤0.01).
Bioinformatics
Identification of differential expression genes (DEGs), functional enrichment and signaling pathway enrichment analysis
The relevant cDNA microarray data were obtained using the Affymetrix microarray technique based on our previous work [9]. Over-expression of TAGLN in RKO human colon cancer cells resulted in 256 downstream transcripts that were differentially expressed with at least 2-fold change (P<0.05). Among these, transcripts without gene symbols, gene database codes and duplicates were excluded. The remaining DEGs were screened for further bioinformatics analysis.
Using the Metascape tool (www.metascape.org/), the screening parameters were set as follows: P < 0.01 or 0.001 (Biological Process), participating genes ≥3 and enrichment factor > 1.5. We conducted functional and signaling pathway enrichment analysis of the DEGs referring to the gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Reactome databases.
Construction of the protein-protein interaction (PPI) network, topological analysis and key gene screening
The DEGs were simultaneously translated into proteins and the search tool for retrieval of interacting genes (STRING 10.0, https://string-db.org/) [16] was used for PPI analysis. Subsequently, relevant data was imported into the Cytoscape online software (www.cytoscape.org/) [17] and a PPI network was constructed. In this study, the degree centrality and intermediate centrality of the DEGs were calculated using the CytoHubba plug-ins. Those with values 2-fold higher than the overall average value were selected as the core genes in the network. In addition, the core modules were obtained with an MCODE plug-in (k-core = 2). The core genes and the genes included in the core modules were defined as the key genes. Key genes were further analyzed with Metascape for signaling pathway enrichment in KEGG and Reactome database using the same parameters previously mentioned.
Prediction of the TFs for the key genes
The TF evaluation model within the Gene-cloud of biotechnology information (GCBI) tools (https://www.gcbi.com.cn/) was used to predict the TFs for the key genes. Those with medium or high recommendations were selected and potential TFs were selected for further analysis. We then compared these potential TFs to the DNA-binding proteins identified in the mass spectrometry analysis.
Nuclear localization signal analysis
The sequences of the selected potential TF(s) were obtained from the Uniprot database (https://www.uniprot.org/)[18]. The classical nuclear localization signals (cNLS) Mapper (www.nls-mapper.iab.keio.ac.jp/) [19] was used to detect the nuclear localization signal of the potential TF(s).
Statistical analysis
Statistical analysis was performed using the SPSS 20.0 software (IBM Corp., Armonk, USA). The relevant values were expressed as mean ± standard deviation (SD), and the significance of the difference between two groups was determined with the Student’s t-test. Pearson correlation analysis was used to analyze the expression level of the key genes obtained from cDNA microarray and RT-PCR. P<0.05 (bilateral) was considered statistically significant.