Journal: Molecular Medicine Reports

Article Title: Role of IFT88 in icariin-regulated maintenance of the chondrocyte phenotype

doi: 10.3892/mmr.2018.8486

Figure Lengend Snippet: Primer sequences.

Article Snippet: The PVDF membranes were incubated with antibodies against IFT88 (cat. no. AP11138b; 1:200 dilution; Abgent Inc.), ERK and phosphorylated ERK (cat. nos.

Techniques:

Journal: Molecular Medicine Reports

Article Title: Role of IFT88 in icariin-regulated maintenance of the chondrocyte phenotype

doi: 10.3892/mmr.2018.8486

Figure Lengend Snippet: Icariin promotes ciliary assembly and IFT88 expression. (A) Primary cilia were stained for acetylated-α-tubulin (red coloration, denoted by the white arrows; magnification, ×200). (B) The histograms reveal that, compared with the control group, 10 µmol/l icariin increased primary ciliary assembly (Control group, 27.91±9.95% cf. Icariin, 34.06%±10.06; *P<0.05), and icariin moderately increased the ciliary length from 3.07±0.74 to 3.34±1.34 µm. (C) Icariin increased IFT88 gene expression, peaking at 10 µmol/l (*P<0.05 cf. 0 µmol/l icariin). (D) Icariin upregulated production of the ciliary protein, IFT88, in a concentration- and time-dependent manner (*P<0.05 cf. 0 µmol/l icariin, or treatment at 0 h). IFT88, intraflagellar transport protein 88.

Article Snippet: The PVDF membranes were incubated with antibodies against IFT88 (cat. no. AP11138b; 1:200 dilution; Abgent Inc.), ERK and phosphorylated ERK (cat. nos.

Techniques: Expressing, Staining, Concentration Assay

Journal: Molecular Medicine Reports

Article Title: Role of IFT88 in icariin-regulated maintenance of the chondrocyte phenotype

doi: 10.3892/mmr.2018.8486

Figure Lengend Snippet: Icariin enhances primary ciliary assembly and maintains the phenotype of the primary chondrocytes. (A) Primary cilia were stained for acetylated-α-tubulin (red). Functional IFT88 (green) was widely dispersed in the cytoplasm, although it was principally centralized along the ciliary axonemes, which also contained acetylated α-tubulin (denoted by the white arrows; magnification, ×200). (B) Icariin increased the proportion of primary chondrocytes with cilia, from 29.54±8.24 to 38.48±10.36%, and led to a slight elongation of the cilia from 2.48±0.85 to 2.65±0.63 µm (*P<0.05). (C) Toluidine blue staining revealed that icariin promoted cartilage matrix secretion by primary chondrocytes. (D) Expression of phenotype-associated genes in primary chondrocytes (*P<0.05). IFT88, intraflagellar transport protein 88.

Article Snippet: The PVDF membranes were incubated with antibodies against IFT88 (cat. no. AP11138b; 1:200 dilution; Abgent Inc.), ERK and phosphorylated ERK (cat. nos.

Techniques: Staining, Functional Assay, Expressing

Journal: Molecular Medicine Reports

Article Title: Role of IFT88 in icariin-regulated maintenance of the chondrocyte phenotype

doi: 10.3892/mmr.2018.8486

Figure Lengend Snippet: Icariin regulates IFT88 expression via the ERK signalling pathway. (A) Quantitative polymerase chain reaction measuring the expression levels of the IFT88 and various phenotype-associated genes after transformation of IFT88 siRNA (*P<0.05 compared with the control group; ## P<0.01 compared with the icariin group). (B) IFT88 knockdown reduced the expression of phosphorylated ERK (**P<0.01 compared with the siCon group). (C) Icariin promoted expression of IFT88 and phosphorylated ERK, and slightly increased ERK phosphorylation, after transformation of IFT88 siRNA (**P<0.01 compared with the control group). (D) An ERK inhibitor reduced IFT88 expression. Icariin upregulated IFT88 expression, but could not restore such expression in the presence of the ERK inhibitor, PD0325901 (*P<0.05; **P<0.01 compared with the control group). ERK, extracellular signal-regulated kinase; IFT88, intraflagellar transport protein 88; Ica, icariin; PD, PD0325901, Con, control; T-ERK, total ERK; P-ERK, phosphorylated ERK.

