Heat Shock Proteins

The pSSPF2/PfHsp60-GFP-Link plasmid described in [23] was used to construct transfection plasmids. Overlapping synthetic oligonucleotides corresponding to the wild-type and “M9” mutated B. subtilis glmS ribozyme sequences reported in [21] (link) were combined in gene synthesis-PCR and the resulting 166 bp ribozyme element cloned at the 3′-UTR position downstream of the GFP gene in pSSPF2/PfHsp60-GFP-Link via the XhoI and PstI sites. Oligonucleotides syn1-syn8 were used to construct wild-type glmS and oligonucleotides syn1-syn7 and syn9 were used to construct the M9 variant (Table S1).
The ribozyme sequences are positioned upstream of the Plasmodium berghei dihydrofolate reductase-thymidylate synthase 3′-transcription terminator sequence (PbDT-3′). The resulting plasmids pGFP_glmS and pGFP_M9 express GFP with a mitochondrial transit peptide from P. falciparum heat shock protein 60. This open reading frame is under the control of 5′ and 3′ flanking sequences of P. falciparum heat shock protein 86 (Pfhsp86 5′) and PbDT-3′, respectively. The P. falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) gene was amplified by PCR from a previously described plasmid [24] (link) using oligonucleotide primers dhfr-F and dhfr-R and cloned into pGFP_glmS and pGFP_M9 via the unique BglII and KpnI sites. The resulting plasmids pDHFR-TS-GFP_glmS and pDHFR-TS-GFP_M9 for study of episomal reporter gene activity (Fig. 2 and 3) contain a PfDHFR-TS-GFP open reading frame under the control of Pfhsp86 5′, glmS ribozyme and PbDT-3′ flanking sequences. The pJRTS_GFP_glmS plasmid for integration at the endogenous P. falciparum PF3D7_0417200 locus encoding PfDHFR-TS was made by restriction digestion of pDHFR-TS_GFP_glmS with SpeI, followed by re-ligation of the plasmid backbone.

PCR was performed using the touchdown protocol described by Sargent et al. [35 (link)] in a 20 μl reaction containing HotStart Taq Master Mix (Qiagen, Valencia, CA), 0.4 μM each primer, and 1.0 ng genomic DNA. PCR products were separated by electrophoresis through a 1.5% TAE agarose gel and visualized by ethidium bromide staining. Primers were designed to span the intron in the N-terminal domain of a low molecular weight heat shock protein gene identified as an EST (GenBank accession number CX661743.1) from a "Yellow Wonder" (Y2) heat-treated seedling cDNA library. Template DNA for Y1, Y2, F. iinumae J-17, and F. vesca subsp. americana Pawtuckaway was obtained from 50-100 mg young leaf tissue using a DNeasy Plant Mini kit (Qiagen). Y1 and F. vesca subsp. americana Pawtuckaway plants were obtained from T. Davis (University of New Hampshire) and F. iinumae J-17 plants were obtained from the US National Plant Germplasm collection.

Colon tissues were homogenized in PRO-PREP™ Protein Extraction Solution (Intron, Seongnam, Korea) and mixed with 5× sample buffer (Genscript, Piscataway, NJ, USA) containing a protease inhibitor (Gendepot, Katy, TX, USA) and phosphatase inhibitor (Gendepot). The protein concentration was quantified using the BCA protein assay (Abbkine, Wuhan, China). The antibodies used for western blotting were PERK (clone C33E10), p-PERK (clone 16F8), eIF-2α (9722 S), p-eIF-2α (9721 S), GRP78/BiP (clone C50B12), CHOP (clone L63F7), IKK (2682 S), p-IKK (clone 16A6), IκB (9242 S), NF-κB p65 (clone D14E12), p-NF-κB p65 (clone 93H1), and anti-rabbit IgG-HRP (7074P2), all of which were purchased from Cell Signaling Technology (Danvers, MA, USA); ATF4 (clone H-290), β-actin (clone C4), and ERdj5 (clone 66.7), which were purchased from Santa Cruz Biotechnology; and anti-mouse IgG-HRP (ADI-SAB-100-J), which was purchased from Enzo Life Sciences (NY, USA). Protein bands were visualized using a chemiluminescence detection system for horseradish peroxidase (GBioscience, St. Louis, MO, USA).
The lysates of MODE-K cells were used to determine the expression of ER stress marker proteins, including PERK, p-PERK, eIF-2α, and p-eIF-2α, using western blotting. MODE-K cells were activated with Pam₃CSK₄ for a specified time, and NF-κB pathway signaling was analyzed. MODE-K cells were treated with 5 µM GSK 2606414 (Tocris), a PERK inhibitor, for 2 hr before Pam₃CSK₄ treatment.

