Pcr primers

Total RNA was extracted from cells using the RNeasy™ Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions^{6 (link)}. First-strand synthesis and RT-PCR were performed using the QuantiTect™ Reverse Transcription Kit (Qiagen) and Rotor-Gene™ SYBR Green PCR Kit (Qiagen), according to the manufacturer’s protocol. Amplification and detection were performed using Rotor-Gene™ Q (Qiagen). PCR primers were purchased from Qiagen. The change in expression of each target mRNA was calculated relative to the level of 18S rRNA.

Genomic DNA was extracted from a single specimen with DNeasy tissue kit (Qiagen, Hilden, Germany) following the directions of the manufacturers. The primer pairs 16SA/16SB [28 (link)] and LCO1490/HCO2198 [29 (link)] were used to PCR amplify a 512-base pair (bp) fragment of 16S and a 512-bp fragment of COI, respectively. Primer pair 28SF/28SR [30 (link)] was used to obtain sequences from 28S (1022 bp). For the amplification of 769 bp of EF1α, the primer pair HaF2For1/2R53ST [10 (link)] was used. The PCR amplifications followed Schwentner et al. [11 (link)]. The PCR products were gel purified using the QIAquick Gel Extraction Kit (Qiagen) and then sequenced in both directions with PCR primers by Sangon Biotech Co. (Shanghai, China). All sequences were submitted to GenBank (accession numbers MZ313265-MZ313268 and MZ318668-MZ318671; Table S1).

Total RNA was extracted from cells using the TaKaRa FastPure RNA kit (Takara Bio, Ohtsu, Japan), according to the manufacturer’s instructions. First-strand synthesis and RT-PCR were performed using the One-step SYBR PrimeScript RT-PCR kit (Takara Bio), according to the manufacturer’s protocol. Amplification and detection were performed using the Applied Biosystems 7300 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). PCR primers were purchased from Qiagen. The change in expression of each target mRNA was calculated relative to the level of 18 S rRNA^{44 (link)}.

Total RNA was extracted from A549 cells using the TaKaRa FastPure RNA kit (Takara Bio, Ohtsu, Japan), according to the manufacturer’s instructions. First-strand synthesis and RT-PCR were performed using the One-step SYBR PrimeScript RT-PCR kit (TAKARA, Ohtsu, Japan), according to the manufacturer’s protocol. Amplification and detection were performed using the Applied Biosystems 7300 Real-time PCR System (Applied Biosystems, Foster City, CA). PCR primers were purchased from Qiagen. The change in expression of each target mRNA was calculated relative to the level of 18S rRNA47 (link)48 (link).

Ursolic acid (purity ≥ 98%), thiobarbituric acid (TBA), phenazinemethosulphate (PMS), nitroblue tetrazolium (NBT), 5,5-dithiobis 2-nitrobenzoic acid (DTNB), 3-(4, 5-dimethyl-2-thiaozolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT), 2′7′-diacetyl dichlorofluorescein (DCFH-DA), rhodamine-123, and nicotinamide adenine dinucleotide (NAD) were supplied by Sigma-Aldrich, St. Louis, USA. The mouse monoclonal antibodies anti-TNF-α, anti-NF-κB (p65), anti-p53, anti-Bax, anti-Bcl-2, anti-caspase-3, anti-MMP-2, anti-MMP-9, anti-β-actin, and goat anti-mouse IgG-HRP polyclonal antibody were also purchased from Sigma-Aldrich, St. Louis, USA. RNA isolation kit, cDNA synthesis kit, PCR primers, and PCR master mix were purchased from Qiagen, USA. Bovine serum albumin (BSA), RIPA buffer, low melting agarose (LMPA), normal melting agarose (NMPA), phosphate buffered saline (PBS) and reduced glutathione (GSH) were purchased from Merck, India. All other chemicals, gradient solvents, and other analytical grades were acquired from S.D Fine Chemical and Himedia, India.

Genomic DNA of P. aeruginosa CF isolates was extracted using a DNA Isolation Kit (Qiagen). Primers used for PCR amplification and DNA sequencing are listed in Table S5. PCR amplifications were performed with the following conditions: 8 min at 95°C, 33 cycles of 1 min at 94°C, 1 min 20 sec at 60°C, 2 min at 72°C, and a final extension of 10 min at 72°C. PCR products were cleaned with a Gel Purification Kit (Qiagen), and both strands were sequenced directly using the same PCR primers (DNA Sequencing Facility, Univ. of Chicago, IL, USA). To score mutations within the gene, sequencing results were compared with the corresponding gene sequence of strain PAO1 (www.pseudomonas.com) using the BLAST program of the NCBI database (www.ncbi.nlm. nih.gov/blast/).

CD4 T cells were isolated using Human T cell Enrichment Cocktail (STEMCELL Technologies, Canada), followed by purification of naive (CD3⁺CD4⁺CD62L⁺CD45RA⁺CD28⁺), CM (CD3⁺CD4⁺CD62L⁺CD45RA⁻CD28⁺), and effector memory (CD3⁺CD4⁺CD62L⁻CD45RA⁻CD28⁺) subsets by fluorescence-activated cell sorting. ATAC-seq libraries were generated using 50,000 cells for each subset. Cells were first washed with cold PBS and RSB buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl₂), followed by washing with RSB buffer containing 0.1% NP-40 and 0.1% Tween-20. Subsequently, cell pellets were resuspended in a transposase reaction mix (25 μl 2× TD buffer, 2.5 μl transposase (Illumina), and 22.5 μl nuclease-free water) and incubated at 37 °C for 30 min. DNA fragments were purified using Qiagen MiniElute Kit and the library was amplified with Nextera PCR primers. Library quality was checked on bioanalyzer and sequenced on Illumina NextSeq 500. The sequencing reads were processed by trimming adapters and low-quality reads using in-house scripts and aligned to human reference genome hg19. Peaks were identified for each sample using macs2 and a consensus peak set was determined consisting of peaks present in at least three samples. Reads were converted into bigwig format for visualization of the genomic tracks, which were normalized to total reads mapped within the consensus peak set⁶⁴ .