Journal: Neurotherapeutics

Article Title: Inhibitions and Down-Regulation of Motor Protein Eg5 Expression in Primary Sensory Neurons Reveal a Novel Therapeutic Target for Pathological Pain

doi: 10.1007/s13311-022-01263-2

Figure Lengend Snippet: Eg5 is necessary for PI3K/Akt signalling-mediated VR1 membrane trafficking. A Examples of immunofluorescence images of VR1 in the culture of DRG neurons with vehicle, monastrol and the PI3K/AKT signalling activator YS-49. B Statistical analysis of the VR1 density with vehicle, monastrol and YS-49 ( n = 3 mice and 63–73 neurons for each group). C Representative images of fluorescence recovery of FM1-43 bleaching in the culture of DRG neurons with vehicle, monastrol and YS-49. D Statistical analysis of the fluorescence recovery of FM1-43 bleaching with vehicle, monastrol and YS-49 ( n = 10 neurons for each group). E Schematic of monastrol and YS-49 applications and the effects on CFA-induced inflammatory pain behaviour ( n = 6–11 mice for each group). F Representative Western blot of VR1 and pEg5 in the plasma membrane of DRG neurons with vehicle, monastrol and YS-49. G Analysis of VR1 and pEg5 proteins in the plasma membrane of DRG neurons treated with vehicle, monastrol or YS-49 ( n = 4 replicates for each group). H Schematic of the Eg5-dependent VR1 membrane trafficking mechanism. Veh, vehicle control; Mon, monastrol; MT, microtubule. Scale bars, 10 μm. One-way ANOVA with Tukey’s post hoc test in ( B ), ( E ) and ( G ). Two-way ANOVA in ( D ). n.s., nonsignificant, * P < 0.05, *** P < 0.001. Error bars show the mean ± SEM

Article Snippet: Where indicated, neurons were challenged with corresponding dilutions of vehicle, monastrol (100 μM, Santa Cruz, USA, sc-202710A), and the PI3K inhibitor LY294002 (10 μM, MCE, HY-10108).

Techniques: Membrane, Immunofluorescence, Fluorescence, Western Blot, Control

Journal: Arthritis Research & Therapy

Article Title: E2F2 directly regulates the STAT1 and PI3K/AKT/NF-κB pathways to exacerbate the inflammatory phenotype in rheumatoid arthritis synovial fibroblasts and mouse embryonic fibroblasts

doi: 10.1186/s13075-018-1713-x

Figure Lengend Snippet: MyD88 mediates regulation of the PI3K/AKT/NF-κB pathway by E2F2. a , b E2F2 can regulate expression of PI3K/AKT/NF-κB. E2F2-silenced RASFs and E2f2 −/− MEFs were cultured with or without lipopolysaccharide (LPS). Western blot was performed to detect phosphorylation of AKT and NF-κB P65 both in E2F2-silenced RASFs ( a ) and E2f2 −/− MEFs ( b ). c , d Effect of E2F2 on translocation of p65. E2F2 knocked-down RASFs ( c ) and E2f2 −/− MEFs ( d ) were cultured under LPS stimulation (10 μg/mL) for 12 h; nuclear and cytoplasmic proteins were extracted separately and then Western blot was performed (Lamin A/C as a reference for nuclear extraction (N); Tubulin as a reference for cytoplasmic extraction (C).) e , f Effects of E2F2 on p65 nuclear translocation both in RASFs (Fig. ) and MEFs (Fig. ) observed using confocal fluorescence microscopy. g – j E2F2-silenced RASFs and E2f2 −/− MEFs were cultured with or without LPS. qRT-PCR ( g , h ) and Western blot ( i , j ) were performed to detect the effect of E2F2 on MyD88. k Schematic representation of the MyD88 promoter, primers for the ChIP assay, and the E2F2 binding motif in the MyD88 promoter. l , m E2F2 was recruited to the MyD88 gene promoter in RASFs in the presence of LPS. ChIP ( l ) and luciferase (Luc) reporter assay ( m ) were performed in RASFs and MEFs in the presence or absence of LPS (10 μg/mL). n MyD88 mediated the effect of E2F2 on PI3K/AKT/NF-κB pathways. Western blot showed that knockdown of MyD88 could significantly inhibit the phosphorylation of AKT and P65 in the presence or absence of E2F2 overexpression. qRT-PCR showed that inhibition of MyD88 can inhibit the expression of inflammatory factors and significantly reduce the upregulation of interleukin (IL)-1α ( o ), IL-1β ( p ), and tumor necrosis factor (TNF)-α ( q ) by E2F2. The effect of inhibitors of PI3K/AKT/NF-κB pathways (LY294002 and PDTC) on E2F2; qRT-PCR was used to detect the effect of inhibitors on expression of IL-1α ( r , u ), IL-1β ( s , v ), and TNF-α ( t , w ) in response to LPS. The results shown are means ± SEM of three independent experiments performed in triplicate. ** P < 0.01, *** P < 0.001, versus the control. Ad-GFP adenovirus encoding green fluorescent protein, NC knockdown scramble control, si small interfering, WT wild-type

