Tissue culture and cell treatments
The experiments used human colon cancer cells that were 5-FU-sensitive HCT-8 cells and 5-FU-resistant HCT-8/FU cells, which were obtained from MEIXUAN Bioscience & Technology Co. Ltd. (Shanghai, China). The human colon cancer cells SW480 were obtained from the Cell Resource Centre of Chinese Academy of Medical Science (Beijing, China). HCT-8 and SW480 were maintained at 37°C in a 5% CO2 incubator in RPMI 1640 (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS) (Biological Industries, Kibbutz BeitHaemek, Israel) and 1% penicillin-streptomycin (Keygen Biotech, Nanjing, China). Research grade 5-FU (MCE, Monmouth Junction, USA) was used in the assays to evaluate the cell viability. Apatinib (Hengrui medicine Co, Ltd., Jiangsu, China), raltitrexed (Zhengda Tianqing Pharmaceutical Co., Ltd., Jiangsu, China), artemisinin (National Institutes for Food and Drug Control, Beijing, China), cisplatin (QILU Pharmaceutical Co. Ltd., Shandong, China) were used in the immunofluorescence assays to evaluate the morphology changes of CBs.
Vector preparation and transfection
The expression plasmid of FLAG tagged UHMK1 were cloned individually into pENTER vectors by Vigene Bioscience Co., Ltd. (Shandong, China). The construct of the UHMK1-K54A mutant was generated from the wild type by Vigene Bioscience Co., Ltd. (Shandong, China). The siRNA targeting UHMK1 duplexes of 5’-AAGCAGUUCUUGCCGCCAGGA-3’ and 5’-CGAGUAUGGUUUCCGCAAATT-3’ were purchased from General Biosystems, Inc. (General Biosystems, Inc, Chuzhou, China). Coilin-targeting siRNA of 5’-GAGAGAACCUGGGAAAUUUTT-3’ was obtained from General Biosystems, Inc. (Chuzhou, China). A scrambled siRNA 5’-UUCUCCGAACGUGUCACGUTT-3’ was used as the control. The cells were transfected with either the UHMK1 plasmids or paired siRNA oligos using a Lipofectamine™ RNAiMAX Kit (Invitrogen, Waltham, Massachusetts, USA) following the vendor’s recommended protocols. Western blotting was performed to detect the protein levels of the corresponded genes at 48 h post transfection.
Cell viability assay
A number of 5,000 cells were seeded in each well of 96-well plates and cultured with 5-FU at 0, 1, 5, 10, 20, 40, 60, 80 μg/ml for 48 h. The cell viability was determined using cell counting kit (CCK8) (KeyGENBioTECH, Jiangsu, China) according to the vendor’s standard protocols. The plates were scanned at 450 nm for absorbance using a spectrophotometer (BioTek, Winooski, VT, USA). Each data point was measured for the average from six duplicates. The experiments were repeated independently for 3 times.
The method has been widely implemented by our laboratories. Cells of 5.0×104 were plated onto a glass coverslip placed into the well of a 12-well plate. The cells on coverslips were fixed, permeabilized, blocked and washed with phosphate-buffered saline (PBS). Anti-coilin (Proteintech Group, Rosemont, USA) was used as the primary antibody and an Alexa Fluor® 594 secondary antibody (Life Technologies, MA, USA) was used for incubation in the dark. The nuclei were stained with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO, USA) prior to the examination and image acquisitionunder a confocal system (Leica Microsystems TCS SP8. Wetzlar, Germany). Control samples without adding the primary antibody were prepared for determining the level of non-specific noise.
mRNA-sequencing and data processing
RNA-seq analyses were performed as earlier described. Cells were scraped off from the surface in trypsin-versene solution and collected by 500 g centrifugation. The pellet was washed with PBS to remove residual media. Total RNA extractions were performed with the RNA-Quick Purification Kit (Yishan Biotechnology Co., Ltd., Shanghai, China) following the manufacturer's protocol. The RNA concentrations were determined using a Nanodrop ND1000 spectrophotometer (Thermo Scientific). The quality assessments and mRNA sequencing libraries were performed in the laboratory of VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (Vazyme Biotech, Nanjing, China), VAHTS RNA Multiplex Oligos Set1- Set2 for Illumina (Vazyme Biotech, Nanjing, China), VAHTS DNA Clean Beads (Vazyme Biotech, Nanjing, China), VAHTS mRNA Capture Beads (N401-01, Vazyme Biotech, Nanjing, China). All prepared samples subjected to paired-end multiplex sequenced (2×150 bp) on the Illumina Hiseq X10 platform. Approximately 8 Gb sequencing data was generated for each sample.
