cDNA encoding for murine Slc16a5 (NM_001080934.1) was purchased from Thermo Fisher Scientific (GeneArt, Rockford, IL, USA). The sequence was designed and optimized to contain 5′ (XmaI) and 3′ (XbaI) restriction sites to flank the open reading frame, a Kozak consensus sequence prior to the start codon, and a C-terminal FLAG tag for immunodetection. Briefly, cDNA was amplified using 5′ and 3′ primers prior to the restriction sites using reverse transcription polymerase chain reaction (RT-PCR) on a BioRad CFX Connect™ RT System. The PCR reaction was purified, and the cDNA and previously used oocyte expression vector (pGH19) [7 (link),34 (link)] were double-digested with XmaI and XbaI. The digested PCR product and linearized vector were further purified and ligated using a T4 DNA ligase reaction mixture. The resulting construct was transformed into chemically competent TOP10 Escherichia Coli cells and the plasmid were isolated, purified, and confirmed via sequencing. The pGH19-mMct6 vector without the FLAG tag was generated using site-directed mutagenesis and used for all activity studies. The pGH19-hMCT6 vector was used and prepared similarly to that described previously [7 (link)].
Cfx connect rt system
Manufactured by Bio-Rad
Sourced in United States
The CFX Connect RT System is a real-time PCR detection system designed for amplification and detection of nucleic acid sequences. It features a compact footprint and supports 96-well microplates or 8-tube strips.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy extraction kit, by Qiagen (2 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Ssoadvanced sybr green supermix, by Bio-Rad (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Bio-Rad (1 mentions)
Rnaeasy plus mini kit, by Qiagen (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Taqman pcr master mix, by Thermo Fisher Scientific (1 mentions)
Rneasy plus kit, by Qiagen (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
Epoch plate reader, by Agilent Technologies (1 mentions)
Miscript 2 rt kit, by Qiagen (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
8 protocols using cfx connect rt system
1
Generation of mMct6 Expression Construct
2
Quantitative RNA Expression Analysis
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RNA was extracted using the RNeasy Extraction Kit (Qiagen, Hilden, Germany) according to manufacturer’s protocol. The TaqMan PCR assay was performed with a 7500 Fast Real-Time PCR System using the universal TaqMan PCR master mix (ThermoFisher, Waltham, MA, USA) and FAMTM-labeled probes for KYNU (Hs00187560_m1) and KEAP1 (Hs00202227_m1) and VICTM-labeled probes for β2M (Hs_00187842_m1). PCR was carried out using a BioRad CFX Connect RT System. Values are reported as 2−∆∆Ct.
3
Quantitative analysis of gene expression
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HeLa Kyoto cells (100,000 cells/well), plated the day before in 6 well plates, were treated with drugs diluted to 10 μM (or 20 μL for N-p Tosyl-L-PCK, Supercynnamaldehyde, and Raloxifene), along with Emetine-treated and untreated cells. Eight hours later, cells were lysed in 350 μL RLT buffer (RNAeasy Plus Mini Kit, Qiagen, UK) supplemented with beta-mercaptoethanol (1:100). Cells were harvested, homogenised with a G25 needle, and their RNA extracted following the manufacturer’s instructions. RNA (500 ng) was reverse-transcribed using Quantitect Reverse Transcription Kit (Qiagen). Complementary DNA (cDNA) was diluted 1:5 in deionised water and quantitative real-time polymerase chain reaction (qRT-PCR) was performed on 2 μL of cDNA using Sybr Green (BioRad, UK) to amplify the endogenous housekeeping genes actin and hypoxanthine- guanine phosphoribosyltransferase (HPRT). Samples were then run on a CFX Connect RT System using the Biorad CFX Manager program. The conditions for qRT-PCR were: 95 °C for 7 min; 40 cycles at 95 °C for 10 s, 56 °C for 30 s and 72 °C for 40 s. Data were converted to CT values and RNA expression was compared to untreated controls. Data were normalised to transcription in DMSO-treated control cells (100%, dotted line) using Microsoft Excel. Statistics were performed in GraphPad Prism using one-way Anova, Fisher’s LDS test.
