Journal: The World Allergy Organization Journal

Article Title: IFITM1 is required for epithelial mesenchymal transition in airway remodeling of allergic asthma ☆

doi: 10.1016/j.waojou.2026.101339

Figure Lengend Snippet: IFN-β increases IFITM1 in airway epithelial cells. (A) RT‒qPCR analysis of IFN-ɑ expression in lung tissues (n = 5/group). (B) RT‒qPCR analysis of IFN-β expression in lung tissues (n = 5/group). (C) ELISA analysis of IFN-β expression in lung tissues (n = 6/group). (D) IFN-β in murine lung tissues costained with DAPI (blue), E-cad (green) and IFN-β (red); original magnification, 200×; scale bar, 100 μm; original magnification, 800×; scale bar, 25 μm. (E) Plasma levels of IFN-β in the control group (n = 19) and asthma group (n = 27). (F) Western blot analysis of IFITM1 expression in BEAS-2B cells treated with different concentrations of IFN-β extract. (G) Western blot analysis of IFITM1 expression in MLE-12 cells treated with different concentrations of IFN-β extract. (H) Western blot analysis of p-STAT1/STAT1 expression in BEAS-2B cells treated with IFN-β extract (1 ng/mL). (I) Western blot analysis of p-STAT1/STAT1 expression in MLE-12 cells treated with IFN-β extract (1 ng/mL). (J) Western blot analysis of p-STAT1/STAT1 and IFITM1 expression in BEAS-2B cells treated with human IFN-β extract (1 ng/mL) with or without fludarabine (1 μM) for 24 h. (K) Western blot analysis of p-STAT1/STAT1 and IFITM1 expression in MLE-12 cells treated with mouse IFN-β extract (1 ng/mL) with or without fludarabine (1 μM) for 24 h. The values are presented as the mean ± SD. The results were obtained from at least 3 independent tests. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: MLE-12, a murine pulmonary epithelial cell line, was obtained from ATCC (VA, USA) and cultivated in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Control, Western Blot

Journal: Frontiers in Pharmacology

Article Title: 1-Nitropyrene induces acute lung injury via SYVN1/Caspase-11-mediated apoptosis and pyroptosis in pulmonary epithelial cells

doi: 10.3389/fphar.2026.1723593

Figure Lengend Snippet: The effects of acute 1-NP on apoptosis in mouse lungs and MLE-12 cells. (A) The common genes associated with both ALI and 1-NP-evoked changes were explored by GeneCards and CTD databases. (B) Differential gene expression was analysed by KEGG. (C,D) Apoptotic cells were measured in mouse lungs by TUNEL. (E–H) Representative bands of apoptosis (F) and quantitative analyses of Bad (F) , Bcl-2 (G) , and Caspase-3 (H) expression. (I,J) The colocalization of SP-C with Caspase-3 was analysed by IF (I) , and quantitated by the Mander colocalization coefficient (MCC) (J) . (K–N) The influence of acute 1-NP on apoptosis was explored. (K) The expressions of apoptosis-related proteins were estimated with Western blot and quantitative analyses of Bad (L) , Bcl-2 (M) , and Caspase-3 (N) . All data are displayed as means ± S.E.M.s of six samples. The molecular experiments were repeated twice. * P < 0.05, ** P < 0.01.

Article Snippet: Mouse lung epithelial (MLE-12) cells were purchased from American Type Culture Collection (ATCC).

