Fig. 6

TJSR regulators are potential therapeutic targets. A Functional genomic siRNA screen for TJSR regulators in LNCaP cells. The indicated genes were knocked down, and the level of Cldn4 protein was measured by western blot. One hundred nM MLN was used as a reference (MLN100). B Hit validation using four individual siRNAs. Western blots show the depletion of the target proteins and the corresponding Cldn4 level. Double black arrowheads indicate native and neddylated CUL2 protein. Double white arrowheads indicate DCNL4 isoforms. C Effect of hit depletion on actin polymerization in LNCaP cells. Scale bar = 50 µm. The genes were knocked down using SmartPool siRNAs, and the effect on cell morphology was evaluated by automated fluorescence microscopy and quantified (the histogram in the middle). The histogram on the right shows the validation of the UBE2F hit using four individual siRNAs. Statistical significance: *- p < 0.05 and ***-p < 0.001. D General scheme showing distinct outcomes of neddylation inhibition in PCa cells. E Effect of gene knockdown on PC3 and LNCaP tumoroid growth measured with ViaLight™ reagent. The histogram on the right shows the validation of UBE2F and CUL2 hits using four individual siRNAs and comparison with CUL5 in PC3 tumoroids. F Effect of gene knockdown on PC3 tumoroid morphology. Scale bar = 200 µm