Supplemental Figure 1. CAD, Pgl2, and GAPDH Sirnas Were Transfected in Replicon Cells For

Supplemental Figure 1. CAD, pGL2, and GAPDH siRNAs were transfected in replicon cells for 24 hours followed by the addition of 100 M uridine (as UTP) and cytidine (as CTP) into the cell culture media. At 72 hours, the replicon luciferase levels (A) and cell viability (B) was determined.

Supplemental Figure 2. The siRNAs targeting PI4KA-1, PI4KB-1, and GAPDH were mixed in a 1:1 ratio (25nM:25nM, etc.) as a dose response in the Luc-1b cells for 72 hours and luciferase levels were quantified (diamonds, pGL2:GAPDH, squares, PI4KA-1:GAPDH, triangles, PI4KA:PI4KB, circles PI4KB:GAPDH).

Supplemental Figure 3. RTPCR of PI4KA and PI4KB in replicon cells. (A & B) Replicon cells were transfected with a dose response of PI4KA-1 (black bars) & PI4KA-2 (grey bars) (A) or PI4KB-1 (black bars) & PI4KB-2 (grey bars) (B) siRNAs and mRNA levels were quantified using RTPCR. The RTPCR data is presented as normalized to GAPDH and is representative of three independent experiments with standard deviations.

Supplemental Figure 4. Synthesis of the PI4K binding affinity matrix using LCJ217. Based on the reported inhibitory activity of the arylthiazole PIK93 towards PIK4 and other lipid kinases, we aimed to find a suitable tool compound that could affinity capture and purify PIK4 isoforms, and therefore provide a good method for the identification and selectivity profiling of PIK4 interacting small molecules. Structure-activity relationship (SAR) data analysis (data not shown) of internal and published data led to an arylthiazolylamide that, upon extending an optimal alkyl linker bearing a primary amine in the appropriate position (R2, reaching out of the binding pocket towards the solvent), retained activity and allowed coupling onto NHS-activated Sepharose beads.

Supplemental TABLE 1. The siRNA collections were run in sets of n = 2 at 25 nM. The screening data was Z-score transformed to pick siRNAs that scored ≤ 50% below the median (≤ 0.5 or Z-score of -2).

Supplemental TABLE 2. The siRNA collections were run in sets of n = 2 at 25 nM. The screening data was Z-score transformed to pick siRNAs that scored ≥ 100% (≥ 2.0 or Z-score of 2) above the median.

Supplemental TABLE 3. Details of the siRNA and shRNAs used in the study.

Supplemental TABLE 4. Complete list of peptides from Huh-Luc/neo-ET lysates competed by PIK93 from the LCJ217 affinity matrix.

Supplemental Materials and Methods

CAD siRNA rescue with pyrimidines

Luc-1b cells were transfected with 25 nM siRNA targeting CAD (NM_004341), GAPDH and pGL2 (luciferase). After incubating for 48 hours, uridine or cytidine was added to the cells at a final concentration of 100 µM (stock solutions of uridine (Acros Organics #14077025) and cytidine (Acros Organics #111810500) were made at 500 mM each in deionized water). The 500 mM stocks were diluted to 2.5 mM in complete media and 5 µl of this solution was added to the cells for a final concentration of 100 µM. Cells were incubated for an additional 24 hours and then assayed for luciferase activity and cell viability.

Chemical Proteomics

As described in the paper’s materials and methods.