pdk1 inhibitor ar 12  (MedChemExpress)

Journal: bioRxiv

Article Title: Ubiquitin ligase CHFR impairs Tie2 signaling via K 48 -linked ubiquitylation and degradation of Akt1 in endothelial cells

doi: 10.64898/2026.03.31.715582

Figure Lengend Snippet: a-b CHFR deficiency in EC prevents LPS-induced ubiquitylation of Akt1. HLMVEC transfected with sc-siRNA or CHFR-siRNA. At 72 h post-transfection, cells were challenged with LPS (5 μg/ml) for 6 h in the presence of the proteasomal inhibitor MG132 (10 μM). Cell lysates were immunoprecipitated with anti-Akt1 mAb and blotted with antibodies specific to K 48 -linked poly-Ub or K 63 -linked poly-Ub ( n = 2 independent experiments) ( a ). Chfr fl/fl (WT) and Chfr ΔEC mice were challenged with LPS (10 mg/kg; i.p.) for 6 h. After the LPS challenge, lungs harvested were used to determine ubiquitylation of Akt1 as above in a (b) . c-f CHFR induces ubiquitylation of activated Akt1. c Control and CHFR-depleted HLMVEC were pretreated with inhibitors of PDK1 and mTORC2 for 30 min and then exposed to LPS (5 μg/ml). Thereafter, cell lysates were used for IB to assess phosphorylation of Akt1. d-e HLMVEC pretreated with or without PDK1 and mTORC2 inhibitors for 1 h and stimulated with LPS (6 h) in the presence of MG123 (10 μM) showed that CHFR binds and ubiquitylates phosphorylated Akt1. f HEK-293T cells were transfected with HA-tagged ubiquitin (HA-Ub) (0.5 μg/ml) alone or co-transfected with N-terminal GFP-tagged WT-CHFR (1.5 μg/ml), N-terminal pmCherry-tagged WT-Akt1 (1.5 μg/ml), and phosphorylation-defective Akt1 mutant (Akt1T308A/S473A) (1.5 μg/ml) plasmids. Thirty-six hours after transfection, cells were incubated with MG123 (10 μM) for 3h, and cell lysates were used to determine phosphorylation-dependent ubiquitylation of Akt1.

Article Snippet: Mammalian targets of rapamycin (mTOR) inhibitor (catalog #WYE-354), and 3-Phosphoinositide-Dependent Kinase 1 (PDK1) inhibitor (catalog #GSK2334470) were from Selleckchem.com.

Techniques: Transfection, Immunoprecipitation, Control, Phospho-proteomics, Ubiquitin Proteomics, Mutagenesis, Incubation

Journal: Scientific Reports

Article Title: Disease-specific alterations of the enteric nervous system in precancerous colonic mucosa and their implications for mucin regulation

doi: 10.1038/s41598-025-24480-z

Figure Lengend Snippet: EGC–mediated mucin gene suppression is dependent on the PDK1-RSK pathway. ( A ) Semiquantitative Western blot analysis shows that co-culture with EGCs (co-EGC) increases phosphorylated forms of ERK, RSK1, Akt, and PDK1 in HT-29 cells. n = 3–4. * p < 0.05, ** p < 0.01, paired, two-tailed t test. ( B ) Quantitative RT-PCR shows that RSK inhibitors (LJH685, BI-D1870), PDK1 inhibitor (GSK2334470), and PI3K inhibitor (LY294002) counteract the suppressive effect of EGCs on forskolin (FOK)-mediated mucin gene upregulation. In contrast, RAF inhibitor (Donafenib) and MEK1/2 inhibitor ( PD184352 ) have no effect. n = 3–7. * p < 0.05, ** p < 0.01, *** p < 0.001, ANOVA . ( C ) Quantitative RT-PCR shows that forskolin-stimulated MUC2 upregulation in Caco-2 colon carcinoma cells is attenuated by co-culture with EGCs. This suppressive effect is counteracted by the RSK inhibitor (LJH685). n = 3–4. * p < 0.05, ** p < 0.01, ANOVA.

Article Snippet: After 6 days of co-culture, cells were stimulated with either recombinant VIP (10 nM, Tocris Bioscience, Bristol, UK) or forskolin (10 μg/mL, Nakarai Tesque, Kyoto, Japan) and harvested after 6 h. To inhibit intracellular signaling, pan-RSK inhibitor LJH685 (10 nM, Selleck Chemicals, Kanagawa, Japan) or BI-D1870 (60 nM, Selleck), MEK1/2 inhibitor PD184352 (10 μM, Selleck), RAF inhibitor donafenib (100 nM, Selleck), PI3K inhibitor LY294002 (2 μM, Selleck), and PDK1 inhibitor GSK2334470 (50 nM, Selleck) were applied 30 min before the start of forskolin stimulation.

