Journal: International Journal of Molecular Sciences

Article Title: Primary Cilia Restrain PI3K-AKT Signaling to Orchestrate Human Decidualization

doi: 10.3390/ijms232415573

Figure Lengend Snippet: Primary cilia are indispensable for human decidualization. ( A ) ARL13B immunofluorescence in (NH 4 ) 2 SO 4 -treated stromal cells for 24 h under in vitro decidualization for 48 h. Scale bars, 5 μm. ( B ) Ciliated cells of stromal cells after (NH 4 ) 2 SO 4 treatment for 24 h under in vitro decidualization for 48 h. ( C , D ) Relative mRNA levels of IGFBP1 and PRL of stromal cells after (NH 4 ) 2 SO 4 treatment for 24 h under in vitro decidualization for 48 h. ( E ) Effects of CBA treatment for 24 h on ARL13B immunofluorescence under in vitro decidualization of stromal cells for 48 h. Scale bars, 5 μm. ( F ) Effects of CBA treatment for 24 h on the percentage of ciliated cells under in vitro decidualization of stromal cells for 48 h. ( G , H ) Effects of CBA treatment for 24 h on relative mRNA levels of IGFBP1 and PRL under in vitro decidualization of stromal cells for 48 h. ( I ) ARL13B immunofluorescence after stromal cells were transfected with si IFT88 for 24 h then in vitro decidualization for 48 h. Scale bars, 5 μm. ( J , K ) Ciliated cells and IFT88 protein levels after stromal cells were treated with si IFT88 for 24 h then in vitro decidualization for 48 h. ( L – N ) Relative mRNA levels of IFT88 , IGFBP1 , and PRL after stromal cells were transfected with si IFT88 for 24 h followed by in vitro decidualization for 48 h. In ( A , E , I ), the representative images of three biologically independent experiments are shown. In ( B – D , F – H , J – N ), data are presented as means ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by two-tailed Student’s t test for comparing two groups or one-way ANOVA test for comparing more than two groups.

Article Snippet: The primary antibodies used were as follows: rabbit anti-IFT88 antibody (1:2000, 13967-1-AP, Proteintech), rabbit anti-PTEN (1:1000, 9188s, Cell Signaling), rabbit anti- p-AKT (ser473) (1:1000, 4058s, Cell Signaling), rabbit anti-T-AKT (1:1000, 9272s, Cell Signaling), rabbit anti-FOXO1 antibody (1:1000, 2880s, Cell Signaling), rabbit anti-Phospho-Stat3 (Tyr705) (1:1000, 9131s, Cell Signaling), mouse anti-Stat3 antibody (1:1000, 9139s, Cell Signaling), rabbit anti- Aurora A (1:1000, 14475s, Cell Signaling), rabbit anti-Phospho-Aurora A (Thr288) Antibody (1:1000, AF3011, Affinity Biosciences), rabbit anti-TCHP antibody (1:1000, 25931-1-AP, Proteintech), mouse anti-GAPDH antibody (1:1000, SC-32233, Santa Cruz), and rabbit anti-α-Tubulin antibody (1:1000, 2144s, Cell Signaling).

Techniques: Immunofluorescence, In Vitro, Transfection, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Primary Cilia Restrain PI3K-AKT Signaling to Orchestrate Human Decidualization

doi: 10.3390/ijms232415573

Figure Lengend Snippet: Primer sequences used in this study.

Article Snippet: The primary antibodies used were as follows: rabbit anti-IFT88 antibody (1:2000, 13967-1-AP, Proteintech), rabbit anti-PTEN (1:1000, 9188s, Cell Signaling), rabbit anti- p-AKT (ser473) (1:1000, 4058s, Cell Signaling), rabbit anti-T-AKT (1:1000, 9272s, Cell Signaling), rabbit anti-FOXO1 antibody (1:1000, 2880s, Cell Signaling), rabbit anti-Phospho-Stat3 (Tyr705) (1:1000, 9131s, Cell Signaling), mouse anti-Stat3 antibody (1:1000, 9139s, Cell Signaling), rabbit anti- Aurora A (1:1000, 14475s, Cell Signaling), rabbit anti-Phospho-Aurora A (Thr288) Antibody (1:1000, AF3011, Affinity Biosciences), rabbit anti-TCHP antibody (1:1000, 25931-1-AP, Proteintech), mouse anti-GAPDH antibody (1:1000, SC-32233, Santa Cruz), and rabbit anti-α-Tubulin antibody (1:1000, 2144s, Cell Signaling).

