murine lung epithelial cells  (ATCC)

Journal: Biochemistry and Biophysics Reports

Article Title: Dual knockdown of Alox15 and TGF-β1 by lipid nanoparticle-delivered siRNA in bleomycin-induced pulmonary fibrosis

doi: 10.1016/j.bbrep.2026.102534

Figure Lengend Snippet: LNP-mediated delivery of siRNAs into lung epithelial and fibroblast cells effectively reduces mRNA levels both in vitro and in vivo . (A, B) MLE-12 cells and murine lung fibroblasts were treated with negative control (NC), Alox15, or TGF-β1 siRNA for 24 h, followed by treatment with bleomycin for an additional 24 h, then subjected to real-time PCR assay. Fluorescence images of (C) MLE-12 cells and (D) murine lung fibroblasts treated with 50 nM naked siRNA-Cy5 or siRNA-Cy5 encapsulated with LNPs (siRNA@LNP) for 6 h, followed by staining with Hoechst 33342. Scale bar = 20 μm. (E) Flow cytometry analysis showing the mean fluorescent intensity (MFI) of Cy5 positive cells. (F) Mice were injected with saline, naked siRNA, or siRNA@LNP and sacrificed at 24 h after treatment. Cy5 fluorescence signal in the heart, lung, liver, spleen and kidney was measured by The PhotonIMAGER Optima system immediately. Fluorescence intensity in the lung expressed as total radiant efficiency. n = 3 per group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Scatter plots representing individual mice.

Article Snippet: Murine lung epithelial cells (MLE-12 cells) were obtained from ATCC (Manassas, VA, USA) and cultured in DMEM/F12 medium (Corning) containing 4% FBS, 0.005 mg/mL insulin, 0.01 mg/mL transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM β-estradiol, 10 mM HEPES, 2 mM l -glutamine, 100 U/mL penicillin G, and 100 μg/mL streptomycin.

Techniques: In Vitro, In Vivo, Negative Control, Real-time Polymerase Chain Reaction, Fluorescence, Staining, Flow Cytometry, Injection, Saline

Journal: The Journal of Biological Chemistry

Article Title: Divergent pathways of surfactant protein C maturation for disease-associated isoforms

doi: 10.1016/j.jbc.2026.111252

Figure Lengend Snippet: Generation and characterization of SFTPC -expressing MLE-12 cell lines. A , schematic of GFP-tagged surfactant protein C constructs cloned into the lentiviral expression vector pCW57.1 with a Tet-responsive element (TRE). B , schematic of SP-C proprotein processing: Initial C-terminal cleavage events generate intermediate species (∼16 and ∼6 kDa), followed by N-terminal processing mediated by the lysosomal protease cathepsin H, a step that has been experimentally defined. Final N-terminal trimming yields the mature SP-C peptide (∼3.7 kDa). Putative involvement of additional proteases, including pepsinogen C (PGC) and/or napsin, in downstream processing steps is indicated in gray to denote predicted activity. Sites of palmitoylation and O-glycosylation associated with the I73T threonine substitution are annotated. C , immunoblot of GFP-tagged SP-C WT, I73T, and C121G constructs expressed in MLE-12 cells for 24 h with 2.5 μM doxycycline induction. Arrowheads and asterisk denote C-terminal processing intermediates and the palmitoylated pro-form, respectively. D , live-cell fluorescent widefield microscopy of GFP-SP-C localization in MLE-12 cells. GFP signal ( green ) and Hoechst-stained nuclei ( blue ) are shown. White arrow heads denote WT localization of SP-C on membranes of vesicles. The scale bar represents 10 μm ( left ), 5 μm ( zoom inset ). E , colocalization of GFP-SP-C WT, I73T, and C121G isoforms following 24 h of doxycycline induction in live cells co-stained with organelle markers: LysoTracker (acidic LROs), Wheat Germ Agglutinin (WGA) (plasma membrane), and ER Tracker (endoplasmic reticulum). Merged images show organelle marker ( red ) and GFP-SP-C ( green ). The scale bar represents 10 μm. F , quantification of colocalization by Pearson’s correlation coefficient between GFP signal and each organelle marker in WT, I73T, and C121G-expressing cells. Data represent mean ± SD from n ≥ 10 cells per condition, pooled from three independent experiments. LRO, lysosome-related organelle; MLE-12, mouse lung epithelial-12.

Article Snippet: The parental murine lung epithelial cell line MLE-12 (CRL-2110) ( ) was originally obtained from the American Type Culture Collection and maintained in culture at 37 °C and 5% CO 2 in HITES medium [Ham's F-12 medium (50:50 mixture) containing 0.005 mg/ml insulin, 0.01 mg/ml transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM β-estradiol, 10 mM Hepes buffer, and 2 mM l-glutamine] supplemented with 2% fetal bovine serum and antibiotics.

