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

Journal: Cell reports

Article Title: Intraflagellar Transport Complex B Proteins Regulate the Hippo Effector Yap1 during Cardiogenesis.

doi: 10.1016/j.celrep.2020.107932

Figure Lengend Snippet: Figure 4. IFT88 and IFT20 Are Physically Associated to YAP1, and IFT88 Modulates YAP1 Activity (A) Co-IP experiment using HeLa cells transfected with IFT88-GFP, HA-Amotl1, and YAP1-Myc (l.e., long exposure; s.e., short exposure). Co-IP experiment using HEK293 cells transfected with Flag-Amotl1 and IFT20-GFP. Endogenous levels of Yap1 are monitored. (B) Schematic representation of the IFT88 auxin-inducible degron (AID) system. (C) Western blot analysis of IFT88 AID DLD-1 cells after 2-h auxin treatment. (C0) Western blot analysis of IFT88 and YAP1 degradation after auxin treatment (0, 0.5, and 2 h). (D) Graph shows the increase in normalized YAP1 nuclear signal in cells treated with auxin (6 h) (Mann-Whitney, p value < 0.0001) (controls: 2 replicates, n = 204 cells; Auxin 2 h: 2 replicates, n = 261 cells). (E) Graph shows the increase in YAP/WWTR1 (TAZ) nuclear signal in IFT88-siRNA (48 h)-treated MDCK cells (n = 5 replicates, average cell number analyzed for each condition = 382; t test, p value, 0.004). Box and whiskers (5th–95th percentile). Outliers are represented as red dots (NT-siRNA) or blue squares (IFT88- siRNA).

Article Snippet: We performed IPs using GFP-Trap (ChromoTek) agarose beads in two conditions: Control IP (YAP1-Myc (Boin et al., 2014), pEGFPC1 and HA-Amotl1 (Ragni et al., 2017)) and IFT88 IP (YAP1-Myc, IFT88-GFP (He et al., 2014) and HA-Amotl1).

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Transfection, Western Blot, MANN-WHITNEY