Techniques:

Journal: The Journal of Cell Biology

Article Title: The β-cell primary cilium is an autonomous Ca 2+ compartment for paracrine GABA signaling

doi: 10.1083/jcb.202108101

Figure Lengend Snippet: The primary cilium of intact islets of Langerhans. (A) Confocal microscopy image of a mouse islet where cilia are visualized by immunofluorescence staining against acetylated tubulin (magenta) and nuclei with Dapi staining (blue). (B) Confocal microscopy image of a small mouse islet showing insulin in green, acetylated tubulin in magenta, and nuclei in blue. (C) Maximum intensity projection of part of a mouse islet (outlined by dashed line) immunostained against acetylated tubulin. (D) Epifluorescence microscopy images of pancreas sections immunostained for acetylated tubulin (magenta), insulin (green, top panel), and glucagon (green, bottom panel). (E) 3D reconstruction of confocal micrographs of a section of a mouse islet that contains a cluster of primary cilia (pseudo-colored for clarity). (F) Quantification of the number of cilia found within a radius of 2.5 µm of another cilia compared to at random locations. *** P < 0.001 (two-tailed Student's unpaired t test). (G) Example STED images showing islet cell cilia morphologies (green) and the corresponding distribution of GABA B1 receptors (magenta). (H) Distribution of cilia morphologies in mouse islets ( n = 101 cilia from 6 islets). (I) Length of cilia and GABA B1 receptor compartment in mouse islets separated based on cilia morphology (means ± SEM; n = 101 cilia from 6 islets; two-tailed unpaired Student’s t test). (J) Immunofluorescence images showing the localization of Patched, Smoothened, IFT88, and SSTR3 (all green) to the primary cilium (acetylated tubulin; magenta) of mouse islet cells. (K) Immunofluorescence images (top) and quantification of fluorescence intensity (bottom) of acetylated tubulin (green) and Patched (magenta) in mouse islet cell cilia ( n = 146 cilia, 15 islets, 3 animals). (L) Confocal microscopy image of a large human islet showing acetylated tubulin in green.

Article Snippet: The following antibodies were used in the study: acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), IFT88 (F41236, host: rabbit, 1:200; NSJ Bioreagents), SSTR3 (E-AB-1607, host: rabbit, 1:200; Elabscience), Patched (ab53715, host: rabbit, 1:200; Abcam), Smoothened (ab113438, host: rabbit, 1:200; Abcam), GABA-B1 receptor (NBP1-52389, host: goat, 1:200; Novus Biologicals and AGB-001, host: rabbit, 1:200; Alamone Labs), GABA-B2 receptor (ab230136, host: rabbit, 1:200; Abcam), GABA-A receptor (224-103, host: rabbit, 1:200; Synaptic Systems), CNGA3 (ab2519323002150000, host: rabbit, 1:200; Abcam), γ-tubulin (T5326, host: mouse, 1:200; Sigma-Aldrich), Arl13b (ab136648, host: mouse, 1:300; Abcam), CaV1.2 (ACC-022, host: rabbit, 1:100; Alamone labs), CaV1.3 (ACC-005, host: rabbit, 1:100; Alamone labs), and CNGB1 (bs-12093R, host: rabbit, 1:100; Bioss antibodies).

Techniques: Confocal Microscopy, Immunofluorescence, Staining, Epifluorescence Microscopy, Two Tailed Test, Fluorescence

Journal: bioRxiv

Article Title: Mesoderm-specific transcript reduces ciliary sphingomyelin levels to promote tendon stem/progenitor cells osteochondrogenesis in traumatic heterotopic ossification

doi: 10.1101/2025.03.01.640937

Figure Lengend Snippet: (a) GO enrichment anaylsis of differential expressed genes in tHO mice injury site shows top 10 enriched functions that are important for tHO. (b) GO enrichment analysis of differential expressed genes in TSPCs treated with shIft88 shows top 10 enriched biological processes. (c) GSEA enrichment of TSPCs treated with shIft88 or shNC . The enriched pathway with the absolute value of normalized enrichment score (NES) greater than 1 and P-value less than 0.05 was considered significant. (d) KEGG enrichment analysis of differential expressed genes in Mesenchymal subcluster of tHO injury site identified by scRNA-seq shows top 10 enriched pathways. (e) KEGG enrichment analysis of differential expressed genes in TSPCs treated with shIft88 showed top enriched pathways. (f) qPCR showed relative Hh signaling genes (Shh, Ptch1) mRNA expression in TSPCs with or without shIft88 or shARL3 treatment after 3 weeks of in vitro osteogenic or chondrogenic differentiation. (g) Western blot showed Hh signaling (SHH, PTCH1) and PI3K-Akt signaling (PI3K, p-PI3K, AKT, p-AKT) protein expression levels in TSPCs with or without shIFT88 or shARL3 treatment during in vitro osteogenic or chondrogenic differentiation. (h) The expression level of Smo in each cluster are shown by violin plot. (i) UMAP plot shows the expression of Smo at different time points (Day0 ,3, 7, 21) during tHO development. Data are presented as means ± SD of three independent assays. Statistical analyses were performed by one-way ANOVA analyses with Tukey’s post hoc test for multiple comparisons. ***P < 0.005; ****P < 0.001.

