Additional file 1 of FOXD1 regulates cell division in clear cell renal cell carcinoma

Additional file 1 Table S1: Summary of patient clinical data used for FOXD1 immunohistochemistry analysis. Table S2: Primers used for FOXD1 genotyping and off-target analysis. * Blue indicates primers used for sequencing mismatch region. ** Red bases indicate mismatches from FOXD1. Table S3: Primers for reference genes, FOXD1 targets, and mitochondrial metabolism components. Table S4: Nuclear morphology analysis of 786-O and 786-OFOXD1null. *Immunocytochemistry analysis of cells in different phases of the cell cycle after low density plating, based on nuclear and actin morphologies and localization. Percentage values are based on analysis of 100 cells per group across several fields. **Defects panel indicates collection of possible mitotic defects including lack of chromosome condensation (prophase), loss of spindle polarity (metaphase/anaphase), incomplete sister chromosome separation (cytokinesis), and death of daughter cell (post-mitosis G1). Figure S1: FOXD1 grade and stage analyses. (A) FOXD1 expression level comparisons at different ccRCC tumor grades based on transcriptome data from The Cancer Genome Atlas (B) FOXD1 expression level comparisons at different ccRCC tumor stages based on transcriptome data from The Cancer Genome Atlas. (C-D) Kaplan-Meier survival analyses for ccRCC patients with high versus low tumor expression of FOXD1, analyzed based of tumor stage. *p < 0.05. Figure S2: FOXD1 antibody validation. (A-B) TSA amplification staining for FOXD1 (green) on E12.5 mouse kidneys on normal and FOXD1-null backgrounds. Nuclei counterstained with DAPI (blue), (C-D) Immunohistochemistry staining of adult human kidney tissue (C) and FOXD1-null E12.5 mouse kidney (D). Figure S3: FOXD1 qPCR reference gene selection. (A) Stability scores generated by comparing candidate reference gene assays (Table S3) on equivalent mRNA quantities of 786-O versus 786-OFOXD1null analyzed using the BioRad Reference Gene Selector Tool. Colors of bars denote genes that are over (green) or under the acceptable stability threshold between samples. (B-D) Standard curves for reference genes selected for this study. Figure S4: FOXD1-null cells difficulty in tumor establishment. (A) Xenograft of 786-O and 786-OFOXD1null into flanks of 6-week old NCG mice. Red dotted line outlines tumors. Resected tumors from shown mice are shown in right-hand corner of image (B) Xenograft growth rate analysis. Figure S5: Histone H3 phosphorylation analysis. (A-B) Full membranes used for Western Blot analysis, with proteins visualized using the Bio-Rad Stain-Free gel system. Protein loading was quantified in each lane for normalization. (C-F) Full protein blots for histone H3 and phosphorylated histone H3 (Ser10) of synchronized 786-O and 786-OFOXD1null cells at designated time points after release from thymidine block. Blue boxes indicate areas used for quantification. (G) Densitometry analysis (Ser10), normalized to total protein in well. Figure S6: CDC2 Phosphorylation analysis. Full membranes and quantification used for western blot analysis of CDC2 (A-B) and phosphorylated forms at T161 (C-D) and Y15 (E-F). All protein levels were normalized to total protein loaded. Figure S7: CyclinB1 and MICU1 protein analysis. Full membranes and quantification used for western blot analysis of CyclinB1 (A-B) and MICU1 (C-D). All protein levels were normalized to total protein loaded. Figure S8: EdU pulse-chase analysis. (A) Labeling schematic of pulse-chase experiment. Cells in S phase incorporate EdU and are labeled (yellow circles). After 8 h EdU treatment, cells in G2 are labeled with EdU. After 2 h (2 h chase), labeled cells in G2 progress into mitosis (M). After an additional 2 h (4 h chase), labeled cells in M divide and enter G1. (B) Representative images showing staining of pH 3 and γH2AX at 2 h intervals following EdU pulse of both 786-O and 786-OFOXD1null. (C) EdU pulse-chase cell cycle progression analysis. Cells in G2 were labeled with EdU following 6 h or 10 h incubation with EdU. Labeled cells were analyzed for pH3 expression every 2 h post-labeling using immunocytochemistry. One hundred EdU+ cells were analyzed (N = 3). (D) Analysis of EdU labeled cells, for 6 h or 10 h inncubations, for γH2AX expression every 2 h post- labeling by immunocytochemistry. One hundred EdU+ cells were analyzed (N = 3). Figure S9: FOXD1 correlated genes involved in cell cycle regulation. Leading edge analysis of FOXD1 correlated genes from TCGA and top scoring candidates. *indicates kinases linked to phosphorylation of histone H3.