Anti il 6

To establish primary mammospheres, cells were plated on poly-HEMA coated dishes as single cell suspension in standard mammosphere medium containing phenol red-free DMEM/F12 (GIBCO), B27 supplement (50x, no vitamin A; Life Techonologies) and recombinant epidermal growth factor (rhEGF, 20 ng/ml; SIGMA). Where indicated, cells were plated in medium containing phenol red-free DMEM/F12, B27 supplement and 5% WF. In a subset of experiments, blocking antibody anti-IL6 (R&D Systems, 0.2μg/ml) or STAT3 inhibitors (S3I-201; STA-21; Stattic; Galiellalactone, purchased from Santa Cruz Biotechnology, Inc.) were added to the medium. After ten days, primary mammospheres were counted. To establish secondary mammospheres, primary mammospheres were collected, disaggregate in trypsin using 25-gauge needle fitted to a syringe. Cells were plated at the same seeding density of the primary generation. Mammosphere forming efficiency (MFE%) was calculated as follows: number of mammospheres per well/number of cells seeded per well × 100. Mammosphere self-renewal was calculated as follows: total number of secondary mammospheres /total number of primary mammospheres.

Isotype-IgG, rhIL-4, rhIL-13, M-CSF, rhIL-6, anti-IL-6 and human IL-6 Quantikine ELISA were purchased from R&D Systems. Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma. Bisindolylmaleimide I (protein kinase C (PKC) inhibitor) was purchased from Calbiochem. Matrigel was from BD Biosciences. The primary human antibodies in this study were used: mouse anti-CD163, rabbit anti-CD31 (ZSGB-BIO); rabbit anti-MMP14, rabbit anti-LAMC2 (Abcam); rabbit anti-MMP9, rabbit anti-Phosphorylated PKC (pan) (Cell Signaling Technology); mouse anti-β-actin (Beyotime).

To prepare homogenates, the forebrains were homogenized in 50 mM phosphate buffer, pH 7.4 containing 10 mM EGTA, 10 mM EDTA, 0.1 mM PMSF and 100 mM NaCl in the presence of a protease inhibitor cocktail (1 μg/mL leupeptin, 0.1 μg/mL pepstatin and 1 μg/mL aprotinin). The protein concentration in homogenates was measured according to the method of Lowry et al. (1951 (link)) using bovine albumin as a standard.
Samples of 20–40 μg of protein/lane were subjected to SDS-PAGE in 10% acrylamide mini-gels, transferred further onto nitrocellulose membranes (Hybond-ECL, Amersham, UK) and examined for the expression of proinflammatory cytokines. Blots were incubated with primary antibody: anti-IL-1β (1:500, R&D System, MN, USA), anti-IL-6 (1:250; R&D System, MN, USA), anti-TNF-α (1:500, R&D System, MN, USA). Monoclonal anti-actin antibody (specific towards α, β, γ forms of the actin) was used as internal standard (1:1000; MP Biomedicals, Warsaw, Poland). Thereafter, the respective secondary anti-goat or anti-mouse HRP-conjugated antibody (Sigma Aldrich, Inc., St. Louis, MO, USA) was applied at a dilution of 1:10,000. Bands were visualized using the ECL kit and quantified by densitometric analysis using ImageScanner III (GE Healthcare) and the ImageQuant TL v2005 program.

Colonic tissues were cut on silanized glass slides and deparaffinized three times from 10% formalin fixed, paraffin-embedded colon tissue. Staining against anti-IL-6, anti-IL-1β (R&D Systems, USA), and anti-Nrf2 (BS1258) (1 : 100) was performed according to the kit protocol (KeyGEN, NJ, China). Rabbit polyclonal antibody phospho-Nrf2 (Ser40) (ab76026) (1 : 100) was obtained from Abcam (Cambridge, UK). Briefly, the slides were deparaffinized. Microwaves are used for antigen retrieval. The slides were microwave-boiled twice in 10 mM sodium citrate buffer containing 0.1% Tween 20 for 10 minutes. Each section was treated with 5% hydrogen peroxide and 4% peptone casein blocking solution for 20 min to diminish nonspecific staining. The slides were incubated with primary antibodies in PBS containing 5% BSA and 10% goat serum. Biotinylated secondary anti-rabbit antibodies were added and incubated at room temperature for 40 min. Streptavidin-HRP (horseradish peroxidase) was added, and after 30 min, the sections were stained with DAB substrate. Finally, counterstaining was performed using hematoxylin [26 (link)].

Immunocytochemistry and immunohistochemistry were performed according to published protocols [34 (link)]. The following primary antibodies were applied overnight at 4°C: anti-SARS-CoV-2 (G2) monoclonal antibody (at a dilution 1:300), anti-SARS-CoV-2 monoclonal antibody (1A9 clone; Genetek), anti-angiotensin-converting enzyme (ACE)-2 (Abcam), anti-thyroid transcription factor (TTF)-1 (Dako), anti-surfactant protein (SP)-B (1B9 clone; Zeta Corporation), anti-p16^INK4A (Santa Cruz), anti-IL-1β (Abcam), anti-IL-6 (R&D systems), anti-phospho-histone (Ser¹³⁹) H2AX (γΗ2ΑΧ) (Cell Signaling), anti-Ki67 (Abcam) and anti-p21^Waf1/Cip1(Cell Signaling).