Fig 2: Immunoblot analyses of proteins regulating glutaminolysis and lipogenesis in nonadherent MIAPaCa-2 (mutant KRAS) and BxPC3 (wild-type KRAS) cancer cells treated with CPT. A total of 5×105 cells were seeded into poly-HEMA-coated 60 mm dishes and treated with CPT at 10 and 20 µM for 48 h. GAPDH was used as the internal control. KRAS, Kirsten rat sarcoma viral oncogene homolog; CPT, cryptotanshinone; GOT, glutamic-oxaloacetic transaminase; GLUD, glutamate transport system permease protein; GLS, glutaminase; IDH, isocitrate dehydrogenase; FASN, fatty acid synthase; ACC1, acetyl-CoA carboxylase 1; ACLY, ATP-citrate lyase; p-, phosphorylated.

Fig 3: Targeting GLS1 inhibits alkali burn-induced neovascularization in the mouse corneas. (a) Representative images of the macroscopic CoNV appearance after DMSO or CB-839 treatment on day 7 after alkali burn injury. (b) Immunofluorescence staining of CD31 (red) in the mouse corneas from different groups. Scale bars: 100 µm. (c) Statistical analysis of the clinical assessments for CoNV. (d) Statistical analysis of CD31-positive cell number. ELISA assay was performed to examine the concentration of VEGF-A (e) and PDGF-BB (f) in the normal corneas (control) and the alkali-injured corneas treated with DMSO or CB-839 (200 mg/kg) for 1, 3, and 7 days. n = 5 - 6/group for ELISA or immunofluorescence. Each experiment repeated three times, and data in graphs represent the means ± SEM. *P < 0.05.

Fig 4: Targeting glutaminase 1 (GLS1) inhibits M2 polarization of macrophages in the alkali-injured mouse corneas. (a) RT-PCR analysis of M1 and M2 marker genes in the alkali-injured mouse corneas treated with DMSO or CB-839 (200 mg/kg) for 7 days. (b) Immunofluorescence double staining of F4/80 (red) and M2-specific marker Mrc1 (green) in the alkali-injured mouse corneas treated with DMSO or CB-839 for 7 days. Scale bars: 50 µm. (c) Representative Western blot and quantitative analysis of STAT1, phospho-STAT1 (Tyr701), STAT3, phospho-STAT3 (Tyr705), STAT6, and phospho-STAT6 (Tyr641) in the normal corneas (control) and the alkali-injured corneas treated with DMSO or CB-839 for 7 days. ß-Actin served as an endogenous reference for normalization. n = 6 - 8/group for RT-PCR or Western blotting and n = 4 - 6/group for immunofluorescence. Each experiment repeated three times, and data in graphs represent the means ± SEM. ns: no significance. *P < 0.05.

Fig 5: Targeting glutaminase 1 (GLS1) changes the basic biology of macrophages in vitro. (a) Glutamine consumption rates for BMDMs measured after DMSO or different concentrations of CB-839 treatment. Medium was collected after 6 hours of treatment and analyzed for glutamine. (b) [3H]-Thymidine incorporation in DNA in BMDMs treated with different doses of CB-839 for 24 h. (c) The percentage of apoptotic BMDMs 24 h after treatment with DMSO or different concentrations of CB-839 was determined by Annexin-V/PI staining and flow cytometry. (d) Representative Western blot and quantitative analysis of ITGAM, ITGB2, and ICAM-1 protein expression in BMDMs treated with CB-839 (1 µM) for 24 h. ß-Actin served as an endogenous reference for normalization. (e) After stimulating BMDMs with TNF-a (10 ng/ml) alone or combined with CB-839 (1 µM) treatment for 1 h, fluorescence microscope was applied to observe the number of adhesion of calcein-AM-labeled BMDMs to monolayers of MRMECs. Each experiment repeated three times, and data in graphs represent the means ± SEM. *P < 0.05.