Cell lines and clinical samples
The human lymphocytic cell lines (CCRF-SD) and diffuse large B-cell lymphoma cell lines (DOHH2, OCL-LY10, HBL-1 and TMD8) were purchased from the Cell Bank of the Chinese (Shanghai, China). All cells were cultured in the RPMI-1640 medium (Hyclone, USA), supplemented with 1% streptomycin/penicillin (Beyotime, Shanghai, China) and 10% FBS (Gibco, USA), and incubated at 37°C with 5% CO2. A number of 24 DLBCL patients’ RNA samples and 24 normal lymph gland RNA samples and a total of 30 paraffin-embedded DLBCL patients’ tissues with paired adjacent tissues were obtained at Department of Hematology of Southwest Hospital of Third Military Medical University. All DLBCL patients were diagnosed by Lymph node biopsy and confirmed diagnosis by at least two experienced pathologists. The study was approved by the Ethics Committee of Third Military Medical University.
4-5 weeks old male athymic nude mice were obtained from the Center and followed the care and use of Guidelines from Laboratory Animals of the TMMU. Nude mice were kept in a specific pathogen-free condition and all experimental procedures were approved by the TMMU Animal Care and Use Committee.
The paraffin-embedded patients’ tissues were dewaxed and rehydrated and incubated with human PC4 antibody (1: 200; Sigma, St. Louis, Missouri, USA) at 4°C overnight. Then, the slides were sequentially incubated with secondary antibody at 37°C for 1hour, and used DAB to visualize positive staining. PC4 expression in DLBCL patients’ tissues was evaluated by percentage of positive-staining cells. Intensity was graded as follows: 0, no signal; 1, weak (light yellow); 2, moderate (brown); and 3, strong staining. The percentage of positive cells was evaluated quantitatively and scored as: 0 (< 5% positive tumor cells), 1 (5–25% positive tumor cells), 2 (26–50% positive tumor cells), 3(51–75% positive tumor cells) and 4 (> 75% positive tumor cells). The final quantification of each staining was obtained by multiplying these 2 scores. A total staining score of 0–12 was calculated and graded as negative (−, score 0–1), weak (+, score 2–4), moderate (++, score 5–8), or strong (+++, score 9–12).
Total RNA was collected from DLBCL cells using Trizol (Cwbiotech, China). 1μg RNA was reverse transcribed into cDNA according to the recommended protocol by the RevertAid First Strand cDNA Synthesis kit. (#K1622, Thermo Fisher Scientific, Inc.) Quantitative RT-PCR was performed according to the recommended protocol by SYBR Green qPCR master mix (Takara). When the reactions were completed, the relative gene expression was calculated by the comparative threshold cycle (Ct) method. GAPDH expression was used as control. Human-specific primers sequences are shown in Additional file 1: Table S1.
The shRNA lentivirus vector targeting human PC4(shPC4#1:5’-GACAGGUGAGACUUCGAGATT -3’; 5’- UCUCGAAGUCUCACCUGUCTT -3’.shPC4#2:5’-ACAGAGCAGCAGCAGCAGATT-3’;5’-UCUGCUGCUGCUGCUCUGUTT-3’) ; and c-Myc(sh-c-Myc#1:5’-ATGTCAAGAGGCGAACACA-3’;5’-TGTGTTCGCCTCTTGACAT-3’.sh-c-Myc#2:5’-ACGATTCCTTCTAACAGAAAT-3’;5’-ATTTCTGTTAGAAGGAATCGT-3’.) negative control shRNA(5’-UUCUCCGAACGUGUCACGUTT-3’;5’-ACGUGACACGUUCGGAGAATT-3’) were purchased and constructed by GenePharma (Shanghai, China). The human PC4 plasmid and control were purchased and constructed from GeneChem (Shanghai, China). The human GLUT1 plasmid were purchased and constructed from GenePharma (Shanghai, China). GFP-LC3 plasmid was purchased and constructed from Addgene and siRNA targeted to Atg7 was purchased and constructed by GeneChem (Shanghai, China). TMD8 and HBL-1 cells were transfected with shRNA or plasmid according to the recommended instructions. PC4 and c-Myc binding site mutation was conducted by Cas9-sgRNA co-expressed plasmids according to the instruction. Binding site wild type sequence: CCAACAAATGCAATGGGAGT.Binding site mutation sequence: TTGGTGGGCATGGCAAAGAC.
Cell proliferation assay
Cells were cultured in 96-well plates with a density of 3000 cells each well and 100 ul RPMI-1640 medium. Cellular proliferation was measured with the Cell Counting Kit-8 (Dojindo, Japan) at the wavelength of 450 nm. Data were read by a microplate reader (Multiskan Go Multimode Reader; Thermo Scientific).
Cell apoptosis analysis by flow cytometry
Cells were treated with AnnexinV- 7-AAD (BD Biosciences) for 30 min at 37°C in the dark for apoptosis analysis, then analyzed by flow cytometry.
