Chelex

We adhere to the nomenclature rules for radiopharmaceutical chemistry (30 (link)). Panitumumab, trastuzumab, and cetuximab were obtained from the Memorial Sloan Kettering hospital pharmacy. Onartuzumab was provided by Genentech. The antibodies were conjugated with the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine (DFO-Bz-NCS; Macrocyclics) and then radiolabeled with ⁸⁹Zr in accordance with previously reported methods (28 (link)). The antibodies were conjugated with p-SCN-Bn-DFO in a 5:1 DFO:antibody molar ratio at 37°C for 90 min. After reaction, the conjugates were purified via a PD-10 column using Chelex (Bio-Rad) phosphate-buffered saline (0.5 g/L Chelex resin) at pH 7.4. The ⁸⁹Zr-oxalate (supplied in 1.0 M oxalic acid at Memorial Sloan Kettering Cancer Center (28 (link))) was neutralized to pH 7.0–7.5 with 1.0 M Na₂CO₃ followed by addition of the corresponding DFO–antibody conjugate in Chelex phosphate-buffered saline (pH 7.4). The mixture was incubated at 37°C for 1 h on an agitating heating block. [⁸⁹Zr]Zr-DFO-antibody and radiochemical purity was determined by instant thin-layer chromatography, and the product was used for in vivo studies.

HCT116 cells were treated with 5 μM Δ⁹-THC or 5 μM Δ⁹-THC plus 50 μM BODIPY-THIF for 6 h, cross-linked with 2% formaldehyde for 15 min, collected by scraping in PBS and then centrifuged and lysed in 1 mL of IP buffer (150 mM NaCl, 50 mM Tris–HCl pH 7.5, 5 mM EDTA, 0.5% Nonidet P-40 and 1% Triton X-100) containing protease inhibitors. The obtained nuclear pellet was resuspended in IP buffer and sonicated. ChIP assays using control rabbit IgG (Santa Cruz) or anti-STAT1 antibodies (Cell Signaling Technology, Danvers, MA, USA) were performed. The immunoprecipitated DNA and input DNA were extracted by incubating the samples with 100 μL of 10% Chelex (Bio-Rad Laboratories, Hercules, CA, USA), then boiling them to reverse the cross-linking and then centrifuging them to remove the Chelex slurry. PCR was performed using two oligonucleotide primers as follows: GDNF (forward primer, 5’-CAGCATGGAAATGAAGCCTA-3’; reverse primer, 5’- TAGTTTAGTCCCCAGGCTAG-3’); IGFBP6 (forward primer, 5’- TGCTGACAATGAGGTTCGTAT-3’; reverse primer, 5’- GTTATGCAACAGGGACCATC-3’); IGF2 (forward primer, 5’- CTGAATTCTCTAGAACGGGCATTCAGCA-3’; reverse primer, 5’- GGGGGCAGGGAGCCGCAGAG-3’); SCF (forward primer, 5’-ATAGGCTAGCAGCACAGACTTCCCTCCACAAAGT-3’; reverse primer, 5’-CATGGAAGCTTTGTGGCGACTCCGTTTAGCT-3’); and VEGFA (forward primer, 5’-GCGTGTCTCTGGACAGAGTTT-3’; reverse primer, 5’- AGCCTCAGCCCTTCCACA-3’).

A549 cells were treated with 5 μM WJ for 24 hours, cross-linked with 1.42% formaldehyde for 15 minutes, collected by scraping in PBS and then centrifuged and lysed in 1 ml of IP buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.5% Nonidet P-40 and 1% Triton X-100) containing protease inhibitors. The obtained nuclear pellet was resuspended in IP buffer and sonicated. The sonicated lysates were immunoprecipitated with an H3K27-ac or an H3K27-me3 antibody, and the immune complexes were recovered using protein A-Sepharose (Roche). The immunoprecipitated DNA and input DNA were extracted by incubating the samples with 100 μl of 10% Chelex (Bio-Rad), then boiling them to reverse the cross-linking and centrifuging them to remove the Chelex slurry. For ChIP-on-chip assays, the DNA was further purified through phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. Real-time PCR was performed with the purified DNA using the following primers: 5′-GCCAACCTCCCGCCCACAAG-3′ and 5′-GTCTTCCCCGCCCCTA CGCC-3′.

Cells were cross-linked with 1.42% formaldehyde for 15 min and quenched unreacted formaldehyde with 0.125 M Glycine solution for 5 min. Cells were scraped in 1 ml of cold PBS, centrifuged, and lysed in 1mL of IP buffer (150mM NaCl, 50mM Tris-HCl, pH 7.5, 5mM EDTA, 0.5% Nonidet P-40, and 1% Triton X-100) containing protease inhibitors (1mM phenylmethylsulfonyl fluoride, 1μM eupeptin and 1μM aprotinin). The nuclear pellet was resuspended in IP buffer and sonicated to shear chromatin. The sonicated lysates were immunoprecipited with antibodies against phospho-H3^ser10 followed by the pull-down with protein A/G-Sepharose (Thermo). The immunoprecipitated DNA and input DNA were extracted by incubating with 100 μl of 10% Chelex (Bio-Rad), boiling to reverse the cross-link, and centrifuging to remove Chelex slurry. RT-qPCR was performed with the purified DNA using specific primers as shown in Supplementary Table.

Targeted NGS was performed on fresh and formalin-fixed paraffin-embedded tumour tissue (FFPE). For fresh tissue, DNA was isolated using the QIAamp DNA mini kits (Qiagen, Hilden, Germany) according to the manufacturer’s specifications. For FFPE tissues, DNA was isolated using lysis buffer (Promega, Madison, WI) and 5% Chelex (Bio-Rad), as reported previously (Smit et al. 2018 (link)). NGS was performed using the IonTorrent (Thermo Fisher Scientific, Waltham, MA) platform with a custom panel consisting of, among others, GNAQ, GNA11, EIF1AX, SF3B1, and BAP1, as reported previously (Smit et al. 2018 (link)).

For diagnosis of SeMNPV contamination, genomic DNA (gDNA) was extracted from S. exigua larvae using 5%Chelex (Bio-Rad, USA). For diagnosis of SeIfVs, RNA samples were extracted from the whole body or different tissues (hemocytes, fat body, midgut, and epidermis) of L5 larvae of S. exigua using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. Extracted RNA was resuspended in nuclease-free water and quantified using a spectrophotometer (NanoDrop, Thermo Scientific, USA). RNA (1 μg) was used for cDNA synthesis with an RT PreMix (Intron Biotechnology, Korea) containing oligo dT primer according to the manufacturer’s instructions. PCR was conducted using DNA Taq polymerase (GeneALL, Korea) with gene-specific primers (Table S1). After heat treatment at 94°C for 5 min, PCR was performed with 40 temperature cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 1 min. A ribosomal protein, RL32, was used as a reference gene to confirm cDNA preparation.