Imagene software

Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and 1 μg RNA was labeled with the Cy3‐TM ULS labeling kit (Kreatech Biotechnology, Amsterdam, the Netherlands) following the instructions. The RNA was hybridized using the LNA‐based capture probe set 10 (Exiqon, Vedbaek, Denmark) consisting of 1344 probes with 725 human miRNAs (Platform number: GPL16851). Spots were quantified through a Imagene software (BioDiscovery, El Segundo, CA, USA), and quantile normalization was performed. The average miRNA expression in the DDP‐sensitive cell line was compared with that in DDP‐resistant cell line.

The expression profile of profibrotic signalling proteins was analysed with Jak/Stat Pathway Phospho and TGF-β signalling antibody arrays (U.S. Biomax, Inc.). Blocking, labelling, washing and fluorescence detection steps were performed according to the manufacturer's instructions. Slides were scanned with an Agilent DNA Microarray Scanner Model G2505B (Agilent Technologies, Inc., Santa Clara, CA, USA) and fluorescence intensity was measured by Imagene software (BioDiscovery, Marina del Ray, CA, USA).

ABH arrays were generated as outlined previously (63 (link)). For galectin recognition of glycans on the printed glycan microarray, slides were incubated with varying concentrations of Gal-9, Gal-9N, or Gal-9C directly labeled with Alexa Fluor 647 in PBS-T for 1 h at room temperature in a dark and humid chamber. The slide was washed by successive immersion in PBS-T (four times), 1× PBS (four times), and distilled water (four times). The slide was dried by microcentrifugation, and an image of bound fluorescence was obtained using a microarray scanner (Scan Array Express). Integrated spot intensities were determined using Imagene software (BioDiscovery). Apparent K_d values were calculated using GraphPad Prism version 9.0.2 (GraphPad Software, LLC).

Medium was collected from 24 h cultures of KHOS, KRIB, HOS and U2OS cells grown in serum-reduced conditions. Medium was collected, centrifuged, filtered and incubated on a cytokine antibody array (RayBio Biotin Label-based Human Antibody Array 1, 507 proteins) (RayBiotech, GA, USA). ImaGene software (BioDiscovery Inc, CA, USA) was used to quantify the spot intensities on array membranes. Sample intensities were corrected against positive and negative controls on the array and expressed in arbitrary units.

The interaction of CLEC5A.Fc with the microbial glycan array was determined in Dr. Richard Cumming’s laboratory, as described previously^{42 (link)}. The Fc-tagged CLEC5A sample and IgG control were diluted to 200 mg/ml in TSM binding buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, 0.05% v/v Tween-20, and 1% w/v BSA), and 100 ml was added to the surface of the Microbial glycan microarray (MGM) slide. After placing a coverslip over the surface, samples were incubated for 1 h at room temperature in the dark. Slides were washed 4× in TSM wash buffer 1 (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, 0.05% v/v Tween-20) and 4× in TSM wash buffer 2 (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂). Detection of binding was achieved with Alexa Fluor-488-labeled anti-human IgG (Invitrogen) diluted to 5 mg/ml in TSM binding buffer on the slide for 1 h at room temperature in the dark. Slides were washed 4× in TSM wash buffer 1, 4× in TSM wash buffer 2, and 4× in water. Binding was quantitated with a microarray scanner (Scan Array Express, PerkinElmer Lifer Sciences) and processed in terms of average relative fluorescence intensity (RFU) of 4 replicates using ImaGene software (BioDiscovery).

Total RNA was isolated with RNA Bee (BioConnect, Huissen, the Netherlands), and 1 μg RNA was labeled with the Cy3-TM ULS labeling kit (Kreatech Biotechnology, Amsterdam, The Netherlands), according to the manufacturer’s instructions. The RNA was hybridized with the LNA-based capture probe set (Exiqon, Vedbaek, Denmark) version 10 (annotation version 13). This probe set consists of 1344 probes including 725 human miRNAs, which are spotted in duplicate. Spots were quantified with the Imagene software (BioDiscovery), obvious outliers were removed and quantile normalization was performed.
miRNA profiling experiments were performed with RNA isolated at two (HCT8 and T24) or three (A2780) different passages. The average miRNA expression in the sensitive cell line was compared with the average expression in the resistant cell line and the fold-change was calculated. The microarray expression data has been deposited to the Gene Expression Omnibus (GEO) data repository (accession number GSE54665).