Western blotting analysis
The cell lines were harvested, washed, and lysed with RIPA buffer (Beyotime, China) which contain protease inhibitor cocktail (Roche) for 30min on ice. Total protein was collected and quantitated by a BCA kit (Beyotime, China) according to the recommended instruction. The protein samples were separated by electrophoresis in gel, and then transferred onto PVDF membranes (Millipore). Blotted membranes were incubated with primary antibodies overnight at 4°C. The membranes were washed 5min for 3 times with TBST, and then incubated with HRP-linked secondary antibody (Cell Signaling Technology, USA) 1h at room temperature. The band intensities were detected and visualized by an enhanced chemiluminescence detection system (Bio-Rad Laboratories). Primary antibodies against c-Myc, LC3, SQSTM1,ATG7,PARP,CASPASE3,Bcl-2,BAX,PI3K,S6K1,AKT,mTOR,4EBP1,AMPK,P38,P53,HIF-1α,GLUT1,PKM2,HK2,LDHA and β-actin were obtained from Cell Signaling Technology. Primary antibodies against PC4 were obtained from Sigma.
Cells were fixed in 4.0% formaldehyde for 10 min and permeabilized with ice-cold methanol or 0.5% Triton X-100 for 5 min. The following primary antibodies were used, and subsequently using secondary antibodies to detect Primary antibodies. Conjugating to AlexaFluor 488 or 555(Invitrogen). Cells were counterstained with DAPI and mounted in Vectashield (Vector Laboratories).
In vivo tumor growth model
For in vivo tumor growth model, 100 ul PBS containing 1x107 PC4 stable knockdown TMD8 cells or negative control cells or controls were injected subcutaneously at one dorsal site of athymic male nude mice. Tumor growth was measured every 2 days, which was calculated by the following formula: volume (mm3) = (width2 * length)/2. At the endpoint, the mice were sacrificed, and then xenografts were dissected, weighed and fixed in 4% paraformaldehyde.
ECAR, glucose uptake, lactate and ATP assays
Extracellular acidification rate (ECAR) was measured using extracellular flux analyzer (XFp) analyzer (Seahorse Bioscience) according to the recommended instructions. Lactate production (Lactate Assay Kit) was measured according to the manufacturer (BioVision). For glucose uptake analyse, cells were cultured with a fluorescent D-glucose derivative, 2-[N-(7-nitrobenz-2-oxa-1, 3-diazo-l-4-yl)amino] -2-deoxy-D-glucose (2-NBDG; APExBIO) for 30min at 37 °C. The fluorescence intensity of 2-NBDG was measured through flow cytometry (BD FACSCanto II™). ATP production (Enhanced ATP Assay Kit) was measured according to the recommended manufacturer’s protocol (Beyotime).
Transmission electron microscopy
Cells were harvested and immediately fixed in 3% glutaraldehyde overnight at 4°C and postfixed with 2% osmium tetroxide for 1 hour at 37°C. And then, cells were embedded and stained using uranylacetate/lead citrate. The samples were imaged using a TEM (JEM-1400PLUS, Japan).
Colony formation assay
For colony formation assay, Firstly, we used 0.5%gelatin soaked plates for 20minutes, then sucked the gelatin, adding lymphoma cells cultured for 6-8hours to adherence to the plates. TMD8 and HBL-1 cells were collected and seeded in 6-well plates. Cells were cultured up to 14 days until colonies were clearly visible, the medium was changed every two days. At the endpoint, cells were washed twice with PBS, and then fixed with 4% paraformaldehyde, stained with crystal violet (Beyotime, China) for 30 min, and colonies were counted (more than 50 cells).
Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0.Approximately 10 ug of total RNArepresenting a specific adipose type was subjected to isolate Poly (A) mRNA with poly-T oligoattached magnetic beads (Invitrogen). Following purification, the mRNA is fragmented into smallpieces using divalent cations under elevated temperature. Then the cleaved RNA fragments werereverse-transcribed to create the final cDNA library in accordance with the protocol for the mRNASeqsample preparation kit (Illumina, San Diego, USA), the average insert size for the paired-endlibraries was 300 bp (±50 bp). And then we performed the paired-end sequencing on an Illumina sequence platform.
Luciferase reporter assay
TMD8 cells were cultured at a density of 5 × 104 cells/well in 96-well culture plates and transfected with 0.2 μg of dual-luciferase reporter construct SUB1, or co-transfected with 0.2 μg of the luciferase reporter construct c-Myc and the internal control vector pRL-TK, pRL-SV40, or pRL-CMV (Promega, Madison, WI) at a ratio of 20:1 (reporter construct: control vector) using LipofectamineTM 2000 (Invitrogen, Carlsbad, CA) according to instruction of the recommended manufacturer.
Chromatin immunoprecipitation (ChIP-PCR)
TMD8 cells were conducted ChIP assay with the SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads) (#9003; Cell Signaling Technology) according to the recommend manufacturer’s instructions. Chromatin fragments were immunoprecipitated with anti-PC4 (NB10059774; Novus Biologicals), or rabbit IgG (#2729; Cell Signaling Technology) coupled with ChIP Grade Protein G Magnetic Beads (#9006; Cell Signaling Technology).
EMSA (electrophoretic mobility shift assay)
The DNA binding assays were performed using purified GST-PC4 protein and biotin-labelled fragments of the promoters containing the W-boxes, using GST protein as a negative control, and non-labeled fragments were used as competitors. The bands at the upper and lower part of membranes indicate shift (protein-probe complex) and unbound free probes, respectively.
Statistical analysis was carried out using SPSS 22.0 software (SPSS Inc., Chicago, USA), and all data were presented as means ± SD. Comparisons between two groups were performed using the Student’s t-test. Comparisons among three or more groups were performed using a one-way analysis of variance (ANOVA). The survival data was carried out using the Kaplan-Meier method. Correlation between PC4 expression and clinical parameters was determined using the Pearson’s χ 2 method. P <0.05 indicated a statistically significant difference.