Cell viability analysis
VCR-sensitive/resistant human colorectal cancer cell line HCT-8, ADR-sensitive/resistant human breast adenocarcinoma cell line MCF-7 (both from Huiying BioTech), ADR-sensitive/resistant human chronic myelogenous leukemia cell line K562, human colorectal cancer cell line HCT-116 (both from KeyGen Biotech), and EGFR knockout HCT-116 cell line  [kindly provided by Dr. Ningzhi Xu at Chinese Academy of Medical Sciences] were maintained in RPMI-1640 medium (Sigma-Aldrich), supplemented with 10% FBS at 37°C in a humidified atmosphere with 5% CO2. Different concentrations of IVM (Meilun BioTech), VCR (YuanchengGongchuang Tech), ADR (KeyGen Biotech) or mitomycin C (Welson Biotech) were used to treat the cells. After 48 h incubation, cells were subjected to MTT analysis and the absorbance at 570 nm was recorded by a Spectra Max i3 microplate reader (Molecular Devices Corp., Sunnyvale, CA, USA).
Xenograft models in mice
A xenograft colorectal carcinoma mouse model was established by injecting 1×107 VCR-sensitive or resistant HCT-8 cells subcutaneously in the flank region of each female nude BALB/c mice (4-week-old, Vital River Lab). When tumors reached about 100 mm3, the nude mice were randomized into four groups (n = 6) according to tumor volumes and body weights. Drugs were injected intraperitoneally daily for 27 days, including IVM (2 mg/kg/day), VCR (0.2 mg/kg/day), IVM (2 mg/kg/day) plus VCR (0.2 mg/kg/day). To prepare IVM for injection, a stock solution (5 mg/ml in DMSO) was prepared and then diluted by using 0.9% NaCl, which resulted in a homogeneous suspension of IVM. Two hundred microliters (200 μl) of the IVM was injected to each mouse. VCR was also prepared in 0.9% NaCl, and mice injected with only 0.9% NaCl solution served as vehicle control. Tumor volume was measured every three days by using calipers. Tumor volumes were calculated as V = length × width2/2. On the 27th day, the tumors were harvested, weighed, and then fixed in 4% paraformaldehyde for immunofluorescence and immunohistochemistry analysis.
In order to establish the leukemia mouse model with K562 cells, the male non-obese diabetic/severe combined immune deficient NOD/SCID mice (4-week-old, Vital River) were given cyclophosphamide (Meilun BioTech) (2 mg/mouse/day) for three days before the ADR-sensitive/resistant K562 cells were injected (2 ×107 cells/mouse) into tail vein. Then, the mice were randomized into three groups (n = 6). The drugs ADR (0.3 mg/kg/day) and/or IVM (2 mg/kg/day) were injected intraperitoneally daily for 27 days. All of the drugs were prepared in 0.9% NaCl and mice injected with only 0.9% NaCl solution served as vehicle control. On the 27th day, the mice were sacrificed, and spleen was weighed, and then fixed in 4% paraformaldehyde for histopathological examination. The peripheral blood was collected in anticoagulant heparin. Cells within bone marrow were washed out by 10 mM phosphate-buffered saline (PBS, pH 7.4). Blood smears were prepared and stained with May-Grünwald Giemsa (MGG) staining. The peripheral blood cells and bone marrow cells were subjected to flow cytometry after stained with mouse anti-human CD33-FITC (555626), CD13-PE (555748), and isotype-matched FITC- (555394), PE- (555749) conjugated control antibodies (all from BD Biosciences).
HPLC analysis of VCR
One milliliter of distilled water was added to the cell pellets and the cells were subjected to freezing (at -80°C) and thawing for three times. The tumor tissues were homogenized with 600 μl of H2SO4 in a glass homogenizer on ice. After centrifugation, the supernatants were dried by vacuum, and then resuspended in 200 μl of distilled water, and analyzed by an Agilent 1100 series HPLC system (California, USA). The samples were injected into the C18 column (250 mm × 4.6 mm, 5 μm) with the mobile phase containing 20 mM KH2PO4 (pH 6.6) and methanol (30:70, v/v). The detection wavelength was 298 nm.
Western blotting analysis
Cells or tumor tissues were homogenized in the buffer containing 50 mM Tris-HCl with pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM PMSF and 1% protease inhibitors. Lysates were centrifuged at 5, 000 × g for 15 min at 4°C and the loading buffer was added to the supernatants. The protein samples were boiled at 100°C for 10 min and electrophoresed in SDS-polyacrylamide gels. Then the gels were transferred onto PVDF membranes (Millipore, Darmstadt, Germany). The membranes were blocked in 5% bovine serum albumin (BSA) (w/v) or 5% fat-free milk (w/v) in Tris-buffered saline with 0.1% Tween 20 (TBST) buffer for 2 h at RT, incubated with the corresponding antibody at 4°C overnight, then incubated with the horseradish peroxidase (HRP)-labelled secondary antibody for 3 h at RT. The following antibodies were used: anti-EGFR (#2232, 1:1000), anti-p-EGFR (#2234, 1:500), anti-P65 (#8242, 1:1000), anti-p-P65 (#3033, 1:500), anti-p-Akt (#9271, 1:500), anti-p-ERK (#4370, 1:500), anti-Akt (#9272, 1:1000), and anti-ERK (#9102, 1:1000) (All from Cell Signaling); anti-P-gp (517310, 1:500, Calbiochem) and anti-GAPDH (CW0100, 1:1000, Beijing Com Win). Finally, the membranes were stained with standard ECL reagents and then photographs were taken by DNR MicroChemi4.2 system (Bio-Imaging Systems Ltd, Neve Yamin, Israel).