Techniques: Expressing, Construct, Clone Assay, Plasmid Preparation, Activity Assay, Glycoproteomics, Western Blot, Microscopy, Staining, Clinical Proteomics, Membrane, Marker

Journal: The Journal of Biological Chemistry

Article Title: Divergent pathways of surfactant protein C maturation for disease-associated isoforms

doi: 10.1016/j.jbc.2026.111252

Figure Lengend Snippet: Divergence of WT and mutant Pro-SP-C expression patterns. A , Twenty-four hours post doxycycline induction of GFP-SP-C WT and GFP-SP-C I73T in MLE-12s, cells were treated with vehicle (dimethyl sulfoxide) or Pitstop 2 for 2 h. B , quantification of total GFP signal and ( C ) contiguous plasma membrane GFP signal for WT and I73T. D , MLE-12 cells were treated with doxycycline for 24 h to induce SP-C expression, followed by a 2 h cotreatment with Pitstop 2 or vehicle. Surface proteins were biotinylated, enriched via streptavidin pulldown, and immunoblotted for GFP-tagged SP-C. E , GFP-SP-C at the cell surface was assessed via susceptibility to proteolysis by proteinase K protection assay in nonpermeabilized versus Triton X-100–permeabilized cells. F , immunofluorescence analysis in nonpermeabilized MLE-12 cells expressing GFP-SP-C variants. WGA ( red ) marks plasma membrane. Anti-GFP ( green ) detects N terminus of SP-C; anti–C-terminal antibody against SP-C ( cyan ). Asterisks mark nontransfected cells. Yellow arrows denote orientation of line scans at the plasma membrane. G , intensity profiles of line-scan analyses across cell membranes in nonpermeabilized cells. Pearson’s correlation coefficients (R values) between GFP ( green ) and WGA ( red ) channels are shown for each variant. All quantitation data are shown as mean ± SD from three independent experiments. Statistical analyses are performed using two-tailed Student’s t test. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; and n.s., not significant. MLE-12, mouse lung epithelial-12; SP-C, surfactant protein C; WGA, Wheat Germ Agglutinin; WGA, Wheat Germ Agglutinin.

Article Snippet: The parental murine lung epithelial cell line MLE-12 (CRL-2110) ( ) was originally obtained from the American Type Culture Collection and maintained in culture at 37 °C and 5% CO 2 in HITES medium [Ham's F-12 medium (50:50 mixture) containing 0.005 mg/ml insulin, 0.01 mg/ml transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM β-estradiol, 10 mM Hepes buffer, and 2 mM l-glutamine] supplemented with 2% fetal bovine serum and antibiotics.

Techniques: Mutagenesis, Expressing, Clinical Proteomics, Membrane, Immunofluorescence, Variant Assay, Quantitation Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Divergent pathways of surfactant protein C maturation for disease-associated isoforms

doi: 10.1016/j.jbc.2026.111252

Figure Lengend Snippet: First cleavage of the SP-C COOH-propeptide occurs in the trans-Golgi network. A , representative images of MLE-12 cells induced with doxycycline for 3 h followed by trans-Golgi trafficking inhibition at 20°C for 2 h, or after cis -/medial-Golgi collapse induced by brefeldin A for 2 h, showing GFP-SP-C ( cyan ) localization relative to the ER (calreticulin, magenta ) and Golgi (GM130, yellow ) as compared to control condition (37°C). The scale bar represents 10 μm. B , representative immunoblot of GFP-SP-C WT and I73T mutant isoform following treatment ( A and C ) densitometry analysis of WT SP-C first COOH-cleavage intermediate band ( solid arrowhead ) normalized to actin, shown as fold change relative to control. Experiments were performed in triplicate and statistical analyses were performed using one-way ANOVA. ∗, p < 0.05. D , schematic of sucrose gradient-based subcellular fractionation to isolate ER/Golgi-enriched membranes from postnuclear supernatants (PNSs). E , left: Coomassie staining of total protein in input and ER/Golgi fractions. Middle: Immunoblots showing enrichment of ER, Golgi, and absence of markers from other organelles (mitochondria and lysosomes) in purified fractions. Right: representative immunoblot showing enrichment of first COOH-cleavage SP-C intermediate ( solid arrowhead ) in the ER/Golgi fraction of WT SP-C expressing MLE-12, and in contrast sparsely detected from this fraction for the I73T mutant. SP-C, surfactant protein C; MLE-12, mouse lung epithelial-12; ER, endoplasmic reticulum.