Article Snippet: Primary antibodies used in western blot are as follows: MEST (1:1000, #11118-1-AP, Proteintech Group Inc.), GLI2 (1:2000, #28245-1-AP, Proteintech Group Inc.), PTCH1 (1:1000, #ab53715, abcam), SHH (1:2000, #20697-1-AP, Proteintech Group Inc.), IFT88 (1:6000, #13967-1-AP, Proteintech Group Inc.), ARL3 (1:1000, #10961-1-AP, Proteintech Group Inc.), PI3K (1:1000, #ab191606, abcam), p-PI3K (1:2000, #ab278545, abcam), AKT (1:2000, #ab8805, abcam), p-AKT (1:1000, #ab38449, abcam).

Techniques: Expressing, In Vitro, Western Blot

Journal: bioRxiv

Article Title: Mesoderm-specific transcript reduces ciliary sphingomyelin levels to promote tendon stem/progenitor cells osteochondrogenesis in traumatic heterotopic ossification

doi: 10.1101/2025.03.01.640937

Figure Lengend Snippet: (a) Western blot shows Hh signaling (SHH, PTCH1) protein expression levels in TSPCs with or without siMest treatment during in vitro osteogenic or chondrogenic differentiation. (b) Western blot showed PI3K-Akt signaling (PI3K, p-PI3K, AKT, p-AKT) protein expression levels in TSPCs with or without siMest treatment durig in vitro osteogenic or chondrogenic differentiation. (c) The sequence logo of GLI2 transcription factor and two potential binding sites of the GLI2 transcription factor on the MEST promoter region predicted by the JASPAR database. (d) The luciferase reporter assay using pGL4.10 as vector showed that GLI2 can bind to the MEST promoter at both site 1 and 2. (e) Immunohistochemistry images demonstrats GLI2 in tHO tendon tissues at different time post burn/tenotomy injury (scale bar 100 um). (f) qPCR shows relative Gli2 mRNA expression in TSPCs with or without shIft88 or shARL3 treatment after 3 weeks of in vitro osteogenic or chondrogenic differentiation. (g) Western blot shows GLI2 protein expression levels in TSPCs with or without shIFT88 or shARL3 treatment during in vitro osteogenic or chondrogenic differentiation. (h) Immunohistochemistry images demonstrats GLI2, PTCH1, and SMO expression in tHO tendon tissues with or without shMest treatment after injury (scale bar 150 um). Data are presented as means ± SD of three independent assays. Statistical analyses were performed by one-way ANOVA analyses with Tukey’s post hoc test for multiple comparisons. **P < 0.01; ****P < 0.001.

Article Snippet: Primary antibodies used in western blot are as follows: MEST (1:1000, #11118-1-AP, Proteintech Group Inc.), GLI2 (1:2000, #28245-1-AP, Proteintech Group Inc.), PTCH1 (1:1000, #ab53715, abcam), SHH (1:2000, #20697-1-AP, Proteintech Group Inc.), IFT88 (1:6000, #13967-1-AP, Proteintech Group Inc.), ARL3 (1:1000, #10961-1-AP, Proteintech Group Inc.), PI3K (1:1000, #ab191606, abcam), p-PI3K (1:2000, #ab278545, abcam), AKT (1:2000, #ab8805, abcam), p-AKT (1:1000, #ab38449, abcam).