Techniques: Gene Expression, TUNEL Assay, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: 1-Nitropyrene induces acute lung injury via SYVN1/Caspase-11-mediated apoptosis and pyroptosis in pulmonary epithelial cells

doi: 10.3389/fphar.2026.1723593

Figure Lengend Snippet: The effects of acute 1-NP on pyroptosis in mouse lungs and MLE-12 cells. (A–C) The number of apoptotic cells was detected in MLE-12 cells through flow cytometry (A) , and quantitative analyses of early apoptosis (B) and late apoptosis (C) . (D) Cytotoxicity was estimated by LDH release from MLE-12 cells. (E–J) The expressions of GSDMD, Pro-Caspase-11, Cleaved-Caspase-11, Pro-Caspase-1, and Cleaved-Caspase-1 were measured in mouse lungs by Western blot. (K,L) The co-localization between SP-C and GSDMD was determined in mouse lungs via IF. (M,N) NLRP3-positive nuclei were analysed in mouse lungs via IF and quantification. (O–W) The effect of acute 1-NP on pyroptosis was estimated in MLE-12 cells. (O–U) Representative bands of pyroptosis markers (O) , and the levels of NLRP3 (P) , Pro-Caspase-11 (Q) , Cleaved-Caspase-11 (R) , GSDMD (S) , Pro-Caspase-11 (T) , and Cleaved-Caspase-11 (U) were quantified. (V,W) The mRNAs of Il-1β and Il-18 were determined in MLE-12 cells by RT‒PCR. All data are displayed as means ± S.E.M.s of six samples. The molecular experiments were repeated twice. * P < 0.05, ** P < 0.01.

Article Snippet: Mouse lung epithelial (MLE-12) cells were purchased from American Type Culture Collection (ATCC).

Techniques: Flow Cytometry, Western Blot

Journal: Frontiers in Pharmacology

Article Title: 1-Nitropyrene induces acute lung injury via SYVN1/Caspase-11-mediated apoptosis and pyroptosis in pulmonary epithelial cells

doi: 10.3389/fphar.2026.1723593

Figure Lengend Snippet: The influences of Caspase-11 elevation on 1-NP-evoked apoptosis in mouse lungs and MLE-12 cells. (A–H) The inhibitory effect of WED on 1-NP-incurred apoptosis was analysed in mouse lungs. (A,B) The number of apoptotic cells was evaluated via TUNEL. (C–F) The parameters of apoptosis were measured via Western blot, consisting of Bad, Bcl-2, and Caspase-3. (G,H) The number of Caspase-3-positive cells was determined by IHC. (I–P) The antagonistic influence of Caspase-11 siRNA on 1-NP-mediated apoptosis was explored in MEL-12 cells. (I,J) Apoptotic cells were detected using TUNEL. (K–N) Representative bands of apoptosis (K) and quantitative analyses of Bad (L) , Bcl-2 (M) , and Caspase-3 (N) . (O,P) The count of Caspase-3-positive cells was analysed. All data are displayed as means ± S.E.M.s of six samples. The molecular experiments were repeated twice. * P < 0.05, ** P < 0.01.

Article Snippet: Mouse lung epithelial (MLE-12) cells were purchased from American Type Culture Collection (ATCC).

Techniques: TUNEL Assay, Western Blot

Journal: Frontiers in Pharmacology

Article Title: 1-Nitropyrene induces acute lung injury via SYVN1/Caspase-11-mediated apoptosis and pyroptosis in pulmonary epithelial cells

doi: 10.3389/fphar.2026.1723593

Figure Lengend Snippet: The effects of Caspase-11 increase on 1-NP-induced pyroptosis in mouse lungs and MLE-12 cells. (A–H) The antagonistic influence of WED on 1-NP-induced apoptosis was analysed in mouse lungs. (A–D) Pyroptosis markers were tested via Western blot, including GSDMD, Pro-Caspase-11, and Cleaved-Caspase-11. (E,F) GSDMD-positive cells were evaluated by IHC. (G,H) Caspase-11-positive cells were detected with IHC. (I–P) The repressive influence of Caspase-11 siRNA on 1-NP-induced pyroptosis was analysed in MLE-12 cells. (I–L) The indicators of pyroptosis were estimated using Western blot (I) and quantitative analyses of GSDMD (J) , Pro-Caspase-11 (K) , and Cleaved-Caspase-11 (L) . (M,N) The number of GSDMD-positive cells was evaluated using IF. (O,P) The mRNA levels of Il-1β and Il-18 were estimated by RT‒PCR. All data are displayed as means ± S.E.M.s of six samples. The molecular experiments were repeated twice. * P < 0.05, ** P < 0.01.