Article Snippet: The parental murine lung epithelial cell line MLE-12 (CRL-2110) ( ) was originally obtained from the American Type Culture Collection and maintained in culture at 37 °C and 5% CO 2 in HITES medium [Ham's F-12 medium (50:50 mixture) containing 0.005 mg/ml insulin, 0.01 mg/ml transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM β-estradiol, 10 mM Hepes buffer, and 2 mM l-glutamine] supplemented with 2% fetal bovine serum and antibiotics.

Techniques: Inhibition, Control, Western Blot, Mutagenesis, Fractionation, Staining, Purification, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Divergent pathways of surfactant protein C maturation for disease-associated isoforms

doi: 10.1016/j.jbc.2026.111252

Figure Lengend Snippet: I73T SP-C expression disrupts Golgi architecture in MLE-12 cells. A , representative transmission electron microscopy (TEM) images of murine AT2 cells from whole lung mounts. Arrows point to Golgi. B , confocal images of MLE-12 cells expressing GFP-tagged WT or I73T SP-C ( green ) and immunofluorescence stained for the trans-Golgi marker p230 ( red ). Nuclei are stained with Hoechst ( blue ). C and D , quantification of the number of p230-labeled Golgi elements per cell ( C ) and Golgi area/number of Golgi particles within a cell ( D ). All quantitation data are shown as mean ± SD from three independent experiments. Statistical analyses are performed using two-tailed Student’s t test. ∗, p < 0.05; and ∗∗∗, p < 0.001. SP-C, surfactant protein C; MLE-12, mouse lung epithelial-12.

Article Snippet: The parental murine lung epithelial cell line MLE-12 (CRL-2110) ( ) was originally obtained from the American Type Culture Collection and maintained in culture at 37 °C and 5% CO 2 in HITES medium [Ham's F-12 medium (50:50 mixture) containing 0.005 mg/ml insulin, 0.01 mg/ml transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM β-estradiol, 10 mM Hepes buffer, and 2 mM l-glutamine] supplemented with 2% fetal bovine serum and antibiotics.

Techniques: Expressing, Transmission Assay, Electron Microscopy, Immunofluorescence, Staining, Marker, Labeling, Quantitation Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Divergent pathways of surfactant protein C maturation for disease-associated isoforms

doi: 10.1016/j.jbc.2026.111252

Figure Lengend Snippet: Furin-like pro-protein convertases are candidate enzymes for initial SP-C cleavage. A and B , Western blot analysis and quantitation of c-term SP-C cleavage ( arrowheads ) in MLE-12 cells cotreated with DC1 (dicoumarol-related pan-proprotein convertase inhibitor) and doxycycline to induce the expression of GFP-SPC WT for 9 h. C and D , immunoblot and quantitation of C-terminal SP-C cleavage after overnight DC1 treatment in primary murine AT2 isolated from SP-C WT mice shows similar inhibition of SP-C processing. E , immunofluorescence of GFP-SPC WT MLE-12 cells cotreated with doxycycline and DC1 for 9 h, fixed and stained with GM130 (Golgi marker). F , immunofluorescence of GFP-SPC WT after 6 h of doxycycline-induction ( green ) shows colocalization with furin ( magenta ) in the perinuclear region of MLE12 cells. Hoechst stains nuclei. Scale bars represent 10 μm. G , GFP-pulldown in MLE12 cells using GFP-nanotrap or Control Binding-nanotrap beads shows interaction of mature furin with GFP-tagged SP-C. H , furin cleavage assay performed with SP-C pro-translation product as the in vitro substrate in the presence and absence of decanoyl-RVKR-CMK. I , immunoblot after GFP-pulldown enrichment following 9 h of doxycycline showing SP-C processing in MLE-12 cells transduced with individual pre-profragment of PC7 or furin or IRES empty vector control. J , immunofluorescence of GFP-SPC WT after overnight of doxycycline-induction and decanoyl-RVKR-CMK furin inhibitor stained for LAMP3. K , immunoblot of GFP-SPC WT expressing MLE-12 cells after overnight treatment of doxycycline and decanoyl-RVKR-CMK. L , quantification ratio of SP-C cleaved intermediate relative to pro-form SP-C in each condition. SP-C, surfactant protein C; MLE-12, mouse lung epithelial-12; AT2, alveolar type II.

Article Snippet: The parental murine lung epithelial cell line MLE-12 (CRL-2110) ( ) was originally obtained from the American Type Culture Collection and maintained in culture at 37 °C and 5% CO 2 in HITES medium [Ham's F-12 medium (50:50 mixture) containing 0.005 mg/ml insulin, 0.01 mg/ml transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM β-estradiol, 10 mM Hepes buffer, and 2 mM l-glutamine] supplemented with 2% fetal bovine serum and antibiotics.

Techniques: Western Blot, Quantitation Assay, Expressing, Isolation, Inhibition, Immunofluorescence, Staining, Marker, Control, Binding Assay, Cleavage Assay, In Vitro, Transduction, Plasmid Preparation

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