Journal: Scientific Reports

Article Title: KIAA0319 influences cilia length, cell migration and mechanical cell–substrate interaction

doi: 10.1038/s41598-021-04539-3

Figure Lengend Snippet: Generation of a cellular KIAA0319 knockout. ( A ) Top: Structure of Human KIAA0319 (based on and Ensembl release 94 ). The diagram shows the correspondence between protein domains and coding exons in KIAA0319. Signal peptide (SP), MANEC domain (MANEC), PKD domains (PKD), cysteine residues (C6) and transmembrane domain (TM) are indicated. Bottom: full DNA sequence of KIAA0319 exon 6 with target sequences for the gRNAs indicated with blue lines. Red lines show the position of the PAM sequences. Translated sequence of amino acids for the targeted exon is shown below the DNA sequence. ( B ) Chromatograms of the deletions found in Ex6KO and translated corresponding amino acids for wild type and knockout cell line. Asterisks indicate premature stop codons. ( C ) Results of the PCR screening to confirm the deletions in Ex6KO. The cartoon on the left represents the screening strategy. Two sets of primers were designed to give different bands in the WT and KO. The stripped area indicates the 19 and 32 base pair (bp) deletions in the exon 6 of KIAA0319. The first set of primers (Ex_6R and Ex_5F) amplifies the region around the deletion and therefore a smaller band is expected for the KO (105–118 bp) compared to the WT (137 bp). The second pair (Ex9R/Ex6delF) has one primer mapping within the deletion. PCR is expected to give a band of 360 bp in the WT and no product in the KO. Images below confirm the expected results for both pairs. Full-length images are presented in Supplementary Fig. . ( D ) Quantification of KIAA0319 mRNA in WT and Ex6KO by qPCR. KIAA0319 expression is significantly lower in Ex6KO (Student’s t -test: p ≤ 0.0001), consistent with nonsense mediated decay of the mRNA caused by the deletion. Origin 2018 version 95E ( https://www.originlab.com/2018 ) was used for data plotting and significance testing.

Article Snippet: Origin 2018 version 95E ( https://www.originlab.com/2018 ) was used for data plotting and significance testing.

Techniques: Knock-Out, Sequencing, Expressing

Journal: Scientific Reports

Article Title: KIAA0319 influences cilia length, cell migration and mechanical cell–substrate interaction

doi: 10.1038/s41598-021-04539-3

Figure Lengend Snippet: Analysis of the cilia length. ( A ) Representative immunofluorescence images of RPE1 wild type and Ex6KO, stained for cilia marker Arl13b (green), centrosomal marker γ-tubulin (red), and DAPI (blue). Insets show magnifications of the cilia indicated with small white squares. ( B ) Plot of the cilia length for wild type ( n = 129) and Ex6KO cells ( n = 104). Groups were compared using the Student’s t -test (*** p ≤ 0.001). Origin 2018 version 95E ( https://www.originlab.com/2018 ) was used for data plotting and significance testing. Scale bar, 10 μm.

Article Snippet: Origin 2018 version 95E ( https://www.originlab.com/2018 ) was used for data plotting and significance testing.

Techniques: Immunofluorescence, Staining, Marker

Journal: Scientific Reports

Article Title: KIAA0319 influences cilia length, cell migration and mechanical cell–substrate interaction

doi: 10.1038/s41598-021-04539-3

Figure Lengend Snippet: Analysis of mechanical activity of RPE1 WT and Ex6KO cells during migration on an ERISM micro-cavity. ( A ) Phase contrast (upper row) and ERISM micro-cavity displacement maps (lower row) of WT (left) and Ex6KO (right) cells. ( B ) Comparison of the surface area covered by WT ( n = 36) and Ex6KO ( n = 36) cells types. ( C ) Comparison of mean speed of WT ( n = 29) and Ex6KO ( n = 24) cells. ( D ) Comparison of mean indented volume of WT ( n = 29) and Ex6KO ( n = 24) cells. Only cells with free movement for ≥ 2 h were included in analysis of speed and indented volume. Plots in ( B,D,E ) show all measured data points and the mean (line). Groups were compared using the Student’s t -test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Origin 2018 version 95E ( https://www.originlab.com/2018 ) was used for data plotting and significance testing. Scale bar, 50 μm.