Article Snippet: The PVDF membranes were incubated with antibodies against IFT88 (cat. no. AP11138b; 1:200 dilution; Abgent Inc.), ERK and phosphorylated ERK (cat. nos.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transformation Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: The chemosensory function of primary cilia regulates cholangiocyte migration, invasion and tumor growth

doi: 10.1002/hep.30308

Figure Lengend Snippet: (A) Effect of ATP in cell migration comparing normal ciliated cholangiocytes (NHC SCR), normal deciliated cholangiocytes (NHC IFT88) and the CCA cell line HUCCT1. Representative images obtained from the wound healing assay and bar graphs showing the distance migrated by the cells relative to control (vehicle) in 24 h are depicted (**p<0.01, n=3). (B) Cell migration analysis by wound healing assay showing the effects of ATP, ADP, Apyrase and their combinations. Bar graph shows the distance migrated by the cells relative to control (vehicle) in 24 h (*p<0.05, **p<0.01, n=3). (C) Invasion assay, representative pictures and bar graph showing the percentage of invasion in 24 h (**p<0.01, n=3). (D) Proliferation rates were assessed in real time using IncuCyte or (E) by MTS assay. Results are expressed in % proliferation relative to control (vehicle).

Article Snippet: After blocking, the membranes were incubated with the appropriate primary antibodies against IFT88 (ProSci, Poway, CA), LKB1, p-LKB1(S428), AKT, p-AKT(S473), PTEN and p-PTEN(Ser380/Thr382/Thr383) (Cell Signaling), FAK (Cell Signaling) and GAPDH (ProSci) at 4°C overnight.

Techniques: Migration, Wound Healing Assay, Invasion Assay, MTS Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: The chemosensory function of primary cilia regulates cholangiocyte migration, invasion and tumor growth

doi: 10.1002/hep.30308

Figure Lengend Snippet: (A) Expression of LKB1 was evaluated in normal cholangiocytes (NHC, H69), experimentally deciliated cholangiocytes (NHC IFT88, H69 IFT88) and CCA cell lines (KMCH, HUCCT1, OZ, EGI-1) by western blotting. (B) Presence of LKB1 in primary cilia using acetylated α-tubulin or ARL13b as ciliary markers. The expression was assessed by confocal immunofluorescence on scramble normal cholangiocytes (NHC SCR). (C) Western blot comparing the effect of ATP on LKB1 phosphorylation in normal cholangiocyte cell line (NHC SCR), experimentally deciliated cholangiocytes (NHC IFT88) and iCCA cell line (HUCCT1) (**p<0.01, n=3). (D) Western blots for p-LKB1 and total LKB1 showing the effect of pre-treatment (30 min) with suramin 100 μM or H89 20 μM on the treatment with ATP for 30 min. (E) NHC cells transfected with shRNA-LKB1 (NHC LKB1) or shRNA-scramble (NHC SCR). Expression levels of LKB1 protein were evaluated by western blot (**p<0.01, n=3) and the effect of ATP on migration was evaluated by wound healing assay (**p<0.01, n=3).

Article Snippet: After blocking, the membranes were incubated with the appropriate primary antibodies against IFT88 (ProSci, Poway, CA), LKB1, p-LKB1(S428), AKT, p-AKT(S473), PTEN and p-PTEN(Ser380/Thr382/Thr383) (Cell Signaling), FAK (Cell Signaling) and GAPDH (ProSci) at 4°C overnight.

