The human myeloid leukemia mononuclear (THP-1) and embryonic kidney (HEK-293) cell lines and mouse lung cancer (LLC) and colon cancer (MC38) cell lines were purchased from the Chinese Academy of Science Cell Bank (Shanghai, China). Human umbilical vein endothelial cells (HUVECs) and the mouse lymphatic endothelial cell line (SVEC4–10) were obtained from Procell Life Science & Technology Co., Ltd. HUVECs and HEK-293 cells were cultured in DMEM (Gibco, Carlsbad, CA, USA). LLC and SVEC4–10 cells were maintained in RPMI 1640 (Gibco, Grand Island, NY, USA). THP-1 cells were cultured in RPMI 1640 (Gibco, Grand Island, NY, USA) supplemented with 0.05 mM β-mercaptoethanol (Sigma–Aldrich). All culture media were supplemented with 10% fetal bovine serum (FBS, Biological Industries, Israel) and 1% penicillin/streptomycin (Gibco). All cells were cultured in a humidified 37 °C incubator with 5% CO2 .
Generation of stable cell lines
The PFUGW-3FLAG-EGFP-Vector (Vector), PFUGW-3FLAG-EGFP-cyclin G2 (Flag-cyclin G2), PFUGW-EGFP-Nonsense (Nonsense), and PFUGW-EGFP-shcyclin G2 (shcyclin G2#1 and shcyclin G2#2) plasmids were purchased from GeneChem Co., Ltd. (Shanghai, China) and used to package lentiviruses for infecting THP-1 cells. Transfected THP-1 cells were selected with 3 μg/mL puromycin (Sigma–Aldrich, Santa Clara, CA, USA) to generate stable cell lines [16, 18]. Small interfering RNA (siRNA) for the knockdown of PP2Ac (siPP2Ac) and scrambled nontargeting siRNA (siNC) were purchased from Genepharma Co., Ltd. (Shanghai, China). These siRNAs were transfected into cells according to the manufacturer’s instructions.
Total RNA was isolated using TRIzol® Reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was reverse-transcribed using a reverse transcription kit (RR036A, TaKaRa, Tokyo, Japan). Real-time PCR was carried out using a SYBR Green PCR Kit (TaKaRa) and the Roche LightCycler 480 real-time PCR system . The primers used were as follows: human CCNG2 (Forward, 5′-TGC CTA GCC GAG TAT TCT TCT-3′; Reverse, 5′-TGT TTG TGC CAC TTT GAA GTT G-3′); human CXCL9 (Forward, 5′-CTG TTC CTG CAT CAG CAC CAA C-3′; Reverse, 5′-TGA ACT CCA TTC TTC AGT GTA GCA-3′); human IL-1β (Forward, 5′-CCA CAG ACC TTC CAG GAG AAT G-3′; Reverse, 5′-GTG CAG TTC AGT GAT CGT ACA GG-3′); human Arg-1 (Forward, 5′-TCA TCT GGG TGG ATG CTC ACA C-3′; Reverse, 5′-GAG AAT CCT GGC ACA TCG GGA A-3′); human β-actin (Forward, 5′-ATT GGC AAT GAG CGG TTC CG-3′; Reverse, 5′-CGT GGA TGC CAC AGG ACT CC-3′); mouse CXCL9 (Forward, 5′-CCT AGT GAT AAG GAA TGC ACG ATG-3′; Reverse, 5′-CTA GGC AGG TTT GAT CTC CGT TC-3′); and mouse GAPDH (Forward, 5′-CAT CAC TGC CAC CCA GAA GAC TG-3′; Reverse, 5′-ATG CCA GTG AGC TTC CCG TTC AG-3′). Human β-actin and mouse GAPDH were used as internal controls.
Western blotting and immunoprecipitation
Cells were lysed with RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche, Basel, Switzerland), as previously described . Equal amounts of protein were separated by SDS–PAGE and transferred to membranes. For immunoprecipitation, cell lysates were incubated with antibodies and Protein A/G Magnetic Beads (HY-K0202, MedChem Express), followed by western blotting. Western blotting was performed as previously described  with specific primary antibodies and horseradish peroxidase-conjugated secondary antibodies, followed by visualization using chemiluminescence (DNR Bio-Imaging Systems, Jerusalem, Israel). The antibodies used were as follows: anti-cyclin G2 (DF2284, Affinity Biosciences), anti-Flag (M20008XS, Abmart), anti-PP2Ac (2038 T, Cell Signaling Technology), anti-STAT1 (14994S, Cell Signaling Technology), anti-p-STAT1 (Y701) (9167S, Cell Signaling Technology), anti-lamin B1 (sc-6216, Santa Cruz), anti-β-tubulin (M30109XS, Abmart), anti-GAPDH (M20006F, Abmart), and anti-IgG (3900S, Cell Signaling Technology).