Article Snippet: Activation of PI3K/AKT pathways was blocked using LY294002 (Calbiochem, MCE, New Jersey, USA) and NF-κB inhibitor PDTC (M4005, Abmole Bioscience, Hong Kong, China).

Techniques: Expressing, Cell Culture, Western Blot, Phospho-proteomics, Translocation Assay, Extraction, Fluorescence, Microscopy, Quantitative RT-PCR, Binding Assay, Luciferase, Reporter Assay, Knockdown, Over Expression, Inhibition, Control

Journal: Cell Death & Disease

Article Title: EGFR-PI3K-PDK1 pathway regulates YAP signaling in hepatocellular carcinoma: the mechanism and its implications in targeted therapy

doi: 10.1038/s41419-018-0302-x

Figure Lengend Snippet: a, b WB was used to detect the effect of EGF treatment for 4 h on the expression of YAP with the inhibitors of EGFR or its downsream members, including the EGFR inhibitor (10uM Gefitinib), PI3K inhibitor (10 uM LY294002, 5 uM Wortmannin), PDK1 inhibitor (10uM GSK2334470, 10 uM BX-795), pan-Akt inhibitor(10 uM MK-2206) and MEK inhibitor(10 uM Trametinib, 10 uM U0126) in the Si RhoA transfected HepG2 and SMMC7721 cells for 48 h in HepG2 and SMMC7721 cells. c, d CCK8 assays were used to detect the effect of YAP knockdown combined with 50 ng/ml EGF stimulation for 48 h on the proliferation of the two HCC cell lines. e, f WB was used to examine the effect of combined treatment on the core effectors of the EGFR downstream signaling. T -test was used to detect the difference. (* P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: The inhibitors used in this study include the PI3K inhibitor wortamanin, PDK1 inhibitor GSK2334470 and BX-795, AKT inhibitor MK2206, MEK inhibitor Trametinib(Selleck, USA), EGFR inhibitors Gefitinib, PI3K inhibitor Ly294002, and MEK inhibitor U0126(MCE, USA).

Techniques: Expressing, Transfection, Knockdown

Journal: Genes & Diseases

Article Title: PGRN exacerbates the progression of non-small cell lung cancer via PI3K/AKT/Bcl-2 antiapoptotic signaling

doi: 10.1016/j.gendis.2021.05.005

Figure Lengend Snippet: The signaling pathway of MAPK/Erk and PI3K/Akt underlying the effect of PGRN on lung cancer cells. (A) The protein levels of p-Erk, Erk, p-Akt and Akt in A549 were analyzed by western blot. (B) The quantitative statistics of protein expression was shown (∗ P < 0.05, ∗∗ P < 0.005 vs . Scramble group). (C) The protein levels of CyclinD1, PCNA, Bcl-2, Bax, p-Erk, Erk, p-Akt and Akt in H520 after treating with LY294002 were analyzed by western blot. (D) The quantitative statistics of protein expression was shown (∗ P < 0.05, ∗∗ P < 0.005 vs . Each another group). (E) The protein levels of CyclinD1, PCNA, Bcl-2, Bax, p-Erk, Erk, p-Akt and Akt in H520 after treating with PD98059 were analyzed by western blot. (F) The quantitative statistics of protein expression was shown (∗ P < 0.05, ∗∗ P < 0.005 vs . Each another group). All experiments were repeated three times independently.

Article Snippet: MEK inhibitor PD98059 5 μM (MCE, CAS no. 167869-21-8, USA), trametinib 5 nM (MCE, CAS no. 871700-17-3, USA), PI3K inhibitor LY294002 1 μM (MCE, CAS no. 154447-36-6, USA), and copanlisib 10 nM (MCE, CAS no.1032568-63-0, USA) were used to target the PI3K/Akt and MAPK/Erk pathways, and 740Y–P 30 μM (MCE, CAS no. 1236188-16-1, USA) was used to activate PI3K.

