Fig 1: NAC alleviated oxidative stress and UPR in CSE-exposed Calu-3 cells. (A) The protein levels of IRE1α, p-IRE1α, XBP1s and IP3R measure by Western blot after NAC treatment. (B) Total ROS were measured in Calu-3 cells after NAC treatment. Scale bars: 100 µm. (C) Treatment with NAC decreased the levels of intracellular Ca2+. (D) Quantitative analysis of IRE1α and p-IRE1α levels by Western blot. (E) Quantitative analysis of XBP1s levels by Western blot. (F) Quantitative analysis of IP3R levels by Western blot. (G) Quantitative analysis immunofluorescence intensity of ROS levels. (H) Relative immunofluorescence intensity by flow cytometry after treatment with NAC. (I) The expression of MUC5AC in culture supernatants was determined by ELISA after treatment with NAC. (J) The level of MUC5AC mRNA expression determined by qRT-PCR in Calu-3 cells after treatment with NAC. There are three independent experiments (n = 3).
Fig 2: MUC5AC and KDELR2 expression levels are increased in patients with COPD. (A) Genes upregulated in healthy individuals (n = 40) and COPD patients (n = 111) from the GSE76925 dataset. (B) Relative KDELR1, KDELR2, and KDELR3 mRNA levels in healthy controls and patients with COPD in the GSE76925 dataset. (C) Haematoxylin and eosin images, AB‐PAS and immunohistochemistry (IHC) analysis of MUC5AC in lung sections from the control group (n = 19) and COPD group (n = 18). The image parameters were as follows: 1600 × 1200, 72 pixels per inch (PPI). (D) The percentage of goblet cells in the lung field was determined. (E) Quantitative immunohistochemistry (IHC) analysis of MUC5AC in COPD patients (n = 18) and control subjects (n = 19). (F) Coimmunofluorescence staining of MUC5AC and KDELR2 in the lung tissues of controls and patients with COPD (2048 × 2048, 96 PPI). Scale bars: 50 μm. IOD: Integrated optical density. Data are shown as the mean ± SEM. *p < 0.01 compared to controls. ns, not significant. Two‐tailed unpaired Student's t‐tests were used.
Fig 3: GOAC visualization and mucus production. (A) An upper view in light microscopy of a negative control GOAC (seeded with the usual cell load but without prior extra-cellular matrix coating) shows little to no staining after in-GOAC Alcian blue staining protocol compared to a fully confluent 100% Caco-2 cellularized GOAC (B) and a 50%/50% Caco-2–LS 174T bicellular GOAC (C). A general view (D) and close-up view (from the rectangle in D, ° as graphical marker) (E) of a histological section performed in a cellularized segment of a 100% Caco-2 GOAC’s inlet catheter add further validation of the model’s inner tissue architecture. A continuous mono-layer epithelium is seen lining the inner plastic wall (°) of the inlet catheter, seemingly producing a continuous ~2 µm thick layer of an intensely Alcian blue-stained compound, likely mucus. Quantitative ELISA tests targeting mucin MUC2 (F) reveals significantly superior MUC2 production in bicellular GOACs than in both the medium negative control and any mono-cellular GOACs. An identical assay targeting mucin MUC5AC (G) reveals significantly superior MUC5AC production in the bicellular GOACs than in the medium negative control and in the Caco-2 mono-cellular GOACs but significantly inferior MUC5AC production than in the LS 174T mono-cellular GOACs. *: statistically significant difference (p < 0.05) in univariate ANOVA (see Section 2).
Fig 4: KDELR2 is overexpressed and colocalized with MUC5AC in the bronchial epithelium of rats with COPD. (A) Schematic diagram of the rat model of COPD. (B) Haematoxylin and eosin images, AB‐PAS images and immunohistochemistry (IHC) images of MUC5AC and KDELR2 in lung sections from control rats and COPD model rats (n = 5). The image parameters used were 1600 × 1200 and 72 PPI. (C) Coimmunofluorescence staining of MUC5AC and KDELR2 in lung sections from control rats and COPD model rats (n = 5). The imaging parameters used were 2048 × 2048 and 96 PPI. (D) The percentage of goblet cells in the airway was determined. (E, F) Quantitative analyses of immunohistochemical data for MUC5AC and KDELR2 in the airways of control rats and rats with COPD. Scale bars: 50 μm. IOD: Integrated optical density. Data are shown as the mean ± SEM. *p < 0.01 compared to the control group. The p‐value was obtained by two‐tailed Student's t‐test.
Fig 5: Knockdown of KDELR2 decreased the expression of MUC5AC. (A) Schematic diagram showing the protocol for the administration of AAV containing KDELR2 shRNA or NC shRNA to COPD rats. (B, C) Confocal laser immunofluorescence staining of MUC5AC and quantitative analysis of control rats, COPD model rats, and COPD model rats transfected with AAV negative control shRNA and KDELR2 shRNA (n = 6). The imaging parameters were as follows: 2048 × 2048, 96 PPI. Scale bars: 50 μm. (D) Relative mRNA expression of KDELR2 determined by RT–qPCR (n = 6). (E) The relative expression of MUC5AC in BALF was determined using ELISA (n = 6). Data are shown as the mean ± SEM. *p < 0.05 compared with the control group; **p < 0.05 compared with the COPD group. One‐way ANOVA with Tukey–Kramer post hoc test.
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