Human peripheral blood monocytes isolation, culture and identification
Anticoagulant whole blood was diluted 1:1 with PBS (containing 5% FBS), then it was slowly added to the centrifuge tube containing lymphocytes separation medium (LTS10771, TBD), centrifuged at 1000 g for 20 min. The removed PBMC layer was resuspended in PBS, centrifuged at 300 g for 5 min, and discarded the supernatant. The previous step was repeated. Cells were resuspended and cultured in RPMI 1640 supplemented with 10% FBS, 1% penicillin/streptomycin, and 20 ng/mL M-CSF (216-MC-025/CF, R&D Systems). Macrophages were incubated with APC anti-human CD68 antibody (333,810, Biolegend), identified by flow cytometry.
Bone marrow-derived macrophage (BMDM) isolation, culture and identification
The femur and tibia were removed from eight-week-old C57BL/6 mice, and single-cell suspensions were prepared in Hank’s Balanced Salt Solution (PB180323, Procell) containing 5% FBS. Red blood cells were lysed using red blood cell lysis buffer. The samples were centrifuged, and the supernatant was discarded. The cells were resuspended and cultured in RPMI 1640 supplemented with 10% FBS, 1% penicillin/streptomycin, and 20 ng/mL M-CSF (CB34, Novoprotein) . BMDMs were incubated with APC anti-mouse/human CD11b antibody (101,212, Biolegend) and FITC anti-mouse F4/80 antibody (123,108, Biolegend), identified by flow cytometry.
CTL isolation and culture
The spleens of C57BL/6 mice were removed, homogenized in RPMI 1640 medium, and filtered through a sterile 70-μm cell strainer. CD8+ T cells were isolated using the EasySep™ Mouse CD8+ T-Cell Isolation Kit (STEMCELL), according to the manufacturer’s instructions. Cells were cultured in RPMI 1640 medium containing 10% FBS, 1% penicillin/streptomycin, 0.05 mM β-mercaptoethanol (Sigma–Aldrich), 1 mM sodium pyruvate (Sigma–Aldrich), 2 mM L-glutamine (Invitrogen, Carlsbad, CA, USA), 20 ng/mL recombinant IL-2 (R&D Systems, Minneapolis), and 2 μg/mL anti-CD28 (Invitrogen). The cells were seeded into 5 μg/mL plate-bound anti-CD3 vessels (Invitrogen), as previously described .
−/− C57BL/6 mice were described previously. The TALEN-targeted Ccng2 knockout mice (Ccng2−/−) of the C57BL/6 N genetic background were generated by Cyagen (Cyagen Biosciences, Guangzhou, China) [16, 19]. LLC or MC38 cells (1.5 × 106) were mixed with 3 × 105 BMDMs from wild-type (WT) or Ccng2−/− C57BL/6 mice and subcutaneously inoculated into the right flank of eight-week-old C57BL/6 mice. Each mouse was intraperitoneally injected with 2.5 μg IFN-γ (C746, Novoprotein) on Days 6, 9, and 12 after tumor cell inoculation. Tumors were periodically measured with calipers. The mice were euthanized on Day 15, and tumor weights and volumes were measured. All animal experiments were performed in accordance with relevant regulatory standards and approved by the Animal Ethics Committee of China Medical University.
Mouse tissues were fixed and paraffin-embedded. The expression of Ki-67 (652,402, Biolegend), CD31 (AF3628, R&D Systems) and CD8a (98,941, Cell Signaling Technology) was determined using immunohistochemical assays, as previously described . The slides were mounted with coverslips and photographed under a microscope (Nikon 80i).
Cells were grown on coverslips and incubated with an anti-STAT1 antibody (14994S, Cell Signaling Technology). Embedded mouse tissue was incubated with an anti-CD8a antibody (14–0081-82, Invitrogen). Both followed by incubation with corresponding secondary antibodies for 60 min at 37 °C in the dark. Nuclei were counterstained with DAPI. Photographs were taken with an Olympus LEXT OLS4500 Confocal Laser Scanning Microscope .
