*2.2. Human Blood Samples*

The Irish Blood Transfusion Service (IBTS) at St. James's Hospital in Dublin supplied leukocyte-enriched buffy coats for these studies, from donors who provided informed written consent. Ethical approval was obtained from the research ethics committee of the School of Biochemistry and Immunology at Trinity College Dublin, and all experiments were carried out in accordance with the Declaration of Helsinki. Peripheral blood mononuclear cells (PBMC) were isolated by a density gradient centrifugation. The cells were cultured in complete RPMI medium and maintained in humidified incubators at 37 ◦C with 5% CO2.

#### *2.3. Dendritic Cell Culture*

The MagniSort Human CD14 Positive Selection kit was used according to the manufacturer's protocol to positively select for CD14+ monocytes from PBMC. CD14<sup>+</sup> monocytes were then cultured at 1 × 106 cells/mL in complete RPMI and supplemented with GM-CSF (50 ng/mL) and IL-4 (40 ng/mL) to generate monocyte-derived DC. On day three of culture, half of the media was replaced with fresh media supplemented with cytokines at the same starting concentration. On day six, non-adherent and loosely adherent cells were gently removed for use. The purity of CD14loDC-SIGN+ DC was confirmed by flow cytometry and was routinely >95%. DC were cultured at 1 × <sup>10</sup><sup>6</sup> cells/mL for all further assays.

#### *2.4. Western Blot Experiments*

For detection of HO-1 expression, DC were cultured in the presence of HPP or IP (250–1000 μM) for 3, 6, and 24 h, or treated with the Nrf2 inhibitor ML385 (10 μM) for 1 h prior to treatment with HPP or IP at 1000 μM for 24 h. Cell lysates were prepared by washing cells in PBS prior to lysis in RIPA buffer (Tris 50 mM; NaCl 150 mM; SDS 0.1%; Na.Deoxycholate 0.5%; Triton X 100). For detection of Nrf2 expression, DC were cultured in the presence of HPP (1000 μM) or IP (1000 μM) for 6 or 24 h, then washed in PBS and lysed in Laemmli loading buffer. For detection of hexokinase 2 (HK2) expression, DC were cultured in the presence of HPP or IP at 1000 μM for 6 h prior to stimulation with LPS (100 ng/mL) for 12 h, then washed in PBS and lysed in Laemmli loading buffer. For detection of p-AMPK and t-AMPK expression, DC were cultured in the presence of

HPP or IP (both 1000 μM) for 15 min, then washed in PBS and lysed in Laemmli loading buffer. For the detection of p62 and LC3, DC were cultured in the presence of HPP or IP (both 1000 μM) for 6, 12, or 24 h, then washed in PBS, lysed in Laemmli loading buffer, and sonicated. All samples were lysed in the presence of a protease inhibitor cocktail and phosphatase inhibitor cocktail set. Samples were electrophoresed and transferred to PVDF. The membranes were incubated overnight at 4 ◦C with monoclonal antibodies specific for HO-1, Nrf2, HK2, p-AMPK, t-AMPK, p62, and LC3. The membranes were washed in TBS–Tween prior to incubation with an anti-rabbit streptavidin-conjugated secondary antibody for 2 h at room temperature. A Bio-Rad ChemiDoc MP system was used for developing the blots. The membranes were subsequently re-probed with a loading control, β-actin, in order to normalise the protein of interest to the loading control for densitometric analysis.