Techniques: Expressing, Western Blot, Immunofluorescence, Transfection, shRNA, Migration, Wound Healing Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: The chemosensory function of primary cilia regulates cholangiocyte migration, invasion and tumor growth

doi: 10.1002/hep.30308

Figure Lengend Snippet: (A,B) Western blots showing the effect of ATP for 30 minutes on AKT and PTEN phosphorylation in normal ciliated cholangiocytes (NHC SCR), experimentally deciliated cholangiocytes (NHC IFT88), and the iCCA cell line HUCTT1. Bar graph shows densitometry expressed as % of phosphorylated/total ratios in control conditions (*p<0.05, n=3) (**p<0.01, n=3).

Article Snippet: After blocking, the membranes were incubated with the appropriate primary antibodies against IFT88 (ProSci, Poway, CA), LKB1, p-LKB1(S428), AKT, p-AKT(S473), PTEN and p-PTEN(Ser380/Thr382/Thr383) (Cell Signaling), FAK (Cell Signaling) and GAPDH (ProSci) at 4°C overnight.

Techniques: Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: The chemosensory function of primary cilia regulates cholangiocyte migration, invasion and tumor growth

doi: 10.1002/hep.30308

Figure Lengend Snippet: (A) F-actin and filopodia were evaluated by Phalloidin staining (red) in normal ciliated cholangiocytes (NHC SCR), experimentally deciliated cholangiocytes (NHC IFT88), the iCCA cell line (HUCCT1), and normal ciliated cholangiocytes with LKB1 knockdown (NHC LKB1), in the presence or absence of ATP. Nuclei were stained in blue with DAPI (Magnification X600). (B) Quantification of filopodia after treatment with ATP for 30–60 min is shown in bars representing the average number of filopodia per cell (**p<0.01 n=67). (C) The effect of ATP for 60 min on FAK expression was evaluated by western blot (**p<0.01 n=3).

Article Snippet: After blocking, the membranes were incubated with the appropriate primary antibodies against IFT88 (ProSci, Poway, CA), LKB1, p-LKB1(S428), AKT, p-AKT(S473), PTEN and p-PTEN(Ser380/Thr382/Thr383) (Cell Signaling), FAK (Cell Signaling) and GAPDH (ProSci) at 4°C overnight.

Techniques: Staining, Expressing, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: The chemosensory function of primary cilia regulates cholangiocyte migration, invasion and tumor growth

doi: 10.1002/hep.30308

Figure Lengend Snippet: (A) Western blot analysis and graph bar showing the effect of HMC 1 mM for 30 min on LKB1 phosphorylation in normal cholangiocytes (NHC SCR), experimentally deciliated cholangiocytes (NHC IFT88 (shRNA-IFT88)) and iCCA cell lines (HUCCT1) (**p<0.01 n=3). (B) Effect of HMC 1 mM for 24–48 h on proliferation. Rates were assessed by MTS assay (**p<0.01, n=24). (C) Effect of HMC 1mM on migration. Bar graph shows the distance migrated by the cells relative to control (vehicle) in 48 h evaluated in NHC SCR, NHC IFT88 (shRNA-IFT88), and HUCCT1 cells assessed by wound healing assay (**p<0.01 n=3). (D) Effect of HMC 1mM on NHC LKB1 (shRNA-LKB1) migration, bar graph shows the distance migrated by the cells relative to control (vehicle) in 24 h (**p<0.01, n=3). (E) Effect of HMC 1 mM on apoptosis evaluated in 24 h by flow cytometry using (FITC)-annexin V/propidium iodide (PI) staining (**p<0.01 n=3). (F) The anti-tumoral effect of HMC was assessed in vivo using a rat orthotopic syngeneic CCA model. Animals were treated for 8 days with 100mg/kg HMC or vehicle after 6 days of tumor initiation. Bar graph shows tumor/liver rate (%) comparing tumors treated with saline solution (control) and HMC (*p<0.05, n=4). (G) Apoptosis in tumor tissues were assessed by DNA fragmentation detection where green dots are cells in apoptosis. Bar graph shows % nuclei/field in control and treated tumors (*p<0.05, n=4).

