k252a  (Alomone Labs)

Journal: BMC Neuroscience

Article Title: Neurotrophins regulate ApoER2 proteolysis through activation of the Trk signaling pathway

doi: 10.1186/1471-2202-15-108

Figure Lengend Snippet: ApoER2 proteolysis induced by NGF depends on TrkA tyrosine kinase activity and on metalloproteinase activity. PC12-ApoER2 cells were serum-starved and pre-treated with 10 μM DAPT and (A) 100 nM K252a or (C) with 50 μM GM6001 for 1 h. Then, the cells were incubated with 100 ng/mL NGF for 2 h. ApoER2, ApoER2-CTF and the proteolytic fragment of p75 NTR (control) were determined by western blot analysis using antibodies directed against their intracellular regions. α-tubulin is shown as a loading control, and the phosphorylated form of AKT is a control for TrkA activation by NGF. (B and D) The levels of ApoER2 CTF were normalized to the loading control α-tubulin and plotted as the average ± SD of three independent experiments. One way ANOVA, Holm-Sidak post-hoc test, ** P

Article Snippet: For inhibitory agents, we used 100 nM K252a (Alomone Labs), 10 μM DAPT, 25 μM PD98058, 50 μM GM6001 100 nM wortmannin and 50 μM of LY294002 (all from Calbiochem) and 5 μM of ZSTK474 (Selleck Chemicals LLC) Later, the cells were treated with 100 ng/mL NGF (Alomone Lab) for different times in a 5% CO2 incubator at 37°C.

Techniques: Activity Assay, Incubation, Western Blot, Activation Assay

Journal: BMC Neuroscience

Article Title: Neurotrophins regulate ApoER2 proteolysis through activation of the Trk signaling pathway

doi: 10.1186/1471-2202-15-108

Figure Lengend Snippet: NGF induces the proteolytic processing of ApoER2. (A) Serum-starved PC12-ApoER2 cells were pre-treated with 10 μM DAPT (γ-secretase complex inhibitor), 50 μM GM6001 (metalloproteases inhibitor) and/or 100 nM K252a (Trk tyrosine kinase activity inhibitor) for 1 h and then incubated with 100 ng/mL NGF for 2 h. The blot shows full-length p75 NTR , p75 NTR CTF, and α-tubulin as a loading control. As described [ 40 ], NGF induced the proteolysis of p75 NTR and, thus, the accumulation of the CTF. This process depends on TrkA tyrosine kinase activity and the metalloproteinases. (B) Cells were pre-treated with 10 μM DAPT for 1 h and then incubated with 100 ng/mL NGF for 2 h. ApoER2 and the proteolytic fragment ApoER2-CTF were recognized using antibodies against the intracellular region of the receptor. α-tubulin is shown as a loading control. (C) Quantification of blot levels of ApoER2-CTF normalized to the loading control α-tubulin and plotted as the average ± SD of four independent experiments. Student’s t-test, ** P

Article Snippet: For inhibitory agents, we used 100 nM K252a (Alomone Labs), 10 μM DAPT, 25 μM PD98058, 50 μM GM6001 100 nM wortmannin and 50 μM of LY294002 (all from Calbiochem) and 5 μM of ZSTK474 (Selleck Chemicals LLC) Later, the cells were treated with 100 ng/mL NGF (Alomone Lab) for different times in a 5% CO2 incubator at 37°C.

Techniques: Activity Assay, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Dual inhibition of BDNF/TrkB and autophagy: a promising therapeutic approach for colorectal cancer

doi: 10.1111/jcmm.13181

Figure Lengend Snippet: Effects of NT inhibitor (K252a), autophagy inhibitor (CQ) and dual treatment on tumoural growth. Nude mice were subcutaneously engrafted with SW480 or SW620 (1.10 6 cells). When tumours reached a volume of 300 mm 3 , animals were divided into different groups and treated with K252a for 21 days or with CQ for 12 days or with both molecules (see Materials and methods ). ( A ) Tumour growth was determined weekly. Results are expressed in mean tumour volumes (mm 3 ) ± S.D. in comparison with the non treated group (* P

Article Snippet: In a previous study, we demonstrated that CRC cells activate a survival and proliferative loop through BDNF/TrkB signaling ; here we investigated the consequences of NT inhibition in SW480 and SW620 cells cultured for 3 hrs with 100 nM K252a.

