Normal human lung fibroblasts nhlfs

A549, NCI-H358, and NCI-H520 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Roswell Park Memorial Institute (RPMI) 1,640 medium with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in a humidified incubator of 5% CO₂ at 37 °C. Normal human lung fibroblasts (NHLFs) were purchased from Lonza (Walkersville, MD, USA) and maintained using a Fibroblast Growth Media Kit (FGM) (Lonza, Basel, Switzerland) under the same conditions.

The immortalized human small airway epithelial cell line HPL1D was provided by Dr. Takahashi and maintained in Ham’s F12 medium supplemented with 1 % fetal bovine serum (FBS), 5 μg/ml insulin, 5 μg/ml human transferrin, 10⁻⁷ M hydrocortisone, and 2 × 10⁻¹⁰ M thyronine at 37 °C [35 (link)]. The human lung adenocarcinoma A549 cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco’s minimal essential medium (DMEM) (Invitrogen, Eugene‚ OR) supplemented with 10 % FBS, 10 units/ml penicillin, and 10 μg/ml streptomycin (Invitrogen). Normal human lung fibroblasts (NHLFs) were purchased from Lonza (Allendale, NJ) and maintained in FGM-2 medium following manufacturer’s instructions.
siRNAs against human BMAL1(siBMAL1), a mixture of 4 siRNA oligos, and all-star control siRNA (siCtrl) were purchased from Qiagen (Valencia, CA). Cells were seeded onto 6-well cell culture plates and 20 μM of siRNA were transfected by using lipofectamineRNAiMAX (Invitrogen). Cells were treated with TGF-β1 and TNFα one day’s later. During treatment with recombinant human TGF-β1, A549 cells were cultured in DMEM plus 0.5 % FBS and NHLF were cultured in FBM plus 0.2 % bovine serum albumin (BSA). Cells were harvested 48 or 72 h following transfection for isolating RNA or proteins.

Normal human lung fibroblasts (NHLFs) were purchased from Lonza (Allendale, NJ) and maintained in fibroblast growth medium 2 (FGM-2; Lonza) for experiments until passage 6 per the provider’s instruction. Prior to treatment, cells that had reached 80% confluence were serum starved in fibroblast basal medium 2 (FBM-2; Lonza) with 0.2% bovine serum albumin (BSA) overnight [17 (link)].

Human lung adenocarcinoma cells (A549) and mouse alveolar epithelial cells (MLE12) from American Type Culture Collection (Manassas, VA, USA) were cultured in Dulbecco's Modified Eagle Medium (DMEM) and DMEM/Ham's F-12, respectively, supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin). These cells exhibit the cuboidal cell morphology of AEC II. Human pulmonary alveolar epithelial cells (HPAEpiC) from ScienCell Research Laboratories (Carlsbad, CA, USA) were cultured in alveolar epithelial cell medium (ScienCell Research Laboratories) with poly-L-lysine-coated culture vessels (Corning Incorporated, Tewksbury, MA, USA). Normal human lung fibroblasts (NHLFs) from Lonza (Walkersville, MD, USA) were cultured in DMEM supplemented with 10% FBS and antibiotics.

THP-1 human monocyte cells, Jurkat T cells (ATCC, Manassas, VA, USA) were cultured in RPMI media (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (pen/strep). J774 mouse macrophages (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s MEM (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% FBS, 1% pen/strep. Normal human lung fibroblasts (NHLFs) were obtained from Lonza, Walkersville, MD, USA and cultured in Fibroblast basal Medium-2 (Lonza, Walkersville, MD, USA) with supplements (FGM-2 bullet kit, Lonza, Walkersville, MD, USA). The NHLFs were passaged and used till passage 4. Human Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy volunteers from New York Blood Center upon obtaining informed consent and after Institutional Review Board (IRB) approval. were isolated by Ficoll separation and were maintained in RPMI-1640 medium (Gibco, Grand Island, NY, USA) with 10% FBS (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA) prior to the experiment. All the cells were incubated in 5% CO₂ supply at 37 °C.

Lung tissue was enzymatically dissociated by incubation in 2mg/ml collagenase (Sigma, Poole, UK) in serum free phenol red free Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DME-F12, Life Technologies Ltd, Paisley, UK) medium for 2–24 hours at 37°C, with occasional agitation. Cells released were washed in DME-F12 medium containing 10% foetal bovine serum, plated in the same medium and cultured on tissue culture treated plastic at 37°C and 5% CO₂. Adherent cells were seen after 24 hours. Cells were used between passages 1 and 3. 621-101 cells are a TSC2-null cell line derived from a renal angiomyolipoma of a LAM patient and were a gift from Elizabeth Henske [19 (link)]. These cells were maintained in DME-F12 with 10% FCS. Normal Human Lung Fibroblasts (NHLFs) were purchased from Lonza (Slough, UK) and were maintained in DME-F12 with 10% FCS.