Lactate

Cells were cultivated for 7 days and incubated with 0, 20, or 50 mM lactate (Sigma-Aldrich, St. Louis, MO, United States) for 17 h. The pH was verified and adjusted to that of normal culture medium for culture medium containing lactate. Each condition was tested in triplicate and experiments were repeated three times. Cells were scraped off and immediately used for protein extraction.

IPF or control MPCs were cultured under hypoxic (2% O₂) or normoxic (21% O₂) conditions for the duration of the experiment. For lactate stimulation, lactate (MilliporeSigma) in the indicated amounts was added to the media. Cells were cultured for 24 hours for most experiments, except where indicated.

Cells were plated in complete growth medium for 48 h prior to glucose uptake measurement using the Amplex Red Glucose/Glucose Oxidase Assay Kit (Invitrogen). lactate production was measured similarly, but after 96 h of growth in complete medium. Amplex Red Reagent (Invitrogen), lactate (MilliporeSigma, Burlington, MA, USA), and lactate oxidase (MilliporeSigma) were used to determine lactate concentration in the media. Fluorescence was measured at 590 nm. Background fluorescence was corrected for by subtracting the value of the control well. Fluorescence readings were then normalized to cell number.

Peredox is a genetically encoded, circularly permuted fluorescent reporter of the NADH/NAD + ratio. The probe was excited at 387 ± 5 nm and 575 ± 12 nm and emission collected at 530 ± 20 nm and 628 ± 14 nm. Peredox can be calibrated by replacing the glucose in the aCSF with lactate and/or pyruvate (both Sigma), which are the products and substrates of the lactate dehydrogenase (LDH). lactate will cause LDH to produce NADH (hence increase Peredox fluorescence), while adding pyruvate will have the opposite effect. Different lactate:pyruvate ratios were applied to neurons transfected with Peredox in order to obtain a relationship between the lactate:pyruvate ratio and the percentage of maximum Peredox fluorescence, with lactate alone set to 100%. lactate:pyruvate ratios were converted into NADH/NAD + levels by using the LDH equilibrium constant (k), with k = (pyruvate ∗ NADH) / (lactate ∗ NAD +) and k = 1.11 ∗ 10^− 4. This allows to convert the % of maximum fluorescence (achieved by washing on lactate only) into NADH/NAD + ratios. To calibrate single experiments, at the end of each recording, aCSF containing lactate (10 mM) was applied to obtain the maximum fluorescence of Peredox and fluorescence was converted into NADH/NAD + ratios. All measured values were normalised to the signal obtained from mCherry, which is tagged to the Peredox protein. Images were taken every 20 s.

Ethanol, Folin–Ciocalteu’s phenol reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), aluminum chloride hexahydrate, sodium nitrite, (+)-catechin, gallic acid monohydrate, sodium acetate anhydrous, potassium chloride, hydrochloric acid, sodium carbonate anhydrous, oxlalic acid, tartaric acid, malic acid, lactate, acetic acid, citric acid, succinic acid, fumaric acid, fructose, glucose, sucrose, maltose, lactose, sorbitol, gallic acid, protocatechuic acid, catechol, catechin, chlorogenic acid, epigallocatechin gallate, caffeic acid, epicatechin, syringic acid, 4-methycatechol, epicatechin gallate, p-coumaric acid, ferulic acid, and rutin were purchased from Sigma (St. Louis, MO, USA).

PDGF Receptor beta (ab32570), alpha smooth muscle Actin (ab5694), PDGF-BB (ab23914), α-SMA (ab5694), FAP-α (ab53066), β-Tubulin (ab179513), GLUT1 (ab115730), Bcl-2 (ab32124), DAPI, LDH-B (ab240482), LC3β (ab53066), GLUT1 (ab115730), obtained from Abcam. Human Cytokine Antibody Array 1000 Chip (QAH-CAA-1000) purchased from Raybiotech, IgG (A7028) from R&D, Recombinant human PDGF-BB protein from Peprotech. Dylight 488 goat anti-mouse IgG, Dylight 649 goat anti-rabbit IgG from Abbkine. MCT4 (bs-2698R), LDHA (bs-18205R), Hexokinase II (bs-9455R), MCT1/SLC16A3 (bs-10249R) antibodies from Bioss. NF-κB p65 (GB11142), MT-ND1 pAb (GB113284), NF-κB p65 (phospho S536) antibodies from Servicebio. PINK1, Parkin, ULK1, LC3A/B, AMPK, from CST. CCCP, Mdivi-1, SC74751, α-cyano-4-hydroxycinnamate (CHC) from Selleck. Finally, Monodansylcadaverine (MDC) and Lactate from Sigma.