Article Snippet: After blocking, the membranes were incubated with the appropriate primary antibodies against IFT88 (ProSci, Poway, CA), LKB1, p-LKB1(S428), AKT, p-AKT(S473), PTEN and p-PTEN(Ser380/Thr382/Thr383) (Cell Signaling), FAK (Cell Signaling) and GAPDH (ProSci) at 4°C overnight.

Techniques: Western Blot, shRNA, MTS Assay, Migration, Wound Healing Assay, Flow Cytometry, Staining, In Vivo

Journal: Human genetics

Article Title: IFT88 mutations identified in individuals with non-syndromic recessive retinal degeneration result in abnormal ciliogenesis.

doi: 10.1007/s00439-018-1897-9

Figure Lengend Snippet: I. Segregation of IFT88 compound heterozygous mutations in the pedigree RF.C.0804. II. Ophthalmic investigations from the two affected individuals. (A and B) Color fundus images form the affected individuals III:I (aged 52) and III:II (aged 47) showing marked macular neuroretinal atrophy with some foveal sparing. There is modest optic disc pallor with mild attenuation of the arcade vascular arcades. Macular and temporal patches of sub-retinal pigment are also identified. (C and D) Kinetic perimetry from the proband III:I (aged 63) and III:II (aged 58) show a dense macular scotoma with minimal foveal sparing. There is relative sparing of visual fields in the far periphery to a Goldman III4e target in both cases. (E and F) SD-OCT cross-section images across the right (E) and left (F) fovea of the III:I (aged 63) show parafoveal and peripheral atrophy of the outer retina with some sparing centrally and nasally in the left eye. Additionally, the choroid appears thinned and atrophic. III. The sequence of the region containing mutations in IFT88 exons 13 and 20 in members of RF.C.0804 pedigree. The nucleotides altered are indicated by arrows. IV. Domain structure of IFT88 with the location of mutations marked. (a) The numbers indicate the amino acid positions. The black boxes represent coiled coil domains whilst gray boxes indicate tetracopeptide repeats (TPR). (b) A truncated protein was predicted to result from the p.Arg266* mutation.

Article Snippet: Besides its role in intraflagellary transport, IFT88 is also involved in additional biological activities including mitosis ( Delaval et al. 2011 ), hedgehog signaling ( Chang and Serra 2013 ), cell migration ( Boehlke et al. 2015 ) and cell cycle progression ( Kim and Tsiokas 2011 ).

Techniques: Sequencing, Mutagenesis

Journal: Human genetics

Article Title: IFT88 mutations identified in individuals with non-syndromic recessive retinal degeneration result in abnormal ciliogenesis.

doi: 10.1007/s00439-018-1897-9

Figure Lengend Snippet: mIMCD3 cells transiently transfected with wild type IFT88-GFP, p.Arg266* IFT88-Myc and p.Ala568Thr IFT88-Myc were analyzed. Immunostaining was performed with anti Myc antibody (green) and acetylated alpha tubulin (red) a ciliary marker. The nucleus was stained with DAPI (blue). (A-C) Mock transfected HeLa cells were stained acetyl tubulin showed normal cilia. (D-F) Wild type IFT88 (green) was localized to the tip and the base of the cilium (red) (G-I) co-Immunostaining with both acetyl tubulin and IFT88 showed mislocalization of both p.Arg266* IFT88 and (J-L) p.Ala568Thr IFT88 (green) away from the cilia. (M) Ciliary length of cells expressing wild type IFT88-GFP, p.Arg266* IFT88-Myc and p.Ala568Thr IFT88-Myc were measured using image J (NIH, USA).

Article Snippet: Besides its role in intraflagellary transport, IFT88 is also involved in additional biological activities including mitosis ( Delaval et al. 2011 ), hedgehog signaling ( Chang and Serra 2013 ), cell migration ( Boehlke et al. 2015 ) and cell cycle progression ( Kim and Tsiokas 2011 ).