Quantitative PCR analysis
HCT-8 cell pellets, mouse peripheral blood cells and mouse bone marrow cells were suspended respectively and homogenized in 1 ml Trizol reagent (Invitrogen, Carlsbad, CA, USA) on ice. Then, the mixture was placed at RT for 5 min. Two hundred microliters of chloroform were added. The tubes were fiercely shaken for 1 min and centrifuged at 12, 000 × g for 15 min at 4°C. Then the supernatant was transferred into a new centrifuge tube, and 500 μl of propanol was added. The tubes were fiercely shaken for 1 min and centrifuged at 12, 000 × g for 10 min at 4°C. The precipitant was washed with 75% ethanol twice, dried and dissolved in RNase free ddH2O. The total RNA concentration was measured using Biophotometer Plus (Eppendorf, Hamburg, Germany). Total RNA (0.3 ~ 1 μg) was reverse-transcribed into cDNA by using a M-MuLV reverse transcriptase assay kit (Fermentas, Ontario, Canada). The relative mRNA levels of MDR1 and bcr/abl fusion gene were determined by quantitative PCR using a SYBR green Premix Ex TaqTM (Tli RNaseH Plus) PCR kit (TaKaRa, Dalian, China) in a MX3000P real-time thermocycler (Axygen, California, USA). The primer sequences for MDR1 were 5′-GACATGACCAGGTATGCCTA-3′ (sense) and 5′-CTTGGAGACATCATCTGTAAGTC-3′ (antisense); the primer sequences for bcr/abl fusion gene were 5′-GGAGCTGCAGATGCTGACCAAC-3′ (sense) and 5′-TCAGACCCTGAGGCTCAAAGTC-3′ (antisense) and the primer sequences for the control gene GAPDH were 5′-CGCTGAGTACGTCGTGGAGTC-3′ (sense) and 5′-GCTGATGATCTTGAGGCTGTTGTC-3′ (antisense).
Luciferase reporter assay
A 1, 637 bp region encompassing the NF-κB binding site and the annotated transcription start of ABCB1 (-1468 to +168 bp, chr7-: 87713155-87714791) was cloned into a Gaussia luciferase (GLuc) reporter vector (pEZX-PG04, Genecopoeia), which contains a reference reporter gene, secreted alkaline phosphatase (SeAP).
The cells in 24-well plates were co-transfected with the above reporter vector with pcDNA3.1(+)-P65 expression vector or siRNA targeting NF-κB using transfection reagent VigoFect (Vigorous Biotech). After 12 h, the cells were treated with 3 μM IVM and/or 25 nM VCR for 48 h. The activities of GLuc and SeAP were quantified with the secrete-pair dual luminescence assay kit (Genecopoeia).
Immunohistochemistry and immunofluorescence analysis
The fixed tumor tissues in nude mice were frozen and cut into 5 μm thick sections. The sections were fixed in 4% paraformaldehyde for 10 min at RT, perforated by 0.5% Triton-X-100 for 10 min at RT. Then endogenous peroxides were removed, and sections were blocked in TBST containing 3% BSA for 1 h at RT, incubated with anti-P-gp antibody overnight at 4°C and HRP-labelled secondary antibody for 2 h at RT. Then, immunoreactive sites were subsequently identified by using the 3,3′-diaminobenzidine (DAB) substrate kit (Vector Laboratories). The nuclei were stained with hematoxylin for 3 min at RT and the frozen sections were visualized under Olympus IX71 inverted microscope (Tokyo, Japan). For the immunofluorescence analysis, the processes of fixation, perforation and blocking were the same as those of immunohistochemistry. Then slides were incubated with the anti-P-gp antibody at 37°C for 1 h and FITC-labeled secondary antibody for 1 h at 37°C. All images were acquired using a Carl Zeiss LSM710 laser scanning confocal microscope (Oberkochen, Germany).
Two hundred microliters of peripheral anticoagulant heparin-treated blood and 1×106 bone marrow cells were treated using red-blood-cell lysing buffer (BD Biosciences). White blood cells in peripheral blood and bone marrow cells were resuspended in 100 μl of PBS, incubated with 20 μl of mouse anti-human antibodies, which include CD13-PE (555394), CD33-FITC (555626), and isotype-matched FITC- (555748), PE- (555749) conjugated control antibodies (all from BD Biosciences), for 30 min at 4°C. The cells were washed with PBS and resuspended in 300 μl of 2% paraformaldehyde and detected by FACS Aria II flow cytometry (Becton Dickinson, USA).