Article Snippet: Origin 2018 version 95E ( https://www.originlab.com/2018 ) was used for data plotting and significance testing.

Techniques: Activity Assay, Migration, Comparison

Journal: Scientific Reports

Article Title: KIAA0319 influences cilia length, cell migration and mechanical cell–substrate interaction

doi: 10.1038/s41598-021-04539-3

Figure Lengend Snippet: Phenotype recovery through KIAA0319 rescue. ( A ) Western blot confirming the presence of a fusion protein (140 KDa) following transfection with a full length KIAA0319 construct fused to a GFP tag. Full-length blots are presented in Supplementary Fig. . ( B ) Comparison of area covered by RPE1 WT, WT K-GFP, Ex6KO and Ex6KO K-GFP cells attached to ERISM micro-cavity. (WT: n = 16, WT K-GFP: n = 20, Ex6KO: n = 23, Ex6KO KGFP: n = 17). ( C ) Comparison of mean mechanical activity of RPE1 WT, WT K-GFP, Ex6KO and Ex6KO K-GFP cells during migration on ERISM micro-cavity. Only cells with free movement for > 4 h were included in the analysis. Plots in B and C show measured data points and the mean (line). (WT: n = 16, WT K-GFP: n = 19, Ex6KO: n = 24, Ex6KO K-GFP: n = 16) Groups were compared using the Student’s t-test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Origin 2018 version 95E ( https://www.originlab.com/2018 ) was used for data plotting and significance testing.

Article Snippet: Origin 2018 version 95E ( https://www.originlab.com/2018 ) was used for data plotting and significance testing.

Techniques: Western Blot, Transfection, Construct, Comparison, Activity Assay, Migration

Journal: Scientific Reports

Article Title: KIAA0319 influences cilia length, cell migration and mechanical cell–substrate interaction

doi: 10.1038/s41598-021-04539-3

Figure Lengend Snippet: RPE1 KIAA0319 WT and Ex6KO cells use different modes of force exertion. ( A ) Phase contrast images (upper row), Fourier-filtered ERISM displacement maps (middle row) and epi-fluorescence images (lower row, red: actin, green: vinculin, blue: nuclear DNA) of a RPE1 WT cell (left column) and an Ex6KO cell (right column). The insets (i) in the Fourier-filtered ERISM map and the epi-fluorescence image of the WT cell show magnifications of protrusions linked to actin dots. Arrows indicate positions of actin-rich cell protrusions that are counterbalanced by pulling at vinculin-rich positions marked with circles. The insets (ii,iii) show magnifications of vinculin-rich cell-substrate contacts (focal adhesions) for the WT and Ex6KO cell, respectively. ( B ) Temporal evolution of the indentation force applied by different actin dots of the WT cell shown in ( A ). ( C,D ) Temporal evolution of the contraction force applied by different focal adhesions of the WT and Ex6KO cell shown in A, respectively. ( E ) Comparison of the fraction of WT and Ex6KO cells forming actin dot protrusions. Each data point represents the mean value of an independent experiment investigating n = 5 (80%), 3 (67%) and 4 (50%) WT and n = 5 (0%), 6 (0%) and 9 (33%) Ex6KO cells, respectively. The lines depict the means. Groups were compared using the Student’s t-test (* p ≤ 0.05). Origin 2018 version 95E ( https://www.originlab.com/2018 ) was used for data plotting and significance testing. All scale bars: 20 μm.

Article Snippet: Origin 2018 version 95E ( https://www.originlab.com/2018 ) was used for data plotting and significance testing.

Techniques: Fluorescence, Comparison