Heat stress tests were conducted at 37°C or 38.5°C in a hybridization oven (Thermo Electron, ThermoFischer Scientific, Reinach, Switzerland). The flies were raised at 25°C in bottles under optimal crowding conditions. Males were separated from females upon eclosion, staged for 3 days and transferred into empty plastic vials (no more than 30 flies per vial). The vials were sealed with foam plugs soaked in water to mimic a humid environment. The knockdown was scored over time. Both males and females performed equally; data presented for Rictor and Sin1 alleles are for males. For comparison to wild type, the Rictor precise excision allele was shown to perform similarly to the y w and Canton S strains. Oxidative stress (with 5% H2O2) and starvation tests were conducted as described previously (Jünger et al., 2003 (link)). Flies were collected as for heat stress tests.
Hybridization oven
Manufactured by Thermo Fisher Scientific
Sourced in Switzerland, Germany, United States
The Hybridization Oven is a laboratory equipment designed to provide a controlled environment for various hybridization techniques. It maintains a stable temperature and agitation to facilitate the hybridization of nucleic acid samples.
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Lab products found in correlation
Fluidics station, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Command console software 3, by Thermo Fisher Scientific (2 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (2 mentions)
Flashtag biotin hsr rna labeling kit, by Thermo Fisher Scientific (2 mentions)
Genechip human gene 1.0 st array, by Thermo Fisher Scientific (2 mentions)
Poly l lysine coated slide, by Merck Group (1 mentions)
Fmtm 4 64fx, by Thermo Fisher Scientific (1 mentions)
Axio observer z1, by Zeiss (1 mentions)
Agilent gene expression hybridization kit, by Agilent Technologies (1 mentions)
Gene expression wash buffer kit, by Agilent Technologies (1 mentions)
Feature extraction software, by Agilent Technologies (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Agilent microarray scanner, by Agilent Technologies (1 mentions)
Truseq rna sample preparation kit, by Illumina (1 mentions)
Genechip scanner, by Thermo Fisher Scientific (1 mentions)
Genechip eukaryotic poly a rna control kit, by Thermo Fisher Scientific (1 mentions)
Genechip expression hybridization controls, by Thermo Fisher Scientific (1 mentions)
12 protocols using hybridization oven
1
Stress Response Tests in Drosophila
2
Fluorescent Staining of Early Division E. faecalis
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Overnight cultures of E. faecalis OG1RF, EFC3C, and EFC3Py were normalized to OD600 = 0.5 in phosphate buffer saline (PBS, pH 7.4). COE1-3C or COE1-3Py was added to the cultures at the following concentrations: wild type E. faecalis OG1RF (1 and 0.5 μM, respectively), EFC3C (1 and 0.5 μM, respectively), and EFC3Py (4 μM of each), incubated for 60 min at 37°C and followed by addition of 10 μg/mL FMTM 4-64FX (ThermoFisher Scientific). After staining, the cells were washed three times in PBS, and 20 μL of cells was spotted onto poly-L -lysine coated slides (Sigma–Aldrich), dried at 46°C in a hybridization oven (ThermoFisher Scientific) for 15 min, mounted with Vectashield mounting medium (Vecalotor Laboratories, Inc.), and imaged using a Zeiss Axio Observer Z1 fitted with a 100×/1.30 oil immersion objective (Carl Zeiss, Göttingen, Germany).
Quantification of fluorescence distribution along the cell was performed using PSICIC (Guberman et al., 2008 (link)). Cells with a perimeter > 4.8 μm and ≤ 8 μm were defined as early division cells (Kandaswamy et al., 2013 (link)) and chosen for the quantitation of the fluorescence of FM4-64 and COEs. Fluorescence intensity along the circumference of the cell was plotted against the cell perimeter coordinates of 1–100 units to generate a fluorescence distribution profile. Quantitative analysis was performed in two independent experiments.
Quantification of fluorescence distribution along the cell was performed using PSICIC (Guberman et al., 2008 (link)). Cells with a perimeter > 4.8 μm and ≤ 8 μm were defined as early division cells (Kandaswamy et al., 2013 (link)) and chosen for the quantitation of the fluorescence of FM4-64 and COEs. Fluorescence intensity along the circumference of the cell was plotted against the cell perimeter coordinates of 1–100 units to generate a fluorescence distribution profile. Quantitative analysis was performed in two independent experiments.
