Fig 1: INTS13 depletion leads to a loss of primary cilia and broad transcriptional perturbation of ciliary genes.a Western blot of whole cell lysates from RPE cells treated with non-targeting control siRNA (Con.) or either of two siRNA targeting INTS13 (INTS13-1, INTS13-2). Lysates were probed for either INTS13 or tubulin as a loading control and molecular weight markers shown are in kilodaltons. RNAi-depletion and Western blotting was repeated in two independent experiments with a representative image shown. b Immunofluorescence imaging of RPE cells treated with siRNAs as in (a) to visualize primary cilia (PC). DAPI staining in blue indicates the nuclei, ?-tubulin staining is shown in red to visualize the basal body, and ARL13B staining is shown in green to visualize the ciliary axoneme. Representative images show disrupted primary cilia that occurred when INTS13 was targeted by RNAi. The scale bar for the merged image is set at 3 µm while the scale bar in the figure inset present in the center is 1 µm. c Images from (b) were quantified for each condition and graphed as a percentage of the number of control-treated cells with PC (data are mean +/- SD, con. n = 3785 cells, 13-1 n = 2969 cells, 13-2 n = 2675 cells. Raw calculations for each condition were divided by the control mean to adjust control to 1). Statistical significance was calculated using a one-way ANOVA with Tukey’s multiple comparisons test. Control vs INTS13-1 and INTS13-2 p < 0.0001, and INTS13-1 vs INTS13-2 p = 0.0024. d RNA sequencing was done on siRNA treated RPE cells in triplicate. Genes with significant changes in expression are shown by color indicating the log2 fold change. e Results from (d) were used for gene ontology analysis: the top five significant results are shown for upregulated and downregulated genes. All five GO terms for upregulated genes are related to cilia. f Quantitative RT-PCR validating three ciliary genes with expression changes identified using RNA-seq. Results are quantified from independent biological replicates (n = 3, data are mean + /- SD). Statistical significance was calculated using multiple unpaired t-tests with a Holm-Sidak correction. For 13-1 compared to control, CFAP206 p = 0.00287, CFAP53 p = 0.02511, and BBOF1 p = 0.0221. For 13-2 compared to control, CFAP206 p = 0.01878, CFAP53 p = 0.00049, and BBOF1 p = 0.00013 *p < 0.05 **p < 0.01, ***p < 0.001, ****p < 0.0001 respectively. Source data are provided as a Source Data file.