Mouse tumor cell isolation
The tumors were placed in precooled Hank’s Balanced Salt Solution containing 5% FBS and cut to 1 mm with scissors. 1 mg/ mL collagenase I (SCR103, Sigma–Aldrich) and 1 mg/ mL DNase I (10,104,159,001, Roche) were used to digest tumor tissues in 37 °C water bath until tissue block dissolved. The single-cell suspensions were collected through 70 μm filters. Red blood cells were removed by red blood cell lysis buffer. Cells were washed and resuspended in stain buffer (FBS).
Flow cytometry analysis
Cells were pretreated with a phorbol 12-myristate 13-acetate (PMA)/ionomycin mixture (70-CS1001, MultiSciences) and BFA/monensin mixture (70-CS1002, MultiSciences) for 5 h. The cells were then incubated with PE-labeled anti-mouse CD8a antibody (E-AB-F1104D, Elabscience) for 30 min at 4 °C in the dark. Subsequently, the cell membranes were disrupted using the Fixation/Permeabilization Kit (554,714, BD). The permeabilized cells were incubated with antibody at 4 °C for 30 min in the dark. The antibodies used included a recombinant APC-labeled anti-human/mouse granzyme B (372,204, Biolegend) and APC-labeled anti-mouse perforin (S16009B, Biolegend) antibodies. APC-labeled rat IgG2a and κ isotype control (E-AB-F09832E, Elabscience) and APC-labeled mouse IgG1 and κ isotype control (E-AB-F09792E, Elabscience) were used as negative controls. All cells were analyzed by flow cytometry using a BD Accuri C6 Plus Flow Cytometer.
CTL chemotactic assay
Cell chemotaxis assays were performed in 24-well plates using a 5-μm chamber (Corning, NY, USA). A total of 10 × 104 cells/well were suspended in RPMI 1640 containing 0.2% FBS in the upper chamber, and the lower chamber was filled with the conditioned medium of BMDMs from C57BL/6 mice with or without recombinant mouse CXCL9 protein (492-MM-010/CF, R&D Systems). The cells were cultured for 12 h in a humidified incubator at 37 °C with 5% CO2, and cells that invaded the lower surface were stained with 5 nM eBioscience™ Calcein AM Viability Dye (Invitrogen, USA) for 15 min and detected by fluorescence microscopy.
Matrigel HUVEC and SVEC4–10 cell tube formation assays
A 96-well plate was coated with Matrigel (356,230, BD) at 37 °C for 30 min. HUVECs or SVEC4–10 cells (6 × 104) were cultured in 150 μL of conditioned medium with or without recombinant human CXCL9 protein (392-MG-010/CF, R&D Systems), recombinant mouse CXCL9 protein for 2–6 h . The cells were stained with 5 nM eBioscience™ Calcein AM Viability Dye for 15 min and detected by fluorescence microscopy. Tube formation was assessed using AngioTool, including estimating the vessel percentage areas, the total number of junctions, and total vessel length.
Measurement of CXCL9 levels
Cell culture supernatants were collected, and human and mouse CXCL9 levels in the supernatants were quantified using the human and mouse CXCL9 Enzyme-Linked Immunosorbent Assay (ELISA) Kits (E-EL-H6062 and E-EL-M0020c, Elabscience), respectively.
Stimulation of macrophages
THP-1 cells were treated with 100 ng/mL PMA (P8139, Sigma–Aldrich) for 48 h. The macrophages were then stimulated with 100 ng/mL IFN-γ (SRP3058, Sigma–Aldrich), 20 ng/mL LPS (916,374, Sigma–Aldrich), 20 ng/mL IL-4 (SRP3093, Sigma–Aldrich), or 20 ng/mL IL-13 (SRP3274, Sigma–Aldrich) for 48 h.
Cytoplasmic and nuclear protein fractionation
Cytoplasmic and nuclear proteins were separated using a nuclear and cytoplasmic protein extraction kit (P0027, Beyotime), according to the manufacturer’s instructions.
The means of the two groups were compared using the unpaired Student’s t-test. Data are presented as the mean ± SD. The weight and volume of tumors are presented as the mean ± SEM. The growth curves of the tumor volume were analyzed using two-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.