murine lung epithelial cell line  (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: Signal Transduction and Targeted Therapy

Article Title: A nanobody-based proteolysis-targeting chimera offers broad-spectrum protection against diverse influenza virus infections

doi: 10.1038/s41392-026-02666-9

Figure Lengend Snippet: Antiviral efficacy of Nb-PROTACs against H1N1 and H5N1 viruses in mice. a Antiviral activity of mVHL-Nb170 in MLE-12 cells. Cells stably expressing Nb170 or mVHL-Nb170 were infected with SD012 at an MOI of 0.01. The supernatants were collected at the indicated time points and subjected to viral titration in MDCK cells. The data are presented as the means ± SDs from three independent biological samples ( n = 3). b Schematic representation of the experimental design for the in vivo study. BALB/c mice received two intratracheal doses of AAV-LungM3 at an interval of 3 days, each at 5 × 10 10 vg, resulting in a total dose of 1 × 10 11 vg per mouse. Three weeks post-administration, the mice were intranasally challenged with 5 MLD 50 of the PR8 (H1N1) or SD012 (H5N1) virus. c , d Protective efficacy of mVHL-Nb170 in mice. Groups of 14 mice were administered the AAV-LungM3 vector expressing the indicated proteins and subsequently challenged intranasally with 5 MLD 50 of PR8 ( c ) or SD012 ( d ). Nasal turbinates and lungs were collected on day 3 postchallenge for viral titration. The data are presented as the means ± SDs from four animals ( n = 4). Survival and body weight were monitored daily for 14 days ( n = 10). The percentages in c and d denote the mean decrease in viral load compared with that in the controls. Asterisks indicate significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: The human embryonic kidney cell line 293T, murine lung epithelial cell line MLE-12, and human lung epithelial cell line A549 were obtained from the American Type Culture Collection (ATCC).

Techniques: Activity Assay, Stable Transfection, Expressing, Infection, Titration, In Vivo, Virus, Plasmid Preparation

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