The clean reads in compressed FASTQ format were aligned using HISAT2 (verson 2.1.0) to the reference of human genome (Homo_sapiens.GRCh38.dna.primary_assembly.fa) with matched rates over 90%. The resulted SAM files output in the BAM format used with SAMtools (version 1.18, http://samtools.sourceforge.net).The resulted BAM files were sorted with SAMtools. The depth counts were called with HTSeq (version 0.11.2. Linux_x86_64, Simon Anders ([email protected])) with the reference of human genome (Homo_sapiens.GRCh38.94.gtf), European Molecular Biology Laboratory (EMBL)) used to calculate the Fold change (FC) of FPKM and p value among sample groups according to an over-dispersed Poisson model. Differentially expressed (DE) genes were identified with the thresholds of both 1.5 fold change (|log2FC|≥0.58) in mean expression and FDR≤5% using Benjamin-Hochberg procedure. The p-value was identified using DESeq2 (version 1.30.0). The enrichment of DE genes was performed using Gene Ontology (https://go.princeton.edu/) and Cluster 3.0 (Michael Eisen, Stanford University), then displayed with TreeView (version 1.1.6r4, Alok Saldanha). The analyses for alternative splicing events of expressed genes were performed using the rMATS software package (version 4.1.0, http://rnaseq-mats.sourceforge.net/, Xing Lab, Children's Hospital of Philadelphia). The differences in splicing isoforms (SIs) were identified with FDR≤5% and a threshold of p≤0.01 in the mean expression between the samples.
RNA extraction and qRT-PCR
Total RNA was isolated using Trizol (Life Technologies, Carlsbad, CA, USA). HiScript II Q RT Kit (Vazyme, Nanjing, China) was used for reverse transcription. NovoStart® SYBR qPCRSuperMix Plus (Novoprotein, Shanghai, China) was used to quantify gene expression level from the obtained cDNA. The primers for detecting are listed in Table S1. GAPDH was used as the loading reference. The cDNA were determined using a Quantitative Real-time PCR (Archimed X6, Rocgene, Beijing, China)
Western blot analyses were performed as earlier described. Briefly, samples of cell lysates were prepared and separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred onto polyvinylidene fluoride (PVDF) filters. The probing antibodies were against the following antigens: UHMK1 (SC-393605, Santa Cruz Biotechnology, Santa Cruz, CA, USA), coilin (10967-1-AP, Proteintech Group, Rosemont, USA) and GAPDH (TA-08, ZSGB-BIO, Beijing, China).
Co-Immunoprecipitation (Co-IP) assay
Cells were harvested and lysed in 1000 µl of ice-cold lysis buffer (10 mM HEPES, 50 mM NaCl, 5 mM EDTA, 1 mM Benzamidine, 0.5% Triton X-100). The lysate was solubilized via end-over-end rotation at 4°C and clarified via centrifugation at 12,000 rpm for 30 min. A small fraction of the supernatant was taken at this point and incubated with SDS-PAGE sample buffer in order to examine expression of proteins in the whole cell extract. The remaining supernatant was divided equally into two tubes, and then incubated with 2 µg phospho-Ser antibody (SPC-149F, StressMarq Biosciences Inc, Victoria, British Columbia) antibody or 2 µg IgG (C2170, Applygen Technologies Inc, Beijing, China) respectively with end-over-end rotation at 4°C overnight. After incubated with 30 µl of Protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 3 h with end-over-end rotation at 4°C, the immunoprecipitated proteins were eluted from the beads with sodium dodecyl sulfate (SDS) sample loading buffer, resolved by SDS-PAGE and subjected to Western blot analyses using an antibody against coilin (10967-1-AP, Proteintech Group, Rosemont, USA)
The analysis of variance (ANOVA) was used to determine the statistical significance of data in multiple groups. The Student’s t-test was used to compare cell functions between paired groups. Cases of p-value <0.05 was defined as statistically significant. The program of Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA) was used for data plotting.