Mas 5

For microarrays, standard methods were used for cDNA synthesis, fragmentation, and terminal biotin labeling based on Affymetrix protocols. Labeled cDNA was hybridized to the Affymetrix E. coli Genome 2.0 array. Hybridized arrays were stained with streptavidin-phycoerythrin using an Affymetrix Fluidic Station. After staining, arrays were scanned with an Affymetrix GeneChip Scanner 3000 based on the total signal intensity. The resulting microarray data were analyzed using Affymetrix software (MAS 5.0). Consensus “detection p-values”, “change p-values”, and “mean expression ratios” were calculated. All signal intensities with mean expression ratios above 2 were considered significant changes if the p-value was below 0.05. Complete microarray data have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE37780.

Three transcriptome data sets of AML patients, including TCGA,20 GSE1035821 and GSE1159,22 were used to compare the expression of LMO2 between APL and non‐APL patient samples. To perform interarray comparison, the CEL files were analysed by Affymetrix MAS 5.0 software (Affymetrix, Santa Clara, CA, USA). Two‐tailed t‐tests were used to validate the significance of the observed differences, which were considered statistically significant when P < 0.05.

Independent biological replicates of WT (n = 2), calr^−/− (n = 5), and calr truncation mutants NP (n = 3) and PC (n = 3) were cultured on 0.1 % gelatin-coated 10 cm dishes in 7.5 % FBS in DMEM supplemented with L-glutamine, NEAA, penicillin/streptomycin, LIF, and BME. Media was changed regularly every 24 or 48 h. Cells reaching ~80 % confluency were passaged 1:10 to maintain proliferative undifferentiated state of all cell lines, as previously performed [18 (link)]. Total RNA isolation from embryonic stem cells was performed using the Micro-to-Midi Total RNA Purification System (Invitrogen, Carlsbad, CA) for analysis of transcriptome dynamics. Double stranded complementary cDNA and labeled complementary cRNA were obtained from isolated total RNA, with the latter hybridized against the Mouse 430 2.0 GeneChip (Affymetrix). Arrays were scanned using an argon-ion laser, and visualized using MAS 5.0 Affymetrix software to assess quality of hybridization. Data were deposited to the Gene Expression Omnibus (GEO) repository under accession number GSE13805, with relevant updates.

The clinicopathological parameters of the cohort are summarised in Figure S5E,F. The GEO dataset GSE17025 [54 (link)] with heterogeneous distribution of grade and histological subtypes of EC cases (n = 91), was analysed. The GEOquery package [55 (link)] of R was used to retrieve the expression matrices and clinicopathological parameters of the datasets, the expression data of GSE17025 was normalised to a target intensity of 500 using Affymetrix’s MAS5.0 and was log2 transformed. Tumour grade 1 and 2 were grouped as low, grade 3 as high in the analysis.

PAO1, PAO1(p19), ΔbswR and ΔbswR(bswR) cultures were grown overnight in LB medium. Bacterial cells were then diluted to OD₆₀₀ of 0.05 and grown at 37°C with shaking at 250 rpm. When OD₆₀₀ reached ∼1.0, cells were harvested and total RNAs were extracted with RNeasy Protect Bacteria Mini kit (Qiagen). DNA contaminants were removed by DNase I. The quality of purified RNAs was analyzed by electrophoresis and quantified by Nanodrop 1000 Spectrophotometer (NanoDrop Technologies). Reverse transcription, fragmentation and complementary DNA labeling were conducted as described by the manufacturer (Affymetrix). The processed samples were hybridized to the Affymetrix GeneChip of P. aeruginosa, and chips were washed, scanned and analyzed following the instructions from the manufacturer. Hybridization signals were processed using the statistics software MAS-5.0 from Affymetrix.