Pbs 1

Whole abdominal wall samples (n = 30) were decellularized using an already published protocol validated previously in our lab [40 (link)]. Whole rat abdominal wall samples were incubated with decellularization buffers under vigorous agitation at 150 rpm. The decellularization procedure involved the incubation of the samples in CHAPS buffer (pH 7; 8 mM CHAPS, 1 M NaCl, and 25 mM EDTA in PBS 1× (Sigma-Aldrich, Darmstadt, Germany) for 18 h at room temperature (RT). Next, the rat abdominal wall samples were washed 3 times (10 min each time) with PBS 1× (Sigma-Aldrich, Darmstadt, Germany) under continuous agitation (150 rpm) at RT. The samples were incubated in SDS buffer (pH 7; 1.8 mM SDS, 1 M NaCl, and 25 mM EDTA in PBS 1×; Sigma-Aldrich, Darmstadt, Germany) for another 18h at RT. The samples were again washed 3 times (10 min each time) with PBS 1× (Sigma-Aldrich, Darmstadt, Germany) under continuous agitation (150 rpm) at RT. Finally, the samples were placed in a solution containing a-Minimum Essentials Medium (a-MEM; Sigma-Aldrich, Darmstadt, Germany) supplemented with 40% v/v fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) for 36 h at 37 °C and washed again, as described before. The whole decellularization procedure was repeated another 2 times (in total, 3 decellularization cycles, n = 5 samples/decellularization cycle).

Monocyte-derived macrophages were infected with the R5-tropic HIV and the infection was left to progress. At day 11, infection percentage was evaluated by p24 intracellular staining as described in the following paragraph (Figure S1 in Supplementary Material). After that, MDMs were washed twice with PBS 1× (Sigma) and rhMIF was added to a final concentration of 1, 10, or 25 ng/ml. Cells were incubated at 37°C for 8 h until the supernatant was collected. When denoted, pretreatment with the αCD74 blocking antibody (or the appropriate isotype control) was performed at 5 ng/ml for 30 min. In some experiments (TLR4 expression), Fc receptors were blocked for 10 min before the addition of the αCD74 blocking antibody (or its isotype-matched control) with an Fc blocking reagent from BD Biosciences.

For DNA quantification, non-decellularized (n = 10) and decellularized (n = 30, n = 10/decellularization cycle) samples were initially digested using proteinase K (25 μg/mL; Sigma-Aldrich, Darmstadt, Germany) in PBS 1× (Sigma-Aldrich, Darmstadt, Germany) at 56 °C for 12 h, followed by inactivation at 96 °C for 5 min. Next, isolation of the DNA of each sample was performed, and the DNA was finally eluted in 50 μL RNAse-free water (Sigma-Aldrich, Darmstadt, Germany). The DNA content of each sample was spectrophotometrically determined using Nanodrop (Thermo Fischer Scientific, Waltham, MA, USA) at 260/280 nm. Also, the DNA quantification results were verified using DNA electrophoresis on 1% w/v agarose gel. Images were acquired with the UVITEC Imaging System (Cleaver Scientific, Warwickshire, UK).