Techniques: Western Blot, Expressing, In Vitro, Sequencing, Binding Assay, Luciferase, Reporter Assay, Plasmid Preparation, Immunohistochemistry

Journal: bioRxiv

Article Title: Mesoderm-specific transcript reduces ciliary sphingomyelin levels to promote tendon stem/progenitor cells osteochondrogenesis in traumatic heterotopic ossification

doi: 10.1101/2025.03.01.640937

Figure Lengend Snippet: (a) Representative images of alizarin red staining (upper) and alcian blue staining (lower) in TSPCs after 3 weeks of in vitro osteogenic (upper) and chondrogenic (lower) differentiation in control, myriocin, sphingomyelin, myriocin+sphingomyelin, myriocin+siMest, and myriocin+vismodegib treated groups. (b) Quantification of the percentages of (alizarin red) ARS+ and alcian blue (AB+) areas in six groups. (c) qPCR showed relative osteogenic differentiation-related genes (Alpl, Runx2, Bglap ) and chondrogenic differentiation-related genes ( Sox9, Col2a1, Acan ) mRNA expression in TSPCs after 3 weeks of in vitro osteogenic (upper) or chondrogenic (lower) differentiation in six groups. (d) qPCR showed relative Hh signaling genes ( Shh, Ptch1, Gli2 ) mRNA expression in TSPCs after 3 weeks of in vitro osteogenic differentiation in six groups. (e) Micro-CT images show tHO of lower limbs between 1X PBS and sphingomyelin treated groups (n= 4/group). Quantification of tHO degree by bone volume (BV, mm^3) and bone mineral dendity (BMD, g/cc). Data are presented as means ± SD of three independent assays. Statistical analyses were performed by one-way ANOVA analyses with Tukey’s post hoc test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001. ns, P > 0.05.

Article Snippet: Primary antibodies used in western blot are as follows: MEST (1:1000, #11118-1-AP, Proteintech Group Inc.), GLI2 (1:2000, #28245-1-AP, Proteintech Group Inc.), PTCH1 (1:1000, #ab53715, abcam), SHH (1:2000, #20697-1-AP, Proteintech Group Inc.), IFT88 (1:6000, #13967-1-AP, Proteintech Group Inc.), ARL3 (1:1000, #10961-1-AP, Proteintech Group Inc.), PI3K (1:1000, #ab191606, abcam), p-PI3K (1:2000, #ab278545, abcam), AKT (1:2000, #ab8805, abcam), p-AKT (1:1000, #ab38449, abcam).

Techniques: Staining, In Vitro, Control, Expressing, Micro-CT

Journal: bioRxiv

Article Title: Mesoderm-specific transcript reduces ciliary sphingomyelin levels to promote tendon stem/progenitor cells osteochondrogenesis in traumatic heterotopic ossification

doi: 10.1101/2025.03.01.640937

Figure Lengend Snippet: After trauma, the level of SHH within the primary cilia of TSPCs is significantly elevated. This increase in SHH activates the Hh signaling through its interaction with PTCH1 and SMO receptors. Subsequently, the GLI2 transcription factor is activated and translocates to the nucleus, where it promotes the expression of the MEST gene. Concurrently, the phosphorylation levels of PI3K and AKT are enhanced. The upregulation of MEST expression leads to a reduction in sphingomyelin within the cilia, further amplifying the activation of the ciliary Hh signaling. This positive feedback loop dramatically enhances the osteogenic and chondrogenic differentiation potential of TSPCs, resulting in localized ectopic bone deposition and tHO. Conversely, when the ciliary genes Ift88 and Arl3 are knocked down in TSPCs, the production of SHH ligands is markedly reduced. Thus, SMO is inhibited by PTCH1, preventing the activation of GLI2. Additionally, the phosphorylation of PI3K and AKT is suppressed. The reduction in MEST expression subsequently increases the sphingomyelin within the cilia, which further inhibits the ciliary Hh signaling. As a result, the osteogenic and chondrogenic differentiation capacity of TSPCs is significantly diminished, thereby alleviating the occurrence and development of tHO.

Article Snippet: Primary antibodies used in western blot are as follows: MEST (1:1000, #11118-1-AP, Proteintech Group Inc.), GLI2 (1:2000, #28245-1-AP, Proteintech Group Inc.), PTCH1 (1:1000, #ab53715, abcam), SHH (1:2000, #20697-1-AP, Proteintech Group Inc.), IFT88 (1:6000, #13967-1-AP, Proteintech Group Inc.), ARL3 (1:1000, #10961-1-AP, Proteintech Group Inc.), PI3K (1:1000, #ab191606, abcam), p-PI3K (1:2000, #ab278545, abcam), AKT (1:2000, #ab8805, abcam), p-AKT (1:1000, #ab38449, abcam).