3
Microarray Analysis of mRNA Expression
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For mRNA analysis, the array (OE BioTech) was performed using an Agilent expression profile gene chip (Agilent Technologies, Inc.). Two samples were allocated to the control and pressure groups. Total RNA was extracted using TRIzol® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol and quantified using NanoDrop 2000 (NanoDrop Technologies; Thermo Fisher Scientific, Inc.), and detected with an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc.). The Quick Amp Labeling kit (One-Color; Agilent Technologies, Inc.) was used to reverse transcribe total RNA to double-stranded cDNA, prepare the label reaction and transcribe cRNAs from cDNAs, all according to the manufacturer's protocol. Subsequently, an RNeasy Mini kit (Qiagen GmbH) was used to purify the labeled/amplified RNAs and quality control-labeled cRNAs. Each slide was hybridized with cyanine 3-labeled RNA using the Agilent Gene Expression Hybridization kit (Agilent Technologies, Inc.) in a Hybridization Oven (Thermo Fisher Scientific, Inc.) at 65°C for 17 h. After hybridization, slides were washed in staining dishes with Gene Expression Wash Buffer kit (Agilent Technologies, Inc.). Slides were scanned using the Agilent Microarray Scanner (Agilent Technologies, Inc.) and analyzed using the Feature Extraction software (version 10.7; Agilent Technologies, Inc.) with default settings.
4
OncoScan DNA Fragmentation and Hybridization
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Sample DNA was digested by the restriction enzymes NspI, and adapters were ligated to the fragment DNA to perform PCR amplification. Amplified DNA was labeled and further fragmented using an Affymetrix OncoScan array kit and reagent kit bundle, (Cat# 901835, Affymetrix, Santa Clara, CA, US) following the manufacturer’s instructions to obtain biotin-labeled DNA. Hybridization buffers were prepared, and array hybridization was performed at 49 °C in a Hybridization Oven (Cat# 00-0331-220V, Affymetrix, Santa Clara, CA, US). After 16 h of hybridization, arrays were washed in a Fluidics Station (Cat# 00-0079, Affymetrix, Santa Clara, CA, US) according to the manufacturer’s instructions. Arrays were scanned by a GeneChip® Scanner 3000 (Cat# 00-00212, Affymetrix, Santa Clara, CA, US) and Command Console Software 3.1 (Affymetrix, Santa Clara, CA, US) with default settings.
5
High-throughput lncRNA Microarray Analysis
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The purified RNA was used to construct cDNA libraries by using TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) according to manufacturer’s instruction. Following first and second cDNA strands synthesis, samples were then amplified and transcribed into fluorescent cRNA. The labeled cRNAs were further purified, and reverse transcribed to 2nd-cycle cDNA. After purification and fragmentation, 2nd-cycle cDNA was then labeled. The hybridization solution mixed with labeled cDNA, hybridization buffer and other buffers was divided into the gasket slide and arranged on the lncRNA expression microarray slide. The slides were incubated in Hybridization Oven (Affymetrix) at the temperature of 45 °C for 16 h. After hybridization, the arrays were washed, fixed, and last scanned by using a scanner (Affymetrix).
6
Affymetrix Gene Expression Profiling of CBX7 in Thyroid Cancer
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GeneChip Human Gene 1.0 ST Arrays (Affymetrix, Santa Clara, CA), consisting of 764,885 probe sets covering over 28,869 genes, was used to evaluate genes differentially expressed. The whole hybridization procedure was performed following the Affymetrix instructions. The amplification and labeling processes were verified using a GeneChip Eukaryotic Poly-A RNA Control Kit (Affymetrix) with exogenous positive controls. 15 µg of each biotinylated cRNA preparation was fragmented and placed in hybridization mixture containing biotinylated hybridization controls (GeneChip Expression Hybridization Controls, Affymetrix). Samples were then hybridized onto a GeneChip Human Gene 1.0 ST Array at 45 °C for 16 hours at constant rotation (60 rpm) in a Hybridization Oven (Affymetrix). Microarray scanned images were obtained with a GeneChip Scanner (Affymetrix) using the default settings. Images were analyzed with Affymetrix Gene Expression Analysis Software (Affymetrix). Comparisons were made between FRO-EV-1 and FRO-CBX7-1 samples, considering FRO-EV-1 as baseline. The fold change values, indicating the relative change in the expression levels between FRO-EV-1 and FRO-CBX7-1, were used to identify genes differentially expressed.
