Fig 2. Respiratory airway cultures induce a heat stress response upon incubation at elevated temperature.

Ciliated respiratory cultures differentiated from HBEp cells isolated from 3 independent donors (donor 1, male Caucasian aged 63 years; donor 2, Hispanic male aged 62 years; donor 3, Caucasian female aged 16 years; all nonsmokers) were incubated at 37, 39, or 40°C for 72 h prior to fixation or RNA extraction and RNA-Seq. (A) Representative H&E stained tissue sections (donor 1) at 72 h. Scale bars = 20 μm. (B) TEER (Ohm per cm²) measurements of respiratory epithelia integrity (donor 1) at 37 or 40°C. N = 3 tissues per condition; lines, mean; all data points shown; p-values shown, unpaired two-tailed t test. (C) ACE2 expression levels (CPM) in respiratory cultures incubated at 37 or 40°C; means and SD shown; p-value shown, unpaired two-tailed t test. (D) Expression values (log2 CPM) of a reference gene set (24 genes; [34–36]) in respiratory cultures incubated at 37 or 40°C; p-value shown, paired two-tailed t test. (E) Scatter plots showing high confidence (Q < 0.05) DEG transcripts identified between respiratory cultures incubated at 37 or 40°C; up-regulated DEGs, red circles; down-regulated DEGs, blue circles. (F) Reactome pathway analysis of mapped up-regulated DEGs. Top 30 pathways (FDR < 0.05; plotted as −log10 FDR) shown. Dotted line, threshold of significance (−log10 FDR of 0.05). (G) Expression values (log2 CPM) of DEGs associated with cellular response to heat stress pathway (R-HSA-3371556; arrow in F); p-value shown, paired two-tailed t test. (C to H) RNA-Seq data derived from RNA isolated from 3 donors (donors 1 to 3) per sample condition derived from 3 independent experiments per donor. Raw values presented in S1 and S9 Data. ACE2, angiotensin-converting enzyme 2; CPM, counts per million; DEG, differentially expressed gene; FDR, false discovery rate; HBEp, human bronchiolar epithelial; H&E, hematoxylin and eosin; TEER, transepithelial electrical resistance.