Ten microliters of anticoagulant blood were smeared on each glass microscope slide. Then, the slides of blood cells smears were dried and fixed in methanol for 5 min at RT, and then immersed into May-Grünwald solution for 3 min, then immersed into PBS solution (pH 6.8) for 1 min. Finally, the slides were stained with Giemsa solution (diluted 20 times with the PBS) for 10 min and washed with ddH2O for 30 s, air-dried and visualized under Olympus IX71 inverted microscope (Tokyo, Japan).
After the NOD/SCID mice were sacrificed, the spleen was harvested and fixed with 4% paraformaldehyde. Then these tissues were dehydrated in a series of alcohol, embedded in paraffin and sliced into 5-μm sections. Hematoxylin and eosin staining was carried out according to the routine staining method. Briefly, the sections were dewaxed, rehydrated in alcohol, stained with hematoxylin for 15 min, differentiated, and then stained with eosin for 3 min, dehydrated in alcohol and xylene, and mounted. The sections were examined under Olympus IX71 inverted microscope (Tokyo, Japan).
Co-immunoprecipitation (Co-IP) assay
The cells were lysed and then centrifuged. The supernatants were incubated with the anti-avermectins (AVMs) antibody, which had a cross-reactivity of 100% with abamectin (ABM) and 25% with IVM  (provided by Dr. Jianzhong Shen) at 4°C in rotation overnight. Then 80 μl of protein G plus A agarose (Beyotime Biotechnology, Jiangsu, China) was added and the mixture was incubated at 4°C in rotation for another 6 h. Then, the immunocomplexes were washed and the precipitated beads were resuspended in 50 μl of 2 × loading buffer for the electrophoresis.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) was performed using the EZ ChIP kit (EMD Millipore). Briefly, HCT-8 cells treated with IVM for 48 h were collected and cross-linked with formaldehyde. Chromatin was sonicated and then incubated and precipitated with anti-P65, anti-RNA polymerase II (positive control), or normal rabbit IgG (negative control), respectively. The immunoprecipitated DNA fragments were detected by qPCR analysis. The primers for the MDR1 promoter (-1468 to -1319 bp) were 5'-AAACGGATGCATGGGGCGG-3' (sense) and 5'-GAAGATAGACAACTGGTTAGACGAG-3' (antisense).
Plasmids, siRNA and adenovirus
Human full length MDR1 (AF016535.1), human full length EGFR (NM_005228.4) and human full length RELA/P65 (NM_021975.3) were cloned into pcDNA3.1(+) vector (GENEWIZ). HCT-8 cells were transfected using the transfection reagent VigoFect. pGenesil-P-gp vector was used to express shRNA of P-gp in the cells . Three siRNAs targeting EGFR and NF-κB (P65) were synthesized by Shanghai Gene Pharma Co. Ltd (Shanghai, China). The siRNA with the highest gene silencing efficacy was chosen for further use. The recombinant adenoviral vectors expressing LacZ (Ad-LacZ), Akt (Ad-Akt-myr), MKK1 (Ad-MKK1-R4F) or mTOR (Ad-mTOR) (all provided by Dr. Shile Huang at Louisiana State University), were amplified and used as described in the reference  to constitutively activate Akt, ERK and mTOR, respectively. Ad-MKK1-R4F was used for the activation of ERK because MKK1 could phosphorylate and activate ERK in the cells.
The interactions between IVM and EGFR extracellular domain were determined using Super Streptavidin (SSA) biosensors in the Octet RED96 system (ForteBio Inc., Menlo Park, CA, USA). First, the recombinant extracellular domain of human EGFR protein (ab155639, Abcam) was biotinylated and loaded onto the SSA biosensors at 40 μg/mL in PBS containing 0.05% Tween-20 and 0.1% BSA. The biosensors were blocked with biocytin (5 μg/ml) for 60 s. Diluted IVM in PBS solution containing 0.05% Tween-20, 0.1% BSA and 10% DMSO was then added onto the SSA biosensors loaded with EGFR extracellular domain. The real time binding response (Δλ in nanometer, nm) between IVM and EGFR was calculated by subtracting the nonspecific binding of IVM to the SSA biosensors from the binding of IVM with EGFR. The kinetic parameters and affinities were calculated with a non-linear global fit of the data, using Octet data analysis software version 8.5 (ForteBio Inc., Menlo Park, CA, USA)
All experiments were repeated at least three times except that some WB experiments were repeated twice. Statistical significances for survival percentage results in NOD/SCID mice were determined using the log-rank test. In other cases, a one-way analysis of variance (ANOVA) followed by Dunnett’s test was used for multiple comparisons. Values of P < 0.05 were considered significant, and values of P < 0.01 were considered extremely significant. All data are mean ± SD unless otherwise indicated.