Microarray data are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress ) under accession number E-MTAB-2420.
Microarray data are available in the ArrayExpress database (
7
Microarray Analysis of miRNA Expression
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miRNA arrays were performed as described previously (Al-Ghezi et al., 2019 (link)). Purified miRNA samples isolated from testes and ovaries harvested from mice 72 h post-exposure to TCDD were labeled using the Affymetrix® FlashTag™ Biotin HSR RNA Labeling Kit using the manufacturer’s specifications for 100 format miRNA 4.0 arrays. GeneChip™ Hybridization, Wash, and Stain kit was used in conjunction with the Affymetrix® Hybridization Oven and Affymetrix® Fluidics Station on the 450 protocol to prepare Affymetrix® GeneChip® miRNA 4.0 arrays. Prepared arrays were then analyzed using the GeneChip™ Scanner 3,000 7G (All products in this section are sourced from ThermoFisher Scientific, Waltham, MA, United States 02451). Microarray intensity was quantified, and differential expression was analyzed using Applied Biosystems Transcription Analysis Console (TAC) 4.0 software. Expression data were analyzed using Ingenuity Pathway Analysis (IPA) and miRNA targets were determined using the current target prediction database miRDB (Chen and Wang, 2020 (link)), and target prediction algorithm, MirTarget as well as web-based TargetScan. MicroRNA raw data have been deposited to Geo database (accession number is GSE241576 and can be accessed by https://www.ncbi.nlm.nih.gov/geo/info/linking.html ) of National Institute of Health (NIH).
8
Microarray Analysis of SUM-159 Cell Line
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We used GeneChip Human Genome U133A 2.0 Array (Affymetirx) for the microarray analyses as described earlier45 (link). Total RNA was extracted from 1 × 107 SUM-159 cells stably transfected with empty vector or SH3GL245 (link) (in triplicates). Cells were re-suspended in 300 μL of Trizol and total RNA was isolated using the MagMAX™-96 for Microarrays Total RNA Isolation Kit (InvitrogenTM Life Technologies, Carlsbad, CA). Microarray analysis was performed in triplicate45 (link). Hybridization was done in Affymetrix hybridization oven followed by washing and scanning in Fluidic station 450 and Genechip Scanner respectively utilizing GCOS software. Data interpretation and analysis was done through GCOS manager software specifications. Increased or decreased expression of various genes demonstrated through the microarray analysis was presented in Table S1 . All data were submitted to Gene Expression Omnibus Data Base (GEO; #GSE110332).
9
miRNA Expression Profiling with GeneChip
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RNA samples were labelled using a FlashTag™ Biotin HSR RNA Labeling Kit (Affymetrix). The process begins with a brief tailing reaction followed by ligation of the biotinylated signal molecule to the target RNA sample. Afterward, the biotin-labelled RNA was hybridized onto a GeneChip miRNA 4.0 Array for 42 h at 49 °C using an Affymetrix Hybridization Oven. Using the Affymetrix GeneChip system, GeneTitan Arrays were washed and stained in an Affymetrix Fluidics Station 450 and scanned using an Affymetrix GeneChip Scanner 3000 System. The data were analysed with Expression Console Software using RMA analysis. A filtering step excluding probes not reaching the lower quartile of the coefficient of variation was employed, and the total number of obtained human miRNAs was 4993.
10
Affymetrix CytoScan HD Array for Genome-wide CNV Analysis
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The Affymetrix CytoScan HD array with 900,000 probes was applied for screening CNV genes and there were normal genes copy numbers on the slides as controls. For microarray detection, the sample DNA was digested by restriction enzymes Nspl, amplified by the PCR method, labeled and further fragmented to obtain biotin labeled DNA using the Affymetrix CytoScan HD array kit (Cat #901835, Affymetrix, Santa Clara, CA) following the manufacturer's instructions. The array hybridization was performed at 50°C in Hybridization Oven (Cat #00-0331-220V, Affymetrix, Santa Clara, CA). After 16 hours’ incubation, array slides were washed in Fluidics Station (Cat #00-0079, Affymetrix, Santa Clara, CA) according to the user manual. Then, slides were scanned with default settings by the GeneChip Scanner 3000 (Cat #00-00212, Affymetrix, Santa Clara, CA) and Command Console Software 3.1 (Affymetrix, Santa Clara, CA). Data were extracted and further analyzed with Affymetrix Chromosome Analysis Suite (Affymetrix, Santa Clara, CA).
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