Article Snippet: Mouse lung epithelial (MLE-12) cells were purchased from American Type Culture Collection (ATCC).

Techniques: Western Blot

Journal: Frontiers in Pharmacology

Article Title: 1-Nitropyrene induces acute lung injury via SYVN1/Caspase-11-mediated apoptosis and pyroptosis in pulmonary epithelial cells

doi: 10.3389/fphar.2026.1723593

Figure Lengend Snippet: The impacts of acute 1-NP on Caspase-11 ubiquitination and protein degradation in pulmonary epithelial cells. (A) The level of Caspase-11 mRNA was tested by RT‒PCR. At 24 h after 1-NP, MLE-12 cells were incubated with CHX or MG132 for 0 h, 3 h, 6 h, or 12 h. Caspase-11 protein stability was analysed. (B,C) The protein stability of Caspase-11 was estimated by Western blot. At 24 h after 1-NP, MLE-12 cells were incubated with CHX or Bnf A1 for different durations. (D,E) The protein stability of Caspase-11 was evaluated with Western blot. (F) The UbiBrowser database was used to predict the proteins that possibly interact with Caspase-11. (G,H) The interaction between Caspase-11 and SYVN1 was evaluated by Co-IP. (I,J) The interaction between Caspase-11 and ubiquitin was analysed via Co-IP. (K) The interaction between Caspase-11 and SYVN1 was simulated via molecular docking. All data are displayed as means ± S.E.M.s of six samples. The molecular experiments were repeated twice. * P < 0.05, ** P < 0.01.

Article Snippet: Mouse lung epithelial (MLE-12) cells were purchased from American Type Culture Collection (ATCC).

Techniques: Ubiquitin Proteomics, Incubation, Western Blot, Co-Immunoprecipitation Assay

Journal: Frontiers in Pharmacology

Article Title: 1-Nitropyrene induces acute lung injury via SYVN1/Caspase-11-mediated apoptosis and pyroptosis in pulmonary epithelial cells

doi: 10.3389/fphar.2026.1723593

Figure Lengend Snippet: The role of SYVN1 on acute 1-NP-induced Caspase-11 proteasome degradation in pulmonary epithelial cells. (A–D) The effects of acute 1-NP on SYVN1 protein expression were evaluated in mouse lungs and MLE-12 cells via Western blot. (E–L) The effect of SYVN1 reduction on 1-NP-evoked Caspase-11 ubiquitination degradation was observed. SYVN1 overexpression plasmids were transfected, after which the cells were exposed to 1-NP (5 μM). (E,F) The stability of Caspase-11 protein was measured via Western blot. (G,H) The rate of Caspase-11 proteasome degradation was detected using Western blot. (I,J) The interaction between Caspase-11 and SYVN1 was analysed by Co-IP. (K,L) The interaction of Caspase-11 with ubiquitin was explored with Co-IP. (M–S) The effects of SYVN1 decrease on 1-NP-mediated apoptosis and pyroptosis were investigated in MLE-12 cells. (M–Q) The markers of apoptosis and pyroptosis were detected via Western blot. (R,S) The expressions of Il-18 and Il-1β were evaluated through RT‒PCR. All data are displayed as means ± S.E.M.s of six samples. The molecular experiments were repeated twice. * P < 0.05, ** P < 0.01.

Article Snippet: Mouse lung epithelial (MLE-12) cells were purchased from American Type Culture Collection (ATCC).