Techniques: Western Blot, Co-Culture Assay, Two Tailed Test, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Disease-specific alterations of the enteric nervous system in precancerous colonic mucosa and their implications for mucin regulation

doi: 10.1038/s41598-025-24480-z

Figure Lengend Snippet: Proposed hypothetical model of EGC loss and mucin overproduction in SSLs. Under normal conditions, EGCs modulate intracellular signaling of colon epithelial cells to regulate mucin gene expression in response to neuron-derived VIP. EGCs activate both the MAPK/ERK and PI3K/PDK1signaling pathways. While the MAPK/ERK pathway alone may promote mucin gene expression (directly or indirectly), RSKs —activated by both pathways—inhibit PKA signaling, thereby reducing cAMP/PKA-dependent mucin gene induction. In SSLs, loss of EGC results in the attenuation of the PDK1/RSK pathway to enhance VIP-induced mucin gene expression. BRAF V600E mutation may positively impact mucin gene expression by enhancing the MAPK/ERK pathway.

Article Snippet: After 6 days of co-culture, cells were stimulated with either recombinant VIP (10 nM, Tocris Bioscience, Bristol, UK) or forskolin (10 μg/mL, Nakarai Tesque, Kyoto, Japan) and harvested after 6 h. To inhibit intracellular signaling, pan-RSK inhibitor LJH685 (10 nM, Selleck Chemicals, Kanagawa, Japan) or BI-D1870 (60 nM, Selleck), MEK1/2 inhibitor PD184352 (10 μM, Selleck), RAF inhibitor donafenib (100 nM, Selleck), PI3K inhibitor LY294002 (2 μM, Selleck), and PDK1 inhibitor GSK2334470 (50 nM, Selleck) were applied 30 min before the start of forskolin stimulation.

Techniques: Gene Expression, Derivative Assay, Mutagenesis

Journal: International journal of molecular sciences

Article Title: Mechanical Force Triggers Macrophage Pyroptosis and Sterile Inflammation by Disrupting Cellular Energy Metabolism.

doi: 10.3390/ijms26073321

Figure Lengend Snippet: Figure 4. Force disrupts macrophage energy metabolism. (a,b) qPCR and Western blotting analysis of PDK1 and PDH (n = 3). (c,d) qPCR and Western blotting analysis of LDHA (n = 3). (e) Detection

Article Snippet: In some experiments, the macrophages were pre-treated with the PDK1 inhibitor JX06 (10 μM, MCE, Monmouth Junction, NJ, USA) for 1 h before force application.

Techniques: Western Blot

Journal: International journal of molecular sciences

Article Title: Mechanical Force Triggers Macrophage Pyroptosis and Sterile Inflammation by Disrupting Cellular Energy Metabolism.

doi: 10.3390/ijms26073321

Figure Lengend Snippet: Figure 5. Inhibition of PDK1 rectifies the energy metabolism disorders in force-loaded macrophages. (a,b) qPCR and Western blotting analysis of PDK1 and PDH (n = 3). (c,d) qPCR and Western blotting analysis of LDHA (n = 3). (e) Detection results of intracellular acetyl-CoA content (n = 3). (f) Detection results of lactate content in cell culture supernatants (n = 3). (g) Analysis of total ROS levels in cells. Scale bar = 50 µm. (h) Analysis of mitochondrial ROS levels. Scale bar = 50 µm. (i) JC-1 mitochondrial membrane potential analysis, where the ratio of red to green fluorescence intensity reflects the high or low mitochondrial membrane potential. Scale bar = 50 µm. (j) Semi-quantitative fluorescence analysis of DCFH-DA and MitoSOX (n = 6). (k) Analysis of intracellular ATP levels (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: In some experiments, the macrophages were pre-treated with the PDK1 inhibitor JX06 (10 μM, MCE, Monmouth Junction, NJ, USA) for 1 h before force application.

Techniques: Inhibition, Western Blot, Cell Culture, Membrane, Fluorescence

Journal: International journal of molecular sciences

Article Title: Mechanical Force Triggers Macrophage Pyroptosis and Sterile Inflammation by Disrupting Cellular Energy Metabolism.

doi: 10.3390/ijms26073321

Figure Lengend Snippet: Figure 6. Inhibiting PDK1 alleviates force-induced macrophage pyroptosis and sterile inflammation. (a) qPCR analysis of IL-1β (n = 3). (b) Western blotting and quantitative analysis of Pro-IL-1β and IL-1β (n = 3). (c) ELISA analysis of IL-1β in cell culture supernatants (n = 3). (d) Western blotting and quantitative analysis of NLRP3, Caspase1, Cleaved caspase1, GSDMD, and N-GSDMD (n = 3). (e) Schematic diagram illustrating the mechanism by which force triggers macrophage pyroptosis and sterile inflammation by disrupting cellular energy metabolism. ↑indicates upregulation, ↓indicates downregulation. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: In some experiments, the macrophages were pre-treated with the PDK1 inhibitor JX06 (10 μM, MCE, Monmouth Junction, NJ, USA) for 1 h before force application.

Techniques: Sterility, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture