ift88 protein  (DeLaval Inc)

Journal: Journal of Cellular Physiology

Article Title: PTH1R translocation to primary cilia in mechanically‐stimulated ostecytes prevents osteoclast formation via regulation of CXCL5 and IL‐6 secretion

doi: 10.1002/jcp.30849

Figure Lengend Snippet: CM from mechanically‐stimulated MLO‐Y4 cells inhibit the migration of monocytes by a mechanism dependent on the primary cilium and PTH1R. MLO‐Y4 osteocytic cells were transfected with three IFT88, three PTH1R siRNAs, or with a scrambled siRNA for 24 h followed by serum‐deprivation for 24 h. The efficiency of IFT88 and PTH1R silencing was tested by real time PCR (a). Alternatively, osteocyte cells were serum‐deprived for 24 h and treated with 1 mM aqueous chloral hydrate or with 100 nM PTHrP (7‐34) for 1 h. Cells were subsequently stimulated with shear stress (10 dynes/cm 2 ) or with 100 nM PTHrP (1‐37) for 10 min. CM was collected after 18 h (b−d). To evaluate the number of migratory cells, RAW 264.7 (b and c) or human monocytic cells from buffy coat (d) were cultivated in transwell cell culture chamber inserts with an 8 µm pore size. In the lower compartment 20% of each MLO‐Y4 cell‐conditioned medium was added. After 6 h, cells were fixed, stained with crystal violet, and counted with an inverted optical microscope. The number of monocytic cells evaluated with ImageJ software are represented. Results are the mean ± SD of triplicates. * p < 0.05 versus corresponding scrambled siRNA; ** p < 0.01 versus corresponding scrambled siRNA; a p < 0.01 versus SC or corresponding IFT88 siRNA/PTH1R siRNA; b p < 0.01 versus SC or corresponding cilium or PTH1R inhibition. CM, conditioned media; FF; fluid flow; IFT88, intraflagellar transport 88 protein; mRNA, messenger RNA; NC, negative control; PTHrP, PTH‐related protein; PTH1R, PTH 1 receptor; SC, static control; SD, standard deviation; siRNA, small interfering RNA.

Article Snippet: To assess the role of primary cilia and PTH1R on the effect of mechanically‐stimulated migration, we inhibited the formation of primary cilia in osteocytic cells before FF or PTHrP stimulation using two strategies; silencing IFT88, a protein required for ciliogenesis and primary cilia functional competence (Takei et al., ) or preincubation with chloral hydrate, a primary cilia inhibitor (Deren et al., ).

Techniques: Migration, Transfection, Real-time Polymerase Chain Reaction, Shear, Cell Culture, Pore Size, Staining, Microscopy, Software, Inhibition, Negative Control, Control, Standard Deviation, Small Interfering RNA

Journal: Cell Death & Disease

Article Title: Palmitic acid control of ciliogenesis modulates insulin signaling in hypothalamic neurons through an autophagy-dependent mechanism

doi: 10.1038/s41419-022-05109-9

Figure Lengend Snippet: Representative confocal images showing A phospho-IR (pIR) and B phospho-AKT (pAKT) with primary cilium (acetylated α-tubulin, Ac α-tub) double immunostaining in N43/5 hypothalamic neuronal cells. Scale bar: 10 µm. Inserts show a magnification of one cilium within the dotted square. Insert scale bar: 2 µm. Nuclei were stained with Hoechst (blue). Representative blots of N43/5 hypothalamic cells transfected with siRNA against KIF3A followed by insulin (1 nM) or PBS (control) treatment for 3 min to evaluate IR phosphorylation ( C ) or 12 min to evaluate AKT phosphorylation ( E ), with their respective quantifications ( D , F ). G Percentage of ciliated cells in N43/5 hypothalamic cells depleted of KIF3A. H Representative blot of N43/5 hypothalamic cells transfected with siRNA against IFT88 followed by insulin (1 nM) or PBS (control) treatment for 3 min to evaluate IR phosphorylation, with their respective quantifications ( I ). J Percentage of ciliated cells in N43/5 hypothalamic cells transfected with siRNA against IFT88. K Representative images of 2-NBDG uptake in N43/5 hypothalamic cells transfected with siRNA against KIF3A and IFT88 and then stimulated with insulin 1 nM for 30 min, with its quantification ( N ). Scale bar: 10 μm. Representative western blots showing protein levels of N43/5 hypothalamic cells depleted of L KIF3A or M IFT88. As control condition, cells were incubated with Lipofectamine RNAiMAX reagent only (Mock). Data are presented as mean ± SEM. Comparisons between two conditions were made using the unpaired two-tailed Student t -test. Two-way ANOVA was used for comparison of more than 2 groups, followed by Sidak’s post hoc adjustment. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant. n = 3.

Article Snippet: Cells were cultured in six-well plates and transfected at 50% confluence with siRNAs targeting murine Beclin 1 ( Becn1 ) (SASI_Mm01_00048143, Sigma-Aldrich), murine FAK family kinase-interacting protein of 200 kD ( Fip200 ) (SASI_Mm01_00196359, Sigma-Aldrich), murine kinesin family member 3A ( Kif3a ) (SASI_Mm01_00024254, Sigma-Aldrich), murine intraflagellar transport protein 88 homolog ( Ift88 ) (SASI_Mm01_00151435, Sigma-Aldrich), or murine microtubule associated protein 4 ( Map4 ) (SASI_Mm01_00207888).

Techniques: Double Immunostaining, Staining, Transfection, Western Blot, Incubation, Two Tailed Test