Techniques: Expressing, Western Blot, Ubiquitin Proteomics, Over Expression, Transfection, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Notch mediates non-regenerative alveolar repair after staphylococcal lung injury

doi: 10.64898/2026.02.04.703850

Figure Lengend Snippet: (A-J) Cartoons in A and F show the experimental design of the studies used to generate the data shown in B-E and G-J, respectively. As indicated in A and F, we gave intranasal ( I.N. ) instillation of the 12XCSL-DsRed-ExpressDL Notch cleavage reporter plasmid, then I.N. instillation of PBS or S. aureus (SA). SA were SA GFP or SA hla:: Erm as indicated. At 4 h after PBS or SA instillation, we excised the lungs and used confocal microscopy to view DsRed fluorescence ( yellow ) in alveoli. As indicated in F, mice used to generate the data shown in G-J were also intranasally-instilled with siRNA that was non-targeting ( siNT ) or against presenilin1 ( siPS1 ) or ADAM10 ( siADAM10 ). In E and J, circles indicate n and each represent one mouse in which mean DsRed fluorescence was quantified in imaging fields of at least 50 alveoli; bars: mean ± SEM; * p < 0.05 or as indicated by ANOVA with post-hoc Tukey testing. Scale bars: 100 μm. ( K-N ) Immunoblots (K and M) and group band densitometry (L and N) show protein content in cultured alveolar epithelial-like MLE-12 cells. Prior to immunoblot processing, the cells were exposed to PBS, purified Hla ( Hla WT ), or EDTA as indicated for 10 min, then incubated in culture media for 45 min. EDTA served as a positive control. Bands shown in K and M were generated on the same blot but are not contiguous. In L and N, bars show mean ± SEM; * p < 0.05 or as indicated by ANOVA with post-hoc Tukey testing (L) or two-tailed t test (N); circles indicate n and each represent one biological replicate. Replicates were run in the same gel and immunoblotted on the same membrane. Immunoblots were each repeated 2x with the same results.

Article Snippet: MLE-12 cells were grown as monolayer cultures in DMEM/F-12 medium (catalog 30-2006, ATCC) that was supplemented with 10% FBS (Corning, cat. no. 35-011-CV) and 1% penicillin and streptomycin (Gibco, cat. no. 15140122) and maintained in an incubator at 5% CO 2 and 37 C.

Techniques: Plasmid Preparation, Confocal Microscopy, Fluorescence, Imaging, Western Blot, Cell Culture, Purification, Incubation, Positive Control, Generated, Two Tailed Test, Membrane

Journal: bioRxiv

Article Title: Notch mediates non-regenerative alveolar repair after staphylococcal lung injury

doi: 10.64898/2026.02.04.703850

Figure Lengend Snippet: (A-K) Cartoons in A and H show the experimental design of the studies used to generate the data shown in B-G and I-K, respectively. As indicated in A and H, we carried out immunofluorescence determinations of E-cadherin expression ( yellow ) by confocal microscopy in cultured alveolar epithelial-like MLE-12 cells before (B-C) and after (D-F and I-J) cell exposure to PBS, purified Hla ( Hla WT ), vehicle, and the gamma-secretase inhibitor, DAPT as indicated. Note, Hla WT caused rapid loss of E-cadherin from cell-cell junctions. Junctional E-cadherin spontaneously recovered, but DAPT disrupted the recovery. In the group data in G and K, circles indicate n and each represent one biological replicate; bars: mean ± SEM; * p < 0.05 or as indicated by ANOVA with post hoc Tukey testing (G) and two-tailed t test (K). Scale bars: 50 μm. (L-N) Confocal images show immunofluorescence of E-cadherin ( yellow ) in tissue sections of mouse lungs that were fixed at the indicated times after intranasal ( I.N. ) instillation of PBS or SA GFP as indicated. Images were replicated 2x per group. Scale bars: 100 μm.

Article Snippet: MLE-12 cells were grown as monolayer cultures in DMEM/F-12 medium (catalog 30-2006, ATCC) that was supplemented with 10% FBS (Corning, cat. no. 35-011-CV) and 1% penicillin and streptomycin (Gibco, cat. no. 15140122) and maintained in an incubator at 5% CO 2 and 37 C.

