Fig 2: Protein content of MCT1 (A), MCT4 (B), ABCG2 (C) and UGT1A (D) in Caco-2 cells measured by ProteinSimple WES. All antibodies were used in the linear response range following optimization (Fig. A.3). Pherogram and gel blot (lane) image view of duplicate representative samples are shown for treatments where significant changes were found (E–H). NS denotes nonspecific interactions of the antibodies with the fluorescence standards used in the ProteinSimple WES system. A representative view of such interactions (NS) is shown for UGT1A and Claudin 1 antibodies when run in separate lanes with all other components apart from sample [F: mock Claudin 1, mock UGT1A and (in F) in-gel blot image view; lanes 1 and 2]. An initial concentration of 1 mg/ml of Caco-2 whole cell lysate was used for MCT1 and MCT4 detection (A and B), 0.5 mg/ml for UGT1A (C) and 1.5 mg/ml for ABCG2 (and α-tubulin) (D). Statistically significant changes are indicated when compared to control cells (Control) without treatment (*P≤.02, **P≤.01, ***P≤.005). Error bars represent S.E.M. C, control; B, butyrate; A, acetate; P, propionate; L, lactate; M, mix.

Fig 3: Lactate levels are reduced and lactate transporter expressions are increased with AAV9-HA-Slc4a11 in young animals. A, Schematic of the corneal layers and a diagram of lactate movement from the cornea into the anterior chamber with the aid of lactate transporters, monocarboxylate transporter (MCT)1, 2, and 4.1,23B, Quantification of lactate levels in Slc4a11-/- animals. n = 3 (young animals, 2 female and 1 male for each group), n = 5 (older animals, 3 male and 2 female for each group). Mean ± SD. ns = not significant, P ≥ 0.1 (1-way ANOVA with Tukey’s multiple comparisons test). (C) Representative Wes immunoassay of young and older animals for lactate transporters MCT1, MCT2, MCT4, and loading control ɑ-tubulin. D, Quantification of Wes immunoassay results from young animals. n = 3, 1 male and 2 females per group. ns = not significant, P ≥ 0.1 (1-way ANOVA with Tukey’s multiple comparisons test). E, Quantification of Wes immunoassay results from older animals. n = 3, 2 males and 1 female per group. ns = not significant, P = 0.05. For MCT1, the band closest to 66 KDa was used for quantification. Lower molecular weight band was used for MCT4 quantification. AAV9-HA = adeno-associated virus serotype 9 hemagglutinin; MCT = monocarboxylate transporter.

Fig 4: MCT knockdown affects cell viability and metabolic behaviour of PCa cell lines. (A–C) LNCaP and PC3 cells were transfected with two different combinations of siRNA directed against MCT1; PC3 cells were also transfected with two different combinations of siRNA directed against MCT4; successful knockdown was validated using qRT–PCR, 3 days after transfection (n = 4). (D–E) Effects of MCT1 (siRNA #6 + #7) and MCT4 (siRNA #4 + #5) knockdown on cell growth: (D) effect of MCT1 knockdown and MCT4 on different prostate cancer cell lines when compared to the scrambled siRNA after 48 h; (E) effect of MCT1 and MCT4 inhibition on cell growth under hypoxic conditions; ***p < 0.001. (F–G) Percentage of PC3 cells in G0–G1, S and G2–M fraction when MCT1 (F) or MCT4 (G) are silenced compared to scrambled (Scr); data are expressed as mean ± SD (n = 2); ***p < 0.001. (H–K) Oxygen consumption rates (OCRs) and extracellular acidification rates (ECARs) are shown for LNCaP and PC3 cell lines under control conditions (UT) and when MCT1 (siMCT1) or MCT4 (siMCT4) are silenced; *p < 0.05. (L–Q) To assess the effects of MCT1 and MCT4 knockdown on HIF‐target genes, LNCaP and PC3 cells were transfected with siRNAs and grown for 3 days: mRNA expression was assessed by qRT–PCR; values are depicted relative to vehicle control for each siRNA (n = 4); HX, hypoxia; NX, normoxia

Fig 5: P. aeruginosa induces MCT-dependent elevation of lactate and ASL acidification in CFBE.(A) CF HBE ASL pH and G. ASL Lactate concentration after 6 hours in HCO3−-buffered basolateral salt solution containing 5 mM D-glucose in the absence (CF HBE only) or presence of 1 × 106 CFU apical PA01 (+PA01), or 100 nM MCT2 inhibitor AR-C155858 (ARC). *P < 0.05, **P < 0.01, n = 5. Data represent the means ± S.E.M.