Techniques: Western Blot, Expressing

Journal: Journal of Translational Medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: Correlation between DHCR7 expression and macrophage polarization in MASH. ( a ) Relative expression of cholesterol biosynthesis and efflux genes in primary macrophages after ox-LDL (100 µg/ mL) or ox-LDL combined with statin treating for 24 h. ( b ) Expression levels of cholesterol biosynthesis and efflux genes in primary macrophages polarized to M1 with LPS (100 ng/mL) or M2 with IL-4 (20 ng/mL) for 24 h. ( c-d ) Western blot analysis for DHCR7 protein in M1 and M2 macrophages, with densitometry. ( e ) Immunofluorescence staining for DHCR7 in the polarized-macrophages, 200× magnification. ( f-g ) Multiple immunofluorescence co-localization for DHCR7 and CD11B on livers of NCD or CDAHFD mice. ( h-i ) DHCR7 protein and mRNA levels in total liver and primary macrophages isolated from livers of mice on control and CDAHFD. ( j-k ) Immunofluorescence multi-labeling of liver sections from NC and MASH patients for DHCR7 and macrophage marker CD68 to determine the proportion of DHCR7 + CD68 + cells among total CD68 + cells. Data are presented as mean ± SD, with n = 3 per group. * P < 0.05, * * P < 0.01, *** P < 0.001, and **** P < 0.0001 between groups

Article Snippet: For DHCR7-PI3K pathway studies, cells were treated with DHCR7 inhibitor AY9944 (A8658, APExBio, USA) and PI3K inhibitor LY294003 (HY-10108, MCE, USA), or PI3K agonist 740 Y-P (HY-P0175, MCE, USA).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Isolation, Control, Labeling, Marker

Journal: Journal of Translational Medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: The modulatory effect of DHCR7 on macrophage polarization and inflammatory response. ( a ) Relative protein levels of DHCR7 and M2 markers (CD206 and ARG-1) after silencing DHCR7, with densitometry. ( b ) Relative mRNA levels of M1 markers ( Inos , Cd32 ) and inflammatory cytokines ( Tnf-a , Il-6 , Il-1b ) after silencing DHCR7. ( c ) RAW264.7 cells were treated with AY9944 (2.5, 5 or 10 µM) for 24 h to inhibit DHCR7. Relative protein levels of DHCR7, with densitometry. ( d ) Relative mRNA levels of M1 markers ( Inos , Cd32 , Cd16 ) and inflammatory cytokines ( Il6 and Tnfa ) following AY9944 treatment for 24 h. ( e ) CD86 immunofluorescence in AY9944-treated RAW264.7 macrophages, 200× magnification. ( f ) Relative mRNA levels of M1/M2 markers following AY9944 treatment, with densitometry. ( g ) Relative protein quantification after overexpressing DHCR7, with densitometry. ( h ) Relative mRNA levels of M1 markers ( Inos , Cd32 ) and inflammatory cytokines ( Il6 , Il1b , Tnfa ) after overexpressing DHCR7. ( i ) Relative protein levels of M1/M2 markers in LPS-treated RAW264.7 cells overexpressing DHCR7, with densitometry. Data are presented as mean ± SD. * P < 0.05, * * P < 0.01, *** P < 0.001, and **** P < 0.0001 between groups

Article Snippet: For DHCR7-PI3K pathway studies, cells were treated with DHCR7 inhibitor AY9944 (A8658, APExBio, USA) and PI3K inhibitor LY294003 (HY-10108, MCE, USA), or PI3K agonist 740 Y-P (HY-P0175, MCE, USA).

Techniques: Immunofluorescence

Journal: Journal of Translational Medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: Myeloid-macrophage-specific DHCR7 deficiency exacerbates MASH progression. Wildtype (WT) and myeloid-specific DHCR7 knockout (DHCR7 cKO) mice were fed with a normal control diet (CON) or a CDAHFD for 10 weeks ( n = 7). ( a ) Body weight progression, ( b ) liver/body weight ratio, and ( c-d ) serum ALT and AST levels were detected. ( e-f ) Hepatic TG and TC levels were measured. ( g ) ELISA quantification of liver IL-1β and IL-6 levels. ( h ) Histological analysis of liver sections is presented through H&E, ORO staining, and immunohistochemistry for F4/80 + and CD11b + cells (200× magnification). ( i ) NAS score. ( j-k ) Relative protein levels of CD86, ARG-1, CD206 were determined, with densitometry. ( l ) Flow cytometry of primary macrophage in cKO mice and WT with different diets. ( m ) Flow cytometry of TREM2 in macrophages. ( n ) Predominantly of the M1 type in macrophages, including quantification of both the percentage of M1 macrophages and M1 macrophage counts per liver weight. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 between group

Article Snippet: For DHCR7-PI3K pathway studies, cells were treated with DHCR7 inhibitor AY9944 (A8658, APExBio, USA) and PI3K inhibitor LY294003 (HY-10108, MCE, USA), or PI3K agonist 740 Y-P (HY-P0175, MCE, USA).