Techniques: Transfection, Immunostaining, Marker, Staining, Expressing

Journal: Human genetics

Article Title: IFT88 mutations identified in individuals with non-syndromic recessive retinal degeneration result in abnormal ciliogenesis.

doi: 10.1007/s00439-018-1897-9

Figure Lengend Snippet: HeLa cells were immunostained for cilia using acetylated alpha tubulin (red). (A & B) Normal appearing cilia (red) were observed in the control and mock transfected HeLa cells. The absence of acetylated alpha tubulin staining or the presence of an abnormal pattern of staining was noted in cells with the homozygous p.Arg266*(Clone 1) (C) and p.Arg266Glufs*2 (Clone 2) (D) IFT88 mutations.

Article Snippet: Besides its role in intraflagellary transport, IFT88 is also involved in additional biological activities including mitosis ( Delaval et al. 2011 ), hedgehog signaling ( Chang and Serra 2013 ), cell migration ( Boehlke et al. 2015 ) and cell cycle progression ( Kim and Tsiokas 2011 ).

Techniques: Control, Transfection, Staining

Journal: Human genetics

Article Title: IFT88 mutations identified in individuals with non-syndromic recessive retinal degeneration result in abnormal ciliogenesis.

doi: 10.1007/s00439-018-1897-9

Figure Lengend Snippet: A. Expression of IFT88 in HeLa cell lines with homozygous p.Arg266* and p.Arg266Glufs*2 mutations. Lysates of mock transfected and control HeLa cells (lane 1 & 3), cells with p.Arg266Glufs*2 (lane 2) and cells with the p.Arg266* mutation (lane 4) were analyzed by western blot analysis using IFT88 antibodies raised against the C-terminal sequence. Presence of expected size IFT88 bands were noted in lanes 1 and 3 while no bands were detected in lanes 2 and 4. B. Levels of IFT88 transcript in HeLa cells with the homozygous p.Arg266* and p.Arg266Glufs*2 mutations. qRT-PCR analysis showed the expression of IFT88 transcript in control HeLa cells and mock transfected HeLa cells. Whereas the levels of IFT88 transcript were significantly low (P<0.0001) in both mutant cells lines. C. Expression of IFT88 in genome edited cells with homozygous p.Ala568Thr mutation. Whole cell lysates of control HeLa cells and and genome edited cells with p.Ala568Thr mutation were analyzed by western blot for expression of IFT88.

Article Snippet: Besides its role in intraflagellary transport, IFT88 is also involved in additional biological activities including mitosis ( Delaval et al. 2011 ), hedgehog signaling ( Chang and Serra 2013 ), cell migration ( Boehlke et al. 2015 ) and cell cycle progression ( Kim and Tsiokas 2011 ).

Techniques: Expressing, Transfection, Control, Mutagenesis, Western Blot, Sequencing, Quantitative RT-PCR

Journal: Human genetics

Article Title: IFT88 mutations identified in individuals with non-syndromic recessive retinal degeneration result in abnormal ciliogenesis.

doi: 10.1007/s00439-018-1897-9

Figure Lengend Snippet: The levels of IFT88 transcript in mock transfected, control HeLa cells without (IFT88-control-U) or with cycloheximide treatment (IFT88-control-T) and after recovery (IFT88-control-R) were found to be similar. A significant increase in the levels of IFT88 transcript was observed in response to treatment with cycloheximide in cells carrying the IFT88 p.Arg266* mutation (IFT88-R266*-T) compared to untreated cells (IFT88-R266*-U). In these cells, the levels of IFT88 transcript were found to be significantly lower after recovery (IFT88-R266*-R) compared to the levels observed in cycloheximide treated cells (IFT88-R266*-T) (P<0.0001).

Article Snippet: Besides its role in intraflagellary transport, IFT88 is also involved in additional biological activities including mitosis ( Delaval et al. 2011 ), hedgehog signaling ( Chang and Serra 2013 ), cell migration ( Boehlke et al. 2015 ) and cell cycle progression ( Kim and Tsiokas 2011 ).

Techniques: Transfection, Control, Mutagenesis