4
qPCR Analysis of Inflammation and Cancer Markers
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Real-time polymerase chain reaction (qPCR) was used to examine the expression of genes associated with inflammation and cancer progression. Total RNA was isolated from the cells using TRIzol reagent (Invitrogen) according to manufacturer's instructions. 1 μg of RNA was reverse transcribed into complementary DNA (cDNA) to be used for qPCR. qPCR was performed using SsoAdvanced SYBR Green Supermix and CFX Connect RT System (BioRad). The following primers were used for qPCR: Actin, Forward primer (FP): CCCCGCGAGCACAGAG, Reverse primer: ATCATCCATGGTGGTGAGCTGGC; AIM2, FP: CTGCAGTGATGAAGACCATTC, RP: CA TTTCTGATGGCTGCAGATG; CASPASE-1, FP: CAGC ACGTTCCTGGTGTTC, RP: GATGATCACCTTCGGTT TGTC; CD44, FP: CAACCACAAGGATGACTGATG, RP: CTTCTGGGTAGCTGTTTCTTC; CTGF, FP: CCAAC TATGATTAGAGCCAACTG, RP: GTTCTCTTCCAGGT CAGCTTC; IL-1β, FP: GTGGCAATGAGGATGACTTG, RP: GCAGGGAACCAGCATCTTC; IL-6, FP: AGGAG ACTTGCCTGGTGAAA, RP: CAGGGGTGGTTATTG CATCT; IL-10, FP: GCTCAGCACTGCTCTGTTG, RP: CACTCTGCTGAAGGCATCTC; MCP-1, FP: ATGAAA GTCTCTGCCGCCCTTC, RP: CTGCTTGGGGTCAGC ACAGATC; MMP9, FP: CAACTACGACCGGGACAAG, RP: CTTCTTGTCGCTGTCAAAGTTC; PLAU, FP: CCA AAGAAGGAGGACTACATC, RP: GATCTTCAGCAAG GCAATGTC; PLA2G7, FP: GTCTTGGCTCTACCTTA GAAC, RP: CTTCACTGGCTTTCCATGATC; TNFα, FP: TCCTTCAGACACCCTCAACC, RP: AGGCCCCAGTTT GAATTCTT; VEGFα, FP: CAGCTACTGCCATCCAAT CG, RP: GCATAATCTGCATGGTGATGTTG. Actin was used as the internal control.
5
Quantitative RT-PCR Analysis of DUSP16
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Total RNA was isolated from the cells using TRIzol reagent (Invitrogen) according to manufacturer’s instructions. One microgram of RNA was reverse transcribed into complementary DNA (cDNA) to be used for qPCR. qPCR was performed using SsoAdvanced SYBR Green supermix and CFX Connect RT System (BioRad) to examine the expression of DUSP16 with ACTIN as internal control using respective primer pairs (Supplementary Table 2 ).
6
Quantitative RNA Expression Analysis
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RNA was extracted using the RNeasy Extraction Kit (Qiagen, Germantown, MD, USA) according to manufacturer’s protocol. TaqMan PCR assay was performed with a 7500 Fast Real-Time PCR System using universal TaqMan PCR master mix (ThermoFisher, Waltham, MA, USA) and FAMTM-labeled probes for KYNU (Hs00187560_m1) and KEAP1 (Hs00202227_m1) and VICTM-labeled probes for ß2M (Hs_00187842_m1). PCR was carried out using a BioRad CFX Connect RT System (Hercules, CA, USA). Values are reported as 2−ΔΔCt.
7
RNA Extraction from Yeast and Bacteria
8
Quantitative miRNA Expression Analysis
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RNA was extracted from cells using miRNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. The RNA quantification was performed using Epoch plate reader (BioTek) and RNA concentration was adjusted to 20 ng μl−1. Reverse transcription for all RNA extracts was performed using the miScript II RT kit (Qiagen, Germany) according to the manufacturer's instructions using the 96-well T100 thermal cycler (Bio-Rad, Hercules, CA). The cDNA was further utilized as a template for real-time PCR with the miScript SYBR Green PCR Kit and miScript Primer Assays (Hs_RNU6-2_11, MMu_miRNA-129-5p; Qiagen Germany). Quantitative RT-PCR (qRT-PCR) was performed using CFX Connect RT system (Bio-Rad, Hercules, CA) with the following parameters:47 (link) 95 °C for 15 min, then 40 cycles of 94 °C for 15 s, 55 °C for 30 s and 70 °C for 30 s. The results of RT-PCR are presented in Fig. 5c .
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