Techniques: Knock-Out, Control, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: DHCR7 manipulation affects PI3K phosphorylation in macrophages. ( a ) PCA analysis and ( b ) KEGG pathway analysis using RNA sequencing in liver tissues from WT and DHCR7 cKO mice under control diet. ( c ) PCA analysis and ( d ) KEGG pathway analysis using RNA sequencing in liver tissues from NCD and CDAHFD-fed MASH mice. ( e-f ) Protein levels of PI3K and phosphorylated PI3K (P-PI3K) in NCD- and CDAHFD-fed WT and DHCR7 cKO mice. ( g , j ) Protein levels of PI3K, P-PI3K, and DHCR7 in RAW264.7 cells treated with ox-LDL (100 µg/mL) and a co-treatment of ox-LDL (100 µg/mL) and simvastatin (5 µM) for 24 h. ( h , k ) Protein levels of PI3K and P-PI3K in RAW264.7 cells treated with different concentrations of the DHCR7 inhibitor AY9944 (2.5, 5, and 10 µM) for 24 h. ( i , l ) Protein levels of PI3K and P-PI3K in RAW264.7 cells overexpressing DHCR7 and subsequently treated with ox-LDL (100 µg/mL) for 24 h. Data are presented as mean ± SD, with n = 3 per group. * P < 0.05, ** P < 0.01, and *** P < 0.001 between groups

Article Snippet: For DHCR7-PI3K pathway studies, cells were treated with DHCR7 inhibitor AY9944 (A8658, APExBio, USA) and PI3K inhibitor LY294003 (HY-10108, MCE, USA), or PI3K agonist 740 Y-P (HY-P0175, MCE, USA).

Techniques: RNA Sequencing Assay, Control

Journal: Journal of Translational Medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: The DHCR7-PI3K axis in macrophage polarization and inflammatory response. ( a ) RAW264.7 cells were co-treated with LPS (100 µg/mL) with PI3K activator 740 Y-P (5 and 10 µM) for 24 h. Relative mRNA levels of M1 markers ( Inos , Cd32 and Cd16 ) and the inflammatory cytokine ( Il1b and Il6 ) was assessed. RAW264.7 cells were treated with 10 µM AY9944 alone or alongside 5 or 10 µM 740 Y-P for 24 h. ( b ) Relative mRNA levels of M1 markers ( Inos , Cd32 , Cd16 ) and the inflammatory cytokine ( Tnfa and Il-6 ) was assessed. ( c-d ) Relative protein levels of the M1 marker CD86 and M2 markers CD206 and ARG-1 were measured. ( e ) RAW264.7 cells were treated with PI3K inhibitor LY294002 at 2.5, 5, and 10 µM for 24 h, IL-4, or a combination of both for 24 h. Relative mRNA levels of M1 markers ( Cd32 and Cd16 ) and the inflammatory cytokine ( Il-1b and Tnfa) . ( f ) Relative mRNA levels of Mrc-1 were assessed after treating with IL-4 (20 ng/mL) and IL-4 combined with 2.5, 5, 10 µM LY294002 for 24 h. ( g ) RAW264.7 cells were treated with 10 µM AY9944 alone or in combination with 5 or 10 µM 740 Y-P for 24 h, relative mRNA levels of M1 markers ( Cd32 and Cd16 ) and the inflammatory cytokine ( Tnfa and Il-6 ) was assessed. ( h-i ) Relative protein levels of the M1 marker CD86 and M2 markers ARG-1. Data are presented as mean ± SD, with n = 3 per group. * P < 0.05, * * P < 0.01, *** P < 0.001, and **** P < 0.0001 between groups

Article Snippet: For DHCR7-PI3K pathway studies, cells were treated with DHCR7 inhibitor AY9944 (A8658, APExBio, USA) and PI3K inhibitor LY294003 (HY-10108, MCE, USA), or PI3K agonist 740 Y-P (HY-P0175, MCE, USA).

Techniques: Marker

Journal: Journal of Translational Medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: Schematic illustrating how macrophage cholesterol overload drives inflammation via DHCR7-PI3K axis inhibition, contributing to MASH progression

Article Snippet: For DHCR7-PI3K pathway studies, cells were treated with DHCR7 inhibitor AY9944 (A8658, APExBio, USA) and PI3K inhibitor LY294003 (HY-10108, MCE, USA), or PI3K agonist 740 Y-P (HY-P0175, MCE, USA).

Techniques: Inhibition