README: Supplemental Information for "Inhibition of CSF1R and KIT with pexidartinib reduces inflammatory signaling and cell viability in endometriosis"

https://doi.org/10.5061/dryad.66t1g1k7s

Description of the data and file structure

The data provided here is supplemental information derived from the investigative work that led to the primary manuscript. First, work with immunohistochemistry shows the relationship of expression patterns in the protein CSF1R and the macrophage marker, CD68. The following western blots describe dose-response assays using protein phosphorylation in immortalized endometriosis cells (12Z) to assess for upregulation of CSF1R and KIT signaling when exposed to their respective ligands. Next, western blots of control experiments are shown evaluating if pexidartinib is able to reduce protein phosphorylation independent of cell exposure to ligand stimulation. Additional control experiments show the results of gene expression when 12Z cells were exposed to pexidartinib without stimulatory ligands to assess for a ligand-independent effect. Lastly, proof of the 12Z cell line used is shown along with uncropped images of all western blots for quality assessment.

Data is presented as images of immunohistochemistry, western blots, and then results of protein phosphorylation and gene expression comparisons in various experiments.

Figure Legends for Supplemental Figures and Supplemental Information

Supplemental Figure 1. Colocalized expression of CSF1R and the macrophage marker CD68 in endometriosis lesions. Pathology-confirmed endometriosis lesions representing various lesion types were stained with CSF1R antibody (A,C,E) or CD68 antibody (B,D,F). CSF1R was expressed by both stromal and inflammatory cell types and overlapped with CD68+ cells (signifying macrophages). Colocalization appeared more consistent in advanced-stage endometriosis lesions (endometrioma and colorectal lesions). Some regions of epithelium stained for CSF1R, with these tending to occur in regions of tubal metaplasia (indicated by arrowheads in panel A) Scale bar: 100mm in all images. Images represent experiments performed in 3-4 patients per lesion subtype. 

Supplemental Figure 2. Dose response assay evaluating JNK activation at 30 and 60 minutes. At 60 minutes after MCSF stimulation there was evidence of a dose-response (A). After 30 minutes of KITL stimulation, there was a suggestion of a dose response curve, though no clear relationship existed after 60 minutes (B). Ratios shown on the phosphorylated blot reflects the normalized ratio of phosphorylated protein levels against total protein and GAPDH levels. Abbreviations: L-ladder. pJNK-phosphorylated JNK

Supplemental Figure 3. Dose response assay evaluating STAT3 activation at 30 and 60 minutes. There was no strong evidence of a dose-response curve after MCSF stimulation (A). At both 30 and 60 minutes after KITL stimulation there was a suggestion of an escalating increase in phosphorylation ratios (B). Ratios shown on the phosphorylated blot reflect the normalized ratio of phosphorylated protein levels against total protein and GAPDH levels. Abbreviations: L-ladder. pSTAT3-phosphorylated STAT3.

Supplemental Figure 4. Dose response assay evaluating AKT activation at 30 and 60 minutes. While there was not a linear increase in AKT activation, MCSF stimulation at a dose of 25ng/mL increased AKT phosphorylation ~1.3-fold at 60 minutes (A). Similarly, there was not a consistent dose response after KITL stimulation, but a dose of 25ng/mL did increase phosphorylation ~1.3-fold at 30 minutes (B). Ratios shown on the phosphorylated blot reflect the normalized ratio of phosphorylated protein levels against total protein and GAPDH levels. Abbreviations: L-ladder. pAKT-phosphorylated AKT.

Supplemental Figure 5. Histograms showing assessment for changes in phosphorylation in the presence of pexidartinib. Quantification assessment for pJNK 60 minutes after MCSF stimulation (A) and 60 minutes after KITL (B) in the presence of pexidartinib. Quantification of pSTAT3 changes 30 minutes after MCSF (C) and 30 minutes after KITL (D) in the presence of pexidartinib. Histograms represent Mean +/- SEM. Analyzed by One-Way ANOVA. * p<0.05. ** p<0.01. *** p<0.001. Abbreviations: AU-arbitrary units. pJNK-phosphorylated JNK. pSTAT3-phosphorylated STAT3. T-time. Min-minutes.

Supplemental Figure 6. Analysis of pJNK, pSTAT3, and pAKT activity in presence of pexidartinib without ligand stimulation. Western blots assessing for changes in pJNK (A), pSTAT3 (B), and pAKT (C) activity were performed without ligand stimulation to see if pexidartinib inhibition occurred independent of ligand activity. Time points of T=60 and T=90min used to reflect total drug exposure duration in ligand experiments. Abbreviations: Pex-pexidartinib; pJNK-phosphorylated JNK; pSTAT3-phosphorylated STAT3; pAKT-phosphorylated AKT; T-time; Min-minutes. Experiments were replicated using three biological replicates. Displayed western blots representative of overall results.

