Fig. 5: Siah2 supports primary ciliogenesis by inhibiting Pifo and Pard3.

a Schema of the time-lapse ciliogenesis assay. Isolated P7 CGNs were nucleofected with a combination of the indicated expression vectors and cultured overnight in the presence or absence of Shh-N. Cells expressing all three vectors (BFP–NLS-tagged shRNA, Kusabira orange-tagged geminin, and Venus-tagged Arl13b) were selected, and time-lapse confocal imaging (7 min intervals) for 14 h was set up. b Representative frames of the process of primary ciliogenesis or lack thereof are as shown. The cells were scored according to whether ciliogenesis (1) observed on only 1 cell, (2) observed on both cells, or (3) fail to occur. Quantification (Control shRNA, n = 46; Control shRNA + Shh-N, n = 72; Ras(H + N) shRNA + Shh-N, n = 58; Siah2 shRNA + Shh-N, n = 59; independent cells analyzed) shows the frequency of the cells that fall within the 3 observations. Due to limited data points that fit the criteria, the result from this analysis was pooled from n = 9 independent experiments. P values derived from the χ² test (2 degrees of freedom) comparing experimental groups to “Control shRNA + Shh-N conditioned medium”. c Isolated P7 CGNs were co-nucleofected with the indicated constructs and the bicistronic vector expressing Venus-tagged Arl13b and mCherry-tagged histone H2B and cultured in vitro for 48 h. Live-cell imaging was performed followed by quantification of at least n > 1000 cells in each condition from 3 independent experiments, for the proportion of nucleofected cells (H2B-mCherry+) that were ciliated. P values derived from an unpaired one-tail student’s t test comparing experimental groups to “Siah2”. d Western blot analysis of PifoS or PifoS 2NxN stability in the presence of Siah2. HA-tagged PifoS or PifoS 2N×N was co-transfected with expression constructs of LacZ or Siah2 myc into HEK293T cells. Lysates were collected after 24 h post transfection and processed for Western blot analysis. Quantification from n = 3 independent experiments shows fold change in mean intensity measurements (Li-COR Odyssey CLx) compared to LacZ control. P value derived from an unpaired one-tail student’s t test. e Hedgehog response luciferase assay assessing the role of Siah2 in regulating Shh signaling output. Isolated P6 CGNs were nucleofected with the indicated constructs and cultured on matrigel coated white optical bottom plates for 48 h. Luciferase detection was performed using the Pierce Luciferase Glow kit and measured on the Pherastar FSX plate reader. Quantification from n = 4 independent experiments shows the fold change of the luciferase RLU relative to non-stimulated cells. P value derived from an unpaired one-tail student’s t test. Scale bar = 10 μm. Error bars represents mean ± SD.