Techniques: Mouse Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Dual inhibition of BDNF/TrkB and autophagy: a promising therapeutic approach for colorectal cancer

doi: 10.1111/jcmm.13181

Figure Lengend Snippet: Effects of dual treatment (K252a + CQ) on cell proliferation, cell death and vasculogenesis in tumour tissue originating from mice SW480 and SW620 xenografts. After being engrafted subcutaneously by SW480 or SW620, mice were treated by K252a + CQ as described in Materials and methods section. Tumour sample were collected from five xenograft mice for both cell lines. ( A ) Proliferation was determined by Ki67 immunostaining. Magnification was ×100. ( B ) Necrosis was quantified using HES staining. Aeras of necrosis are delimited by N. Magnification was ×50. Results are representative of the ratio between necrosis area and total tumour area obtained with the Nanozoomer Digital Pathology 2.ORS (Hamamatsu) version 2.5.88 software. ( C ) Tumour sample were lysed and total protein were extracted. VEGF and cleaved caspase 3 were assessed by Western blotting. The density of each band was calculated with ImageJ software. Images show representative results performed for each condition, for both xenografts cell lines.

Article Snippet: In a previous study, we demonstrated that CRC cells activate a survival and proliferative loop through BDNF/TrkB signaling ; here we investigated the consequences of NT inhibition in SW480 and SW620 cells cultured for 3 hrs with 100 nM K252a.

Techniques: Mouse Assay, Immunostaining, Staining, Digital Pathology, Software, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Dual inhibition of BDNF/TrkB and autophagy: a promising therapeutic approach for colorectal cancer

doi: 10.1111/jcmm.13181

Figure Lengend Snippet: Metabolic activity, cell death analysis and PARP cleavage in CRC cell lines after K252a, CQ and K252a + CQ treatments. SW480 and SW620 were treated with K252a, CQ or both molecules for 72 hrs (see Materials and methods ). ( A ) Metabolic activity was assessed through MTT testing. Histograms show the means ± S.E.M. of at least three independent experiments (* P

Article Snippet: In a previous study, we demonstrated that CRC cells activate a survival and proliferative loop through BDNF/TrkB signaling ; here we investigated the consequences of NT inhibition in SW480 and SW620 cells cultured for 3 hrs with 100 nM K252a.

Techniques: Activity Assay, MTT Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Dual inhibition of BDNF/TrkB and autophagy: a promising therapeutic approach for colorectal cancer

doi: 10.1111/jcmm.13181

Figure Lengend Snippet: Effect of tyrosine kinase receptor inhibitor (K252a) on TrkB/BDNF expression and autophagy induction. SW480 and SW620 were treated with 100 nM of K252a for 3 hrs, recovered, washed and total RNA and proteins were extracted (see Materials and methods ). ( A ) BDNF, Full Length (FL) and Truncated (Tr) TrkB, transcripts expression was assessed by qPCR. Histograms are representative of three independent experiments. The fold expression was obtained by the comparative cycle threshold method using the GAPDH RNA expression level as internal control. ( B ) BDNF, FL TrkB, Tr TrkB, phospho‐TrkB (P TrkB), AKT, phospho‐AKT (P AKT), mTOR and phospho‐mTOR (P mTOR), proteins expression were assessed by Western blotting. The density of each band representative for protein expression was calculated with ImageJ software. Images show representative results of three experiments. Statistically results are explained in comparison with control cells, normalized at 1 (* P

Article Snippet: In a previous study, we demonstrated that CRC cells activate a survival and proliferative loop through BDNF/TrkB signaling ; here we investigated the consequences of NT inhibition in SW480 and SW620 cells cultured for 3 hrs with 100 nM K252a.

