murine lung epithelial cell line mle 12  (ATCC)

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: Cancer Research

Article Title: ACSL5 Mediates Adaptation to the Palmitic Acid–Enriched Pulmonary Microenvironment to Enhance Metastatic Breast Cancer Cell Survival and Lung Metastasis

doi: 10.1158/0008-5472.CAN-25-0866

Figure Lengend Snippet: Exo-derived USP47 mediates fatty acid synthesis in AT2. A, MLE-12 cells were cocultured with exosomes derived from the control (shNC) and USP47-knockdown (shUSP47) MDA-MB-231/LM3. YAP, FASN and ACLY proteins, as well as the PA levels ( n = 5), in MLE-12 cells were detected. B, MLE-12 cells were cocultured with exosomes derived from the control (vector) and USP47-overexpression (oeUSP47) MDA-MB-231/PRI. YAP, FASN, and ACLY protein levels, as well as the PA levels ( n = 5), in MLE-12 cells were detected. C, Left, Western blotting was performed to detect YAP, FASN, and ACLY protein levels in the control (shNC) or YAP-knockdown (shYAP) MLE-12. Right, relative PA levels ( n = 5) in MLE-12 cells were detected. D, MLE-12 cells were cocultured with the control exosomes (vector) or USP47-overexpression exosomes (oeUSP47) derived from MDA-MB-231/PRI, or YAP-knockdown MLE-12 cells and its controls were cocultured with USP47-overexpression exosomes (oeUSP47) derived from MDA-MB-231/PRI. FASN and ACLY protein levels, as well as the PA levels ( n = 5), in MLE-12 cells were detected. E, MLE-12 cells were cocultured with the control exosomes (shNC) or USP47-knockdown exosomes (shUSP47) derived from MDA-MB-231/LM3, or YAP-overexpression (oeYAP) and its control MLE-12 cells (Vector) were cocultured with USP47-knockdown exosomes (shUSP47) derived from MDA-MB-231/LM3, respectively. FASN and ACLY protein levels, as well as the PA levels ( n = 5), in MLE-12 cells were detected. F and G, Relative PA levels in MLE-12 in indicated group ( n = 5). H, Relative PA levels ( n = 5) in the lung interstitial fluid of BALB/c nude mice injected with the control (shNC) or USP47-knockdown (shUSP47) MDA-MB-231/LM3 cells. I, Representative BLI and metastatic lungs of BALB/c nude mice injected with the control (shNC) or USP47-knockdown (shUSP47) MDA-MB-231/LM3 cells. J, Quantification of BLI and lung metastatic nodules ( n = 7). All data represent the mean ± SD. One-way ANOVA ( A , C–G , H , and J ) and Student t test ( B ) were utilized. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Triple-negative breast cancer cell lines 4T1, MDA-MB-231, and HCC1806 and mouse alveolar epithelial cell line MLE-12 were purchased from the ATCC.

Techniques: Derivative Assay, Control, Knockdown, Plasmid Preparation, Over Expression, Western Blot, Injection

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: 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