Techniques: Immunofluorescence, Expressing, Confocal Microscopy, Cell Culture, Purification, Two Tailed Test

Journal: bioRxiv

Article Title: Notch mediates non-regenerative alveolar repair after staphylococcal lung injury

doi: 10.64898/2026.02.04.703850

Figure Lengend Snippet: (A-D) Confocal images show immunofluorescence of GFP ( magenta ), DAPI ( cyan ), and E-cadherin ( yellow ) in cultured alveolar epithelial-like MLE-12 cells transfected with plasmid DNA encoding a Notch TMD-GFP fusion protein ( TMD-GFP ). Arrowheads in C-D point out locations where GFP and E-cadherin fluorescences merge, signaling TMD-GFP protein localizes to plasma membranes. Scale bars: 20 μm. (E) Immunoblot data show Hes1 and actin protein content in MLE-12 cells transfected with plasmid DNA encoding GFP or TMD-GFP, then either untreated or exposed to purified Hla ( Hla WT ) as indicated. Hla WT exposure was for 10 min, then the cells were incubated in culture media for 45 min prior to immunoblot processing. Immunoblots were each replicated 2x. (F-L) The cartoon in F shows the experimental design of the studies used to generate the data shown in G-L. As indicated in F, we carried out immunofluorescence determinations of E-cadherin expression ( yellow ) by confocal microscopy in MLE-12 cells transfected with plasmid DNA encoding GFP or TMD-GFP. Transfected cells were exposed to Hla WT as indicated. Note, while cells in both groups show a cell junctional pattern of E-cadherin under baseline conditions that is lost in response to Hla WT exposure, only TMD-GFP-transfected cells show E-cadherin recovery. Fluorescence of recovered E-cadherin in GFP-transfected cells appears disorganized and is not located at cell-cell junctions. Images each replicated 2x. Scale bars: 50 μm.

Article Snippet: MLE-12 cells were grown as monolayer cultures in DMEM/F-12 medium (catalog 30-2006, ATCC) that was supplemented with 10% FBS (Corning, cat. no. 35-011-CV) and 1% penicillin and streptomycin (Gibco, cat. no. 15140122) and maintained in an incubator at 5% CO 2 and 37 C.

Techniques: Immunofluorescence, Cell Culture, Transfection, Plasmid Preparation, Clinical Proteomics, Western Blot, Purification, Incubation, Expressing, Confocal Microscopy, Fluorescence

Journal: Inflammation

Article Title: Inhibition of TRPV4 Regulates Mitophagy Through the Sirt1/FoxO1 Signaling Pathway To Alleviate Acute Lung Injury

doi: 10.1007/s10753-025-02433-y

Figure Lengend Snippet: Inhibition of TRPV4 increased mitochondrial autophagy and attenuated damage of MLE12 cells. A CCK-8 assay was detected to evaluate cell viability in each group ( n = 6) B , C Intracellular calcium ion concentrations were measured using laser confocal microscopy (original magnification,×64; scale bar: 50 μm) D , E Fluorescence microscopy was used to assess ROS levels F Cell apoptosis in each group was detected by flow cytometry (original magnification,×64; scale bar: 50 μm) ( n = 6) G , H Changes in mitochondrial membrane potential were detected using laser confocal microscopy with JC-1 staining(original magnification, ×64; scale bar: 50 μm) (×64; scale bar: 20 μm) ( n = 6). Nuclei were revealed using DAPI staining I The expressions of autophagy-related proteins PINK1 and PARK2 and their upstream regulatory factor Sirt1、FoxO1 in mitochondria were measured by Western Blot.

Article Snippet: The mouse lung epithelial cell line MLE12 was bought from American Type Culture Collection (ATCC, CRL-2110, USA).

Techniques: Inhibition, CCK-8 Assay, Confocal Microscopy, Fluorescence, Microscopy, Flow Cytometry, Membrane, Staining, Western Blot