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1.
Figure 2

Figure 2. Deficiency in origin licensing proteins results in increased centrosome and centriole copy number.. From: Deficiency in Origin Licensing Proteins Impairs Cilia Formation: Implications for the Aetiology of Meier-Gorlin Syndrome.

(a–b) Exponentially growing cells were stained with anti-γ-tubulin and anti-Centrin2 to allow visualisation of centrosomes and centrioles, respectively. Cells with >2 centrosomes or >4 centrioles were scored (b). Note that previous studies with ATR-SS cells were carried out using nocodozole to accumulate M phase cells but this analysis was carried out without nocodozole addition to avoid any impact of this drug on spindle assembly17. The inset picture (a) shows the types of abnormalities observed. I: normal G2 phase centrosomes and centrioles in control hTERT fibroblasts. ORC1 deficient (ORC1 P4 hTERT) fibroblasts have defects that include II: supernumerary centrosomes and centrioles, III: highly multiple centrioles, IV: centrioles distal from the centrosome. Control-hTERT-immortalised fibroblasts were subjected to ORC1 siRNA and analyzed as above. Analysis was also undertaken in ORC1-deficient hTERT fibroblasts and in ORC1-hTERT fibroblasts following transfection with ORC1 cDNA. Similar findings were observed using a distinct antibody to mark centrioles (). (c–d) Control fibroblasts were treated with the indicated siRNA and examined as in (b) and by Western Blotting to measure knockdown efficiency using the indicated antibodies.

Tom Stiff, et al. PLoS Genet. 2013 March;9(3):e1003360.
2.
Figure 3

Figure 3. Deficiency in origin licensing proteins dramatically impairs cilia formation.. From: Deficiency in Origin Licensing Proteins Impairs Cilia Formation: Implications for the Aetiology of Meier-Gorlin Syndrome.

a–b) Control (C) or ORC1-deficient cells were induced to enter G0 by serum starvation for 24 or 48 hr and processed to identify cilia using anti-acetylated tubulin and anti-γ-tubulin antibodies to mark the entire cilia or the basal body, respectively. Lower panel shows that in ORC1-hTERT fibroblasts immunostaining with α-acetylated tubulin reveals extended perinuclear microtubular arrays around the centrosome in distinction to the ordered alignment in control cells and as reported for other cilia defective cells . c) Control (C) or ORC1-deficient hTERT cells were monitored for long term cilia formation as above after the indicated numbers of days of serum depletion. d) Origin licensing proteins were knocked down with siRNA in control hTERT cells, serum starved for 24 hrs then analysed for cilia formation as above. Although a marked defect is observed in cilia formation up to 48 h post serum starvation, cilia can form in around 50% of the cells when examined 4–5 days post serum starvation. e) ORC1-P4 hTERT cells were transfected with empty plasmid or plasmid expressing GFP-tagged ORC1 cDNA and positive cells detected with anti-GFP antibodies. The percent of GFP+ cells, representing those that have been successfully transfected, with cilia was assessed as in panel (d). ORC1 cDNA expression resulted in rescue of the defect in cilia formation. shows cilia formation in a gfp+ versus gfp cells.

Tom Stiff, et al. PLoS Genet. 2013 March;9(3):e1003360.
3.
Figure 4

Figure 4. Recruitment of Smo to cilia is deficient in ORC1-deficient fibroblasts.. From: Deficiency in Origin Licensing Proteins Impairs Cilia Formation: Implications for the Aetiology of Meier-Gorlin Syndrome.

