Bleomycin

A549 cells previously silenced for ZNF365 (24 h before) were seeded at 70% confluency and stimulated with 30 mU/mL of bleomycin (Cayman) in serum-free medium for 48 h. Normal human lung fibroblasts were seeded at 70% confluency, previously silenced (48 h before) for ZNF365, and stimulated with [0.1 and 0.5 μM] for 1 h. Cells were harvested and stained with PE Annexin V (556421, BD Biosciences, San Jose, CA, USA) and 7-aminoactinomycin D (7AAD, 559925, Biolegend, San Diego, CA, USA). Cells were analyzed by flow cytometry in a FACSAria flow cytometer (BD Biosciences). Results were analyzed with FlowJo 7.8 software (FlowJo, Ashland, OR, USA, Becton Dickinson, and Company).

bleomycin has been found to induce DNA strand breaks and oxidative stress, resulting in direct injury to the cell [15 (link)]. Subsequently, cell death occurs through either necrosis or apoptosis, accompanied by inflammation and fibrosis. To induce pulmonary fibrosis, bleomycin (Cayman Chemical, Ann Arbor, MI, USA) was applied intratracheally at 80 μg in a total volume of 20 μl to the mice twice a week, and the mice were sacrificed 4 weeks later. This dose was previously confirmed in mice for intratracheal delivery [16 (link)]. For intratracheal injection, the mice were placed in supine position on the operating field and the trachea was exposed with an otoscope. The bleomycin solution was injected into the trachea directly with a syringe through a 25-gauge needle. The lung tissues were isolated for further analyses. Airway responsiveness was expressed using the “enhanced pause” (Penh) as a parameter of altered airway function [17 (link)]. Penh is an empirical parameter that reflects changes in the box flow waveform from both inspiration and expiration. To measure respiratory system resistance, the mice were subjected to whole-body plethysmography for Penh recording (DSI Buxco, St. Paul, MN, USA).

C57BL/6 wildtype mice were purchased from Charles River Laboratories (Shrewsbury, MA). All animal experiments were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee and were performed in accordance with relevant guidelines and regulations. Methods are described in accordance with ARRIVE guidelines. SDC2 transgenic mice were generated using a transgene containing the Hsdc2 coding sequence under the control of the scavenger receptor A enhancer/promoter as described previously^{28 (link)}. CAIA was induced in 8–10 week-old mice by intraperitoneal delivery of collagen type II autoantibodies (Arthritomab, BD BioSciences) at 4 mg/mouse, followed by LPS (50 μg/mouse) stimulation three days later^{37 (link)}. Pulmonary fibrosis was induced by intratracheal injection of a single dose of 0.75 mg/kg body weight of bleomycin (Cayman Chemical, Ann Arbor, MI) three days after collagen type II autoantibody administration. Control mice received an equal volume of sterile saline.

Bleomycin (Cayman Chemical, Ann Arbor, MI, USA) was dissolved in sterile phosphate-buffered saline (PBS) and diluted to 0.5 mg/mL. The upper dorsa of mice were shaved, and a square (about 1.5 cm²) was drawn with a marker. Sevoflurane inhalation was used to anesthetize mice, and 100 μL of Bleomycin was administered by using a 27-gauge needle into the shaved area rotating injection sites, every other day for 4 weeks. On the day following the last injection, CO₂ was used to euthanize the mice, and punch biopsies of 6 mm diameter were taken from the injected skin. Two skin biopsy specimens were weighed and fixed in 10% formalin and used for histological analyses. One specimen was stored in RNAlater solution (Invitrogen, Life technologies, Carlsbad, CA, USA) and processed for RNA extraction. Lesioned skin samples were obtained from age-matched male MKP-1-deficient and wild-type mice (n = 6) injected with Bleomycin. Control skin was collected from MKP-1-deficient and wild-type mice (n = 8) that did not receive Bleomycin treatment.