Supplemental Figure 7. Quantification and comparison of western blot analysis of pJNK, pSTAT3, and pAKT activity in presence of pexidartinib without ligand stimulation. Phosphorylated activity was unchanged across the majority of signaling pathways when cells were exposed to pexidartinib without ligand. Only pSTAT3 (D) was reduced at 90 minutes of exposure at both the 3 and 10mM condition. Time points of T=60 and T=90min used to reflect total drug exposure duration in ligand experiments. Histograms represent Mean +/- SEM. Analyzed by One-Way ANOVA. * p<0.05. ** p<0.01. *** p<0.001. Abbreviations: Pex-pexidartinib; AU-arbitrary units; T-time; Min-minutes. Experiments were replicated using three biological replicates and three technical replicates.

Supplemental Figure 8. Gene expression of IL8, *CCND1 *and GAPDH in the presence of pexidartinib inhibition without ligand stimulation. There were no statistically significant differences between mRNA expressions of IL8 (A) and CCND1 (B) when comparing vehicle to 3 or 10mM pexidartinib doses. The absolute gene expression of GAPDH (C) was similar across all conditions, suggesting no evidence of general suppression of gene expression. Histograms represent Mean +/- SEM. Analyzed by One-Way ANOVA. Abbreviations: Pex-pexidartinib. Experiments were replicated using four biological replicates and three technical replicates.

Proof of validation for 12Z cells. The blocked-out cell lines were used in other, non-related research projects in our lab and not part of this research study.

Complete western blots for JNK pathway assessment of possible dose response to MCSF and KITL. Complete western blots for JNK pathway assessment after MCSF (left blots) and KITL (right blots) to perform dose-response assay (Supplemental Figure 2). No membrane stripping performed after Total Antibody assessment before GAPDH probing. Abbreviations: pJNK-phosphorylated JNK.

Complete western blots for STAT3 assessment of possible dose response to MCSF and KITL. Complete western blots for STAT3 pathway assessment after MCSF (left blots) and KITL (right blots) to perform dose-response assay (Supplemental Figure 3). No membrane stripping performed after Total Antibody assessment before GAPDH probing. Abbreviations: pSTAT3-phosphorylated STAT3

Complete western blots for AKT assessment of possible dose response to MCSF and KITL. Complete western blots for AKT pathway assessment after MCSF (left blots) and KITL (right blots) to perform dose-response assay (Supplemental Figure 4). No membrane stripping performed after Total Antibody assessment before GAPDH probing. Abbreviations: pAKT-phosphorylated AKT

Complete western blots for JNK pathway assessment after MCSF and KITL in the presence of pexidartinib. Complete western blots for figures 3A and 4A of the main paper, evaluating changes in JNK signaling in the presence of pexidartinib after MCSF (left blots) and KITL (right blots). No membrane stripping performed after Total Antibody assessment before GAPDH probing. Experiments were performed in triplicate, with these western blots serving as representative of the other experimental results. Abbreviations: pJNK-phosphorylated JNK.

Complete western blots for STAT3 pathway assessment after MCSF and KITL in the presence of pexidartinib. Complete western blots for figures 3C and 4C of the main paper, evaluating changes in STAT3 signaling in the presence of pexidartinib after MCSF (left blots) and KITL (right blots). No membrane stripping performed after Total Antibody assessment before GAPDH probing. Experiments were performed in triplicate, with these western blots serving as representative of the other experimental results. Abbreviations: pSTAT3-phosphorylated STAT3

Complete western blots for AKT pathway assessment after MCSF and KITL in the presence of pexidartinib. Complete western blots for Figures 3E and 4E of the main paper, evaluating changes in AKT signaling in the presence of pexidartinib after MCSF (left blots) and KITL (right blots). No membrane stripping performed after Total Antibody assessment before GAPDH probing. Experiments were performed in triplicate, with these western blots serving as representative of the other experimental results. Abbreviations: pAKT-phosphorylated AKT.

Complete western blots for JNK pathway inhibition by pexidartinib in absence of inflammatory ligands. Complete western blots for JNK pathway assessment of pexidartinib inhibition in absence of inflammatory ligands (Supplemental Figure 6A). No membrane stripping performed after Total Antibody assessment before GAPDH probing. Experiments were performed in triplicate, with these western blots serving as representative of the other experimental results. Abbreviations: pJNK-phosphorylated JNK.

Complete western blots for STAT3 pathway inhibition by pexidartinib in absence of inflammatory ligands. Complete western blots for STAT3 pathway assessment of pexidartinib inhibition in absence of inflammatory ligands (Supplemental Figure 6B). No membrane stripping performed after Total Antibody assessment before GAPDH probing. Experiments were performed in triplicate, with these western blots serving as representative of the other experimental results. Abbreviations: pSTAT3-phosphorylated STAT3

Complete western blots for AKT pathway inhibition by pexidartinib in absence of inflammatory ligands. Complete western blots for AKT pathway assessment of pexidartinib inhibition in absence of inflammatory ligands (Supplemental Figure 6C). No membrane stripping performed after Total Antibody assessment before GAPDH probing. Experiments were performed in triplicate, with these western blots serving as representative of the other experimental results. Abbreviations: pAKT-phosphorylated AKT.

Sharing/Access information

Data was derived from the following sources and can be accessed online:

• https://endometdb.utu.fi/