a–f) Primary fibroblasts with the indicated deficiency (ORC1, IFT43, WDR35 or PCNT) were induced to enter G0 phase following serum depletion for 48 hr. The Shh pathway agonist SAG was then added for a further 24 hr. a) shows the percentage of cells with cilia after 3 days serum starvation. b) In control cells in the absence of SAG Smo is localised diffusely in the nucleus but not at the cilium. In the presence of SAG a strong uniform Smo staining is visible along the length of the cilium marked with acetylated-tubulin or extending from the basal body marked by γ-tubulin. c) Shows the percentage of cells with cilia and co-localised Smo with or without SAG treatment. d) Examples of abnormal Smo localisation at the cilium: (i) accumulation at distal tip, basal body and weakly along shaft, (ii) localised to distal tip only, (iii) localized to distal tip and partially along cilium shaft, (iv) localised to distal tip only, (v) accumulation at basal body, (vi) non-uniform distribution along length of cilium. e) Shows the percentage of ciliated cells with co-localised Smo. f) shows the percentage of ciliated cells with abnormally localised Smo. g) the percentage of cells with Smo localisation at the cilia following transfection of ORC1-hTERT cells with GFP-taggedORC1 cDNA. Positive GFP cells were detected with anti-GFP antibodies. Smo localisation is quantified in GFP+ cells. Images of Smo localisation in Gfp+ cells is shown in . h and i) percentage of cells with Smo at cilium (h) or percentage of ciliated cells with co-localised Smo (i) following the indicated siRNA. j and k) qRT-PCR analysis of Gli1 transcript levels in the indicated cells with or without SAG for 24 h (j) or (k) following 7 days serum starvation.

Tom Stiff, et al. PLoS Genet. 2013 March;9(3):e1003360.
4.
Figure 5

Figure 5. Deficiency in origin licensing proteins impairs cilia function in response to platelet-derived growth factor (PDGF).. From: Deficiency in Origin Licensing Proteins Impairs Cilia Formation: Implications for the Aetiology of Meier-Gorlin Syndrome.

a–b) Fibroblasts were induced to enter G0 phase following serum depletion for 48 hr. PDGF-AA or –BB and BrdU was then added and the percentage of S phase cells, monitored as BrdU+ cells, was estimated by immunofluorescence 11 (a) and 24 hr (b) later. The receptor for PDGF-AA is located in cilia whilst the –BB receptor is on the cell membrane. c) Analysis as in a) following the indicated siRNA. d) Cellular localisation of PDGFR-α or β. Anti-PDGFR-α or –β antibodies were used to examine the localisation of the two PDGF isoforms in control (C) or ORC1-P4 hTERT cells. PDGFR-α localised to the cilia, identified using anti-acetylated tubulin in control and ORC1-P4 cells although fewer cilia formed in the latter cells. PDGFR-β showed pan cellular localisation but did not co-localise with the cilium. e) Cells were induced to enter G0 phase following serum depletion for 48 hr. Serum was then re-added and the fraction of BrdU+ S phase cells monitored at the indicated times. The top panel shows the results with a control (C) primary fibroblasts, 48BR, primary fibroblasts from Sensenbrenner syndrome patients (IFT43-mutated and WDR35-mutated), PCNT defective fibroblasts and an ORC1 deficient line MGS cells. Both Sensenbrenner syndrome lines and PCNT cells showed a delayed S phase entry, similar to ORC1 defective MGS, compared to the control primary line. f) Analysis of a control hTERT immortalised cell line either without knockdown (C), treatment with control siRNA oligonucleotides (siC) or with oligonucleotides specific (si) for ORC1, ORC4, ORC6, CDC6 or CDT1. Knockdown efficiency was assessed and was similar to that observed in . Note that the control hTERT immortalised line (C) enters S phase more rapidly that the primary fibroblasts line making it difficult to allow a direct comparison between the S phase entry kinetic defects observed in the Sensenbrenner syndrome primary lines in (e).

Tom Stiff, et al. PLoS Genet. 2013 March;9(3):e1003360.
5.
Figure 6

Figure 6. ORC1 Meier-Gorlin syndrome and IFT43 Sensenbrenner syndrome fibroblasts exhibit impaired chondroinduction.. From: Deficiency in Origin Licensing Proteins Impairs Cilia Formation: Implications for the Aetiology of Meier-Gorlin Syndrome.