Techniques: Expressing, Real-time Polymerase Chain Reaction, RNA Expression, Western Blot, Software

Journal: Scientific Reports

Article Title: Long-term depression at hippocampal mossy fiber-CA3 synapses involves BDNF but is not mediated by p75NTR signaling

doi: 10.1038/s41598-021-87769-9

Figure Lengend Snippet: LTD at MF synapses is not mediated via p75NTR or TrkB receptor signaling. ( a ), ( c ) and ( d ) MF LTD is not impaired in the presence of bath applied TAT-Pep5 (1 µM, a ), LM11A31 (100 nM, c ) or K252a (200 nM, d ) in comparison to the respective solvent controls (filled circle: BSA ( a , n = 8/N = 6), ACSF ( c , n = 16/N = 12) and DMSO ( d , n = 12/N = 6); filled gray circle: TAT-Pep5 ( a , n = 11/N = 10), LM11A31 ( c , n = 11/N = 6) and K252a ( d , n = 9/N = 5)). b) Recordings in slices of p75NTR EXIV−/− mice displayed no influence of p75NTR chronic deficiency on LTD magnitudes at MF synapses (filled circle: p75NTR wildtype (n = 8/N = 5); filled gray circle: p75NTR EXIV−/− (n = 12/N = 7)). ( e ) Unaltered MF LTD in the presence of bath applied ANA-12 (20 µM), in comparison to the respective solvent control (filled circle: DMSO (n = 16/N = 11); filled gray circle: ANA-12 ( e , n = 11/N = 6). The figure insets show representative averaged original fEPSP traces. For all MF-LTD experiments, “1” indicates mean fEPSP amplitudes of first 10 min of baseline and “2” depicts mean fEPSP amplitudes between 55 and 60 min after induction of LTD. Data expressed as mean ± SEM. Corresponding scale bars are shown as insets.

Article Snippet: To test for acute effects of BDNF signaling via tropomyosin related kinase (Trk) B, the tyrosine kinase inhibitor K252a (200 nM, Alomone Labs; Israel; dissolved in 0.1% Dimethyl sulfoxide (DMSO; Sigma Aldrich, Germany) was bath applied.

Techniques: Mouse Assay

Journal: Scientific Reports

Article Title: Multiplex coherent anti-Stokes Raman scattering microspectroscopy detection of lipid droplets in cancer cells expressing TrkB

doi: 10.1038/s41598-020-74021-z

Figure Lengend Snippet: K252a inhibition of BDNF-induced TrkB activation. Analysis of HEK-Clone2 cells incubated with BDNF (75 ng/mL), and with or without K252a (100 nM) during 1 h. ( A ) MCARS microspectroscopy of HEK-Clone2 after a dual treatment. HEK-Clone2 cells were incubated with BDNF (75 ng/mL) with or without K252a (100 nM), during 48 and 72 h, the figure includes bright field and fluorescence (Hoechst 33342) images. MCARS images were reconstructed from signal integration at 2850 cm −1 (CH 2 symmetric stretching) and 2930 cm −1 (CH 3 symmetric stretching). Scale bar, 5 µm. ( B ) Quantification of the CH 2 signal intensity in HEK-Clone2 cells by MCARS analysis after a dual treatment for 48 and 72 h. We used HEK-Clone2 with no BDNF treatment as reference. ( C ) Western-blot of Akt and P-Akt (phospho-Akt) realized with 50 µg of extracted protein. Full-length blots for Akt and P-Akt are presented in Supplementary Fig. 7 . *p

Article Snippet: HEK293 and HEK-Clone2 cells were seeded at a density of 104 cells/cm2 and cultured 2 days before treatment with 75 ng/mL recombinant human BDNF (Peprotech) or with 100 nM of K252a (Alomone) Cells were harvested after 1 h of treatment and proteins were extracted.

Techniques: Inhibition, Activation Assay, Incubation, Fluorescence, Western Blot

Journal: Scientific Reports

Article Title: Multiplex coherent anti-Stokes Raman scattering microspectroscopy detection of lipid droplets in cancer cells expressing TrkB

doi: 10.1038/s41598-020-74021-z

Figure Lengend Snippet: MCARS spectroscopy of HT29 cells incubated with K252a. HT29 cell lines were treated with K252a (100 nM), during 48 and 72 h. From top to bottom: bright-field, fluorescence (Hoechst 33342) images, MCARS images reconstructed from signal integration at 2850 cm −1 (CH 2 symmetric stretching) and 2930 cm −1 (CH 3 symmetric stretching), merged images (N = 2). Scale bar, 5 µm.

Article Snippet: HEK293 and HEK-Clone2 cells were seeded at a density of 104 cells/cm2 and cultured 2 days before treatment with 75 ng/mL recombinant human BDNF (Peprotech) or with 100 nM of K252a (Alomone) Cells were harvested after 1 h of treatment and proteins were extracted.

Techniques: Spectroscopy, Incubation, Fluorescence