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: mBio

Article Title: An Aspartate-Specific Solute-Binding Protein Regulates Protein Kinase G Activity To Control Glutamate Metabolism in Mycobacteria

doi: 10.1128/mBio.00931-18

Figure Lengend Snippet: PknG activation by glutamate was indirect, and PknG function required the conserved protein interaction domain (TPR domain). (A) Kinase activity of recombinant PknG toward GarA was independent of glutamate. Kinase reactions in the presence or absence of glutamate were sampled at the indicated times. Phosphorylation of GarA was detected by the known decrease in mobility of phospho-GarA in SDS-PAGE ( , ). The rate of GarA phosphorylation was similar to that seen in previous studies of PknG and was unchanged by the addition of glutamate. Images are representative of three independent experiments. Similar results were obtained with other putative activators: glutamine, aspartate, and α-ketoglutarate. (B) PknG homologues from Actinomycetales have a variable N-terminal domain, sometimes lack the Rdx domain (absent from homologues in the Corynebacteriaceae ), and always have a tetratricopeptide repeat (TPR) domain. (C) Truncated PknG lacking the Rdx was able to restore the ability of pknG -deficient M. smegmatis to grow on minimal medium containing glutamate as the sole nitrogen source, but truncated PknG lacking the TPR domain did not improve growth. Error bars show standard deviations from three wells, and the growth curve is representative of three independent experiments.

Article Snippet: TPR domains have been identified in kinases from multiple bacterial phyla (>1,000 listed in Interpro [ ]), suggesting that TPR-mediated protein interaction could be a widespread regulatory mechanism of bacterial serine/threonine protein kinases.

Techniques: Activation Assay, Activity Assay, Recombinant, Phospho-proteomics, SDS Page