pHBEC were obtained via bronchial brushing from healthy, donors ages 18–40 that were not currently smoking and had no more than a one pack-year of lifetime smoking history. Donors gave their consent after being informed of risks and procedures. The consent and collection protocol were approved by the UNC School of Medicine Committee on the Protection of the Rights of Human Subjects and by the US EPA. Collection of pHBEC from volunteers was performed in accordance with relevant guidelines and regulations. pHBEC were isolated from brush biopsy (“passage 0”) and expanded to passage 3 prior to being plated on 12 mm Transwell^® inserts (Corning #3460; 0.4 μm pore polyester membranes), becoming confluent, and differentiating for 24 days under ALI conditions. Detailed descriptions of the techniques, reagents, and materials used for the culture/expansion of pHBEC and differentiation of pHBEC at ALI used in this study are available as open access methods documents¹³ . Day 24 ALI cultures were visually evaluated for the presence of beating cilia and the production of mucus as indicators of differentiation status to qualify their use in experiments. Donor demographics are listed in Supplementary Table 1. All cultures were at ALI day 24 at the beginning of experiments. The human lung fibroblast cell line IMR90^{14 (link)} was obtained from the American Type Culture Collection (ATCC, No. CCL-186, Batch #64155514), and cultured as described in detail in the open-access methods document¹⁵ . Short tandem repeat (STR) service provided by ATCC (cat #135-XV) was used to authenticate IMR90 cells (Supplementary Fig. 1). As described in Fig. 1A, IMR90 fibroblasts were seeded onto a collagen-coated 12-well plate (Corning, #3513) at a density of 1×10⁵ cells/mL in 800 μL of Pneumacult ALI medium (StemCell Technologies, #05001). The following day, day 23 dpHBEC-ALI inserts were added into the fibroblast-seeded wells (dpHBEC-IMR90 ALI model). The dpHBEC-IMR90 ALI model was used for experiments on the following day. The dpHBEC-IMR90 ALI model was maintained at ALI or subjected to the addition of 250 μL (223 μL/cm²; equal to 73.5 μL in a 6.5 mm Transwell insert) of ALI medium to the apical surface of the culture (“liquid application”). This was the smallest volume of ALI medium that resulted in the most uniform coverage of the dpHBEC cultures during the 24-hour treatment duration as determined by a titration of apical volumes of a 0.5% Crystal Violet (Sigma, #C6158–50G; dissolved in Dulbecco’s Phosphate Buffered Saline (Gibco #14190–144)) stain solution to 12 mm Transwell inserts (Fig. 1B). Inserts were placed on a white light transilluminator (FUJIFILM #IPE4046) and photographed with an iPhone SE (Apple, Model #MX9M2LL/A). The cells were kept submerged in ALI medium for either 6 or 24 hours to represent “early” and “late” effects, respectively. Additionally, the 24-hour treatment is consistent with the liquid application treatment duration utilized in the recent OECD case study on the use of an ALI-differentiated in vitro NAM for an IATA to refine inhalation risk assessment for point of contact toxicity^{12 (link),16 –18} . The effect of liquid application on global gene expression, stress-responsive signaling protein phosphorylation, growth factor secretion, pro-inflammatory cytokine secretion, trans-epithelial electrical resistance (TEER), and permeability to a 20 kDa fluorescent dextran was then determined immediately after 6 and 24 hours of liquid application (Fig. 1A).