Supplementary Figure Legends
Figure 1. Macrophage Markers for Cultured Macrophages. CD11B+, CD11C+,
CD206 and F4/80 were all quantified using FACS analysis. CD11B+ was similar
between all the groups while CD206 and F4/80 were high in the IL4 groups. CD11C+
was also found higher in the IL4 treated macrophages.
Figure 2. Levels of M2 markers in Cultured Macrophages. Several markers of M2
polarization were examined on in vitro polarized and inhibited macrophages.
Macrophages treated with IL4 showed significant upregulation of the genes CCL22,
Hif2Alphag and YM1, while the genes Adenosine Kinase, CCL17 and TGFBeta did not
change.
Figure 3. GB111-NH2 Treatment Reduces Macrophage Viability. Macrophages
viability was determined using WST-1 reagent at day 2 after GB111-NH2 treatment.
Figure 4. ER Stress is Down-Regulated in Response to GB111-NH2 Cathepsin
Inhibition. mRNA levels of ER Stress markers, ATF4, ERDJ, p58IPK and XBP
Spliced/Non-Spliced were measured. Macrophages with Cathepsin inhibition showed
significantly lower ER Stress when compared to controls.
Figure 5. N-Acetyl-Cysteine Lowers Oxidative Stress in Macrophages. Macrophages
in all four experimental groups were treated with NAC for 2 days and then assessed for
levels of DCFDA. NAC treatment effectively lowered DCFDA in all four experimental
groups.
Figure 6. JPM-OEt Macrophage Inhibition Shows Similar Phenotype to GB111NH2. Macrophages treated with JPM-OEt show lower viability, increased Caspase 3 and
increased DCFDA.
Figure 7. Change in protein levels in polarized and non-polarized macrophages. 495
Proteins with differential expression between non-polarized macrophages and IL4polarized macrophages. Proteins were extracted using Welch t-test (FDR=0.05, S0=0.5)
on triplicates. List of the proteins can be found in table S2.
Figure 8. Proteomic Analysis of In Vitro Macrophages with and without Cathepsin
Inhibition
Interaction network of cathepsin-related proteins identified in the study. Network was
constructed using STRING database (http://string-db.org). Circles: cathepsins. Triangles:
Serpins. Hexagons: Cystatins. Grey: no data. * Significants (Welch test, FDR threshold =
0.1)
Figure 9. GB123 Stain of Tumor Activated Macrophages. Frozen sections were
stained with DAPI (Blue) and F4/80 (Green) (Left Panel). Sections were then stained
with DAPI (Blue), F4/80 (Green) and treated with GB111-NH2 inhibitor before staining
with GB123 (Middle Panel). Finally sections were stained with DAPI (Blue), F4/80
(Green) and GB123 (Red) (Right Panel).
Figure 10. Cell Death in Circulating Leukocytes. Blood was extracted from GB111NH2 mice upon sacrificed. WBC’s were extracted and stained with CD11B+ and nucview
to quantify the percent of peripheral leukocytes undergoing apoptosis. Treated and
untreated groups showed similar levels of cell death.