a–b) Phase contrast images (40×) of control (C) hTERT, ORC1-deficient MGS (ORC1-P4hTert) and IFT43-mutated Sensenbrenner (IFT43) patient derived fibroblasts at 0 hr and 24 hr following addition to aggrecan coated plates. Size distribution of aggregates from control (C), ORC1 and IFT43 fibroblasts following 24 hr micromass culture in aggrecan coated plates (n = 350 aggregates scored per line). Larger aggregate size was a feature of control fibroblasts following chondroinduction compared to ORC1 and IFT43 cells. c–d) Semi-quantitative RT-PCR amplification of VEGFA isoform a (upper band) and isoform c (lower band) either uninduced (Und) or during chondroinduction. Both isoforms were induced in control fibroblasts (C) upon chondroinduction. Whilst IFT43 cells exhibited higher endogenous levels of VEGFA isoform c, it was not maintained upon chondroinduction. Isoform a also was not induced after chrondroinduction. Similar findings were observed for ORC1 cells, although the high endogenous level of isoform c reduced less dramatically than that in IFT43 cells but did not increase in as in control cells. ELP4 was used as an amplification control. Panel (d) shows the combined quantification for isoforms a and c from the above panel. Similar findings have been observed in three independent experiments. e) Type I collagen represents a negative marker for chondroinduction as its levels decrease as differentiated chondrocytes secrete a specific extracellular matrix. Consistent with this, COL1A1 levels, as monitored by quantitative RT-PCR were found to decrease in control fibroblasts (C) during chondroinduction. Interestingly, both ORC1 and IFT43 defective patient derived cells exhibited similar levels of endogenous COL1A1 compared to control but by 48 h, the levels had less dramatically diminished compared to control cells. The results represent the mean of three experiments. f–g) Analysis of a control hTERT cell line treated with control siRNA oligonucleotides (siC) or with oligonucleotides specific (si) for ORC1, 4, 6, CDC6 or CDT1. Cells were uninduced (Und) or induced on a chondrogenic matrix then assayed for VEGFA expression as detailed in (c–d). Panel (g) shows the combined quantification for isoforms a and c from the above panel. Similar findings have been observed in two independent experiments.

Tom Stiff, et al. PLoS Genet. 2013 March;9(3):e1003360.
6.
Figure 1

Figure 1. Meier-Gorlin syndrome patients LBLs display impaired origin licensing capacity; some but not all lines show impaired S phase progression.. From: Deficiency in Origin Licensing Proteins Impairs Cilia Formation: Implications for the Aetiology of Meier-Gorlin Syndrome.

(a) EBV uses virally encoded EBNA-1, oriP and the host cell origin licensing complex for replication. ORC activity was assessed by the replicative capacity of plasmid-294, which encodes OriP and EBNA-1 in a control LBL (C) and LBLs derived from MGS patients with mutations in ORC1, ORC4, ORC6, CDC6 and CDT1. Following transfection of the EBV episome into LBLs and incubation to allow replication, episomal DNA was extracted and examined after BamH1 or BamH1+Dpn1 digestion using plasmid-294 as a probe. Dpn1 degrades unreplicated plasmids that retain bacterial Dam-dependent methylation. The EBV episome has a single BamH1 site causing linearization after digestion. Although replication of EBV is less efficient in LBLs compared to hTERT immortalised fibroblasts, ∼5% of the EBV plasmids underwent replication in control cells as shown by the presence of full length episomes (band 1) after Dpn1+BamH1 digestion. The level in MGS patient LBLs is substantially reduced. For quantification, the level of the full length plasmid band (1) was plotted relative to one of the Dpn1 digestion products (2) and normalised to that obtained in the control (C). Efficient episomal transfection was shown by the similar level of digestion products in all samples. Results represent the mean of two experiments. The reduction was highly significant (t-test, 1-tailed equal variance. Nomenclature used throughout: no significance (ns) P>0.05, * P<0.05, ** P<0.01). (b) Control (C) and ORC1 LBLs were BrdU labelled for 30 min and incubated for the indicated times before fluorescence-activated cell sorting (FACS). The percentage of early S phase cells was assessed. The rate of loss of BrdU+ early S phase cells represents the speed of S-phase progression. LBLs with mutations in ORC1, ORC4 and ORC6 show an impaired rate of S phase progression; CDT1-deficient LBLs were similar to control LBLs and the CDC6-deficient LBLs progressed more rapidly through S phase.

Tom Stiff, et al. PLoS Genet. 2013 March;9(3):e1003360.

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