Techniques: Expressing, Activation Assay

Journal: Cell Reports Medicine

Article Title: Patient-derived kidney organoids recapitulate ADPKD and facilitate the identification of Rho pathway inhibitors as candidate therapeutics

doi: 10.1016/j.xcrm.2026.102720

Figure Lengend Snippet: Cilia loss selectively disrupts planar cell polarity and Rho GTPase signaling in PKD organoids (A) Western blot validation of IFT88 knockout efficiency confirmed successful ciliary ablation in the corresponding organoid groups. (B) Mfuzz clustering of transcriptomic profiles from six experimental groups: wild-type control organoids (CON_org), control organoids with IFT88 knockout (CON_ IFT88 KO_org), PKD1 -mutant organoids (PKD1_org), PKD1 mutants with IFT88 knockout (PKD1_ IFT88 KO_org), PKD2 -mutant organoids (PKD2_org), and PKD2 mutants with IFT88 knockout (PKD2_ IFT88 KO_org). Distinct temporal expression patterns were identified, with enrichment of Rho GTPase signaling components in PKD1/PKD2_ IFT88 KO groups. (C) Heatmap summarizing gene set enrichment analysis (GSEA) results across all groups, showing normalized enrichment scores (NES, left) and corresponding p values (right). Cilia-related pathways were significantly downregulated in all IFT88 -knockout organoids. Rho GTPase and cell-cycle-related pathways were specifically suppressed in PKD1/PKD2_ IFT88 KO groups. (D) Comparative transcriptomic profiling revealed coordinated downregulation of genes associated with planar cell polarity and cytoskeletal organization in PKD1/PKD2_ IFT88 KO organoids. (E) RT-qPCR analysis of PCP-associated gene expression in wild-type organoids with or without IFT88 knockout, showing minimal effects of cilia disruption in the absence of polycystin mutations. (F) RT-qPCR analysis in PKD1 organoids with or without IFT88 knockout revealed marked downregulation of PCP-related genes upon cilia disruption. (G) RT-qPCR analysis in PKD2 organoids with or without IFT88 knockout revealed marked downregulation of PCP-related genes upon cilia disruption. (H) Western blot analysis of Rho GTPase signaling components demonstrated stable expression following cilia loss in control organoids, but significant downregulation in PKD1/PKD2_ IFT88 KO organoids, consistent with transcriptomic findings. (I) Immunofluorescence analysis of VANGL2 localization differences among CON tissues, PKD tissues, CON organoids, PKD organoids, and PKD_ IFT88 KO organoids. Scale bars: 25 μm for tissues and 50 μm for organoids. Quantification data are expressed as mean ± SEM (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; and n.s., no statistics, Student’s t test).

Article Snippet: IFT88 , Abclonal , Cat#F2365; RRID: AB_3731500.

Techniques: Western Blot, Biomarker Discovery, Knock-Out, Control, Mutagenesis, Expressing, Quantitative RT-PCR, Gene Expression, Disruption, Immunofluorescence

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: Communications biology

Article Title: FBW7 couples structural integrity with functional output of primary cilia.

doi: 10.1038/s42003-021-02504-4

Figure Lengend Snippet: Fig. 5 FBW7-NDE1 axis affects osteoblast differentiation via effects on primary cilia in C3H10T1/2 cells. a Representative images for ALP staining of wild type, Fbxw7KO, Nde1KO, and Fbxw7Nde1KO C3H10T1/2 cells (n = 3). Images at ×1 or ×2 magnification were taken from 24-well plates. Well diameter: 16 mm. Scale bar: 5 mm. b, c Expression levels of ALP in indicated cells (b) and summary data (c). Data are presented as means ± SEM from four independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test, ****p < 0.0001. d, e mRNA levels of osteoblast differentiation markers Runx2 and Osterix (Osx) in wild type, Fbxw7KO, Nde1KO, and Fbxw7Nde1KO C3H10T1/2 cells. Data are presented as means ± SEM from four or three independent experiments (d or e, respectively). One-way ANOVA with Dunnett’s multiple comparisons test, **p < 0.01, ***p < 0.001. f, g ALP staining after transfection of Fbxw7Nde1KO C3H10T1/2 cells with pcDNA3 or an Ift88-specific sgRNA. Images at ×1 or ×2 magnification were taken from 24-well plates. Well diameter: 16 mm (f). Scale bar: 5 mm. Expression levels of IFT88 in Fbxw7Nde1KO C3H10T1/2 cells transfected with the indicated constructs (g).

Article Snippet: A301‐720A; A301‐721A 1:500), NDEL1 (Abcam, 1:1000), GAPDH (GeneTex, 1:2000), and IFT88 (Proteintech, 1:000).

Techniques: Staining, Expressing, Transfection, Construct