Journal: Disease Models & Mechanisms

Article Title: Cardiac-specific Trim44 knockout in rat attenuates isoproterenol-induced cardiac remodeling via inhibition of AKT/mTOR pathway

doi: 10.1242/dmm.049444

Figure Lengend Snippet: Trim44 KO attenuates ISO-induced cardiac remodeling through inhibition of the AKT/mTOR pathway. (A-E) Expression of phosphorylated (p-) and total AKT, P70S6K, GSK3β and mTOR in heart tissue from control-saline, KO-saline, control-ISO and KO-ISO groups 2 weeks after cessation of ISO treatment, detected using western blotting (A) and quantitatively analyzed using the respective total protein for normalization (B-E) ( n =4 rats per group). (F-J) Expression of phosphorylated and total AKT, P70S6K, GSK3β and mTOR in H9c2 cells from the empty-vector group and Trim44 overexpression group (referred as Trim44-OV) with or without inhibitor treatment (PI3K/AKT inhibitor, LY294002; 25 μM), detected using western blotting (F) and quantitatively analyzed using the respective total protein for normalization (G-J) ( n =4 replicates per group). (K) WGA immunofluorescence (scale bars: 40 μm). (L) Quantitative analysis of cross-sectional area of H9c2 cells for four groups ( n =3-4 replicates per group, 54 cells per group). (M) Quantitative analysis of Nppb expression through real-time PCR for four groups, using Gapdh for normalization ( n =3 replicates per group). * P <0.05, ** P <0.01, *** P <0.001, ### P <0.001 versus respective control group (one-way ANOVA). NS, not significant.

Article Snippet: The membranes were incubated overnight in the hybridization bag with antibodies against Trim44 (11511-1-AP, Proteintech; 1:500), phosphorylated (4060, Cell Signaling Technology; 1:2000) or total (4691, Cell Signaling Technology; 1:2000) AKT, phosphorylated (5536, Cell Signaling Technology; 1:1000) or total (2983, Cell Signaling Technology; 1:1000) mTOR, phosphorylated (9204, Cell Signaling Technology; 1:2000) or total (2708, Cell Signaling Technology; 1:2000) p70S6K, or phosphorylated (5558, Cell Signaling Technology; 1:2000) or total (9832, Cell Signaling Technology; 1:2000) GSK3β.

Techniques: Inhibition, Expressing, Western Blot, Plasmid Preparation, Over Expression, Immunofluorescence, Real-time Polymerase Chain Reaction

Journal: Disease Models & Mechanisms

Article Title: A DUSP6 inhibitor suppresses inflammatory cardiac remodeling and improves heart function after myocardial infarction

doi: 10.1242/dmm.049662

Figure Lengend Snippet: BCI has no effect on CM proliferation, H 2 O 2 -induced CM death, CM hypertrophy and coronary vessel regeneration. (A) Released lactate dehydrogenase (LDH) was comparable in neonatal rat ventricular myocytes (NRVMs) treated with DMSO or BCI for 24 h in the presence or absence of H 2 O 2 ( n =3 per group). (B) Western blots and quantification of Bcl-2 (α-tubulin was used to normalize protein) and p-AKT (AKT was used to normalize protein) in NRVMs treated with DMSO or BCI for 24 h ( n =3 per group). (C) Western blots and quantification of H 2 O 2 -induced cleaved PARP (α-tubulin was used to normalize protein) and cleaved caspase 3 (total caspase 3 was used to normalize protein) in NRVMs treated with DMSO or BCI ( n =3 per group). (D) Immunofluorescent staining showing cTnT + and pH3 + CMs of sham-operated, vehicle-treated or BCI-treated LV tissues at 7 days after MI (scale bar: 100 μm). (E) Immunofluorescent staining showed that the numbers of either α-actinin + Ki67 + or α-actinin + pH3 + CMs were comparable in DMSO- and BCI-treated NRVMs (scale bars: 50 μm; n =3 per group). HPF, high-power field. (F) Immunofluorescent staining and statistics for CD31 + and α-SMA + vessels of sham-operated, vehicle-treated or BCI-treated LV tissues at 7 days after MI (scale bar: 100 μm; n =3 per group). (G) Wheat germ agglutinin staining and statistics for CM size in sham-operated, vehicle-treated or BCI-treated LV tissues at 28 days after MI (scale bar: 20 μm; n =5 per group). Mean±s.e.m.; ns, not significant.

Article Snippet: After blocking, the membranes were incubated with anti-Bcl-2 (1:1000; Cell Signaling Technology, 3498S), anti-AKT (1:1000; Cell Signaling Technology, 9272S), anti-p-AKT (1:1000; Cell Signaling Technology, 4060S), anti-cleaved PARP (1:1000; Cell Signaling Technology, 9548T), anti-caspase 3 (1:1000; Cell Signaling Technology, 9662S), anti-cleaved caspase 3 (1:1000; Cell Signaling Technology, 9664S), anti-NF-κB p65 (1:1000; Cell Signaling Technology, 8242S), anti-p-NF-κB p65 (1:1000; Cell Signaling Technology, 3033S), anti-ERK (1:1000; Cell Signaling Technology, 4695S), anti-p-ERK (1:1000; Cell Signaling Technology, 4370S), anti-p38 (1:1000; Cell Signaling Technology, 8690S), anti-p-p38 (1:1000; Cell Signaling Technology, 4511S), anti-JNK (1:1000; Abcam, Cambridge, UK, ab179461) or anti-p-JNK (1:1000; Abcam, ab76572).

Techniques: Western Blot, Staining

Journal: International Journal of Oncology

Article Title: A disintegrin and metalloprotease 12 contributes to colorectal cancer metastasis by regulating epithelial-mesenchymal transition

doi: 10.3892/ijo.2023.5498

Figure Lengend Snippet: Effects of ADAM12 on oncogenic signaling pathways in the human colorectal cancer cells. Representative western blot images and semi-quantification graphs for the phosphorylation levels of PDK1, GSK-3β and AKT in AV-transfected or AS-transfected DLD1 or SW480 cells. Data are presented as the mean ± SD (n=3). * P<0.05. ADAM12, a disintegrin and metalloprotease 12; PDK1, phosphoinositide-dependent protein kinase 1; GSK-3β, glycogen synthase kinase-3β; EV, empty pcDNA6-myc vector; AV, ADAM12-pcDNA6-myc construct; SS, scrambled siRNA; AS, ADAM12 siRNA; siRNA, small interfering RNA; p-, phosphorylated; T-, total.

Article Snippet: Antibodies against E-cadherin (cat. no. #14472), Snail (cat. no. #3879), vimentin (cat. no. #5741), claudin-1 (cat. no. #4933), integrin α5 (cat. no. #4705), integrin β1 (cat. no. #9699), integrin β3 (cat. no. #13166), phosphorylated (p)-AKT (S473) (cat. no. #4060), p-phosphoinositide-dependent protein kinase 1 (PDK1) (S241) (cat. no. #3438), p-glycogen synthase kinase-3β (GSK-3β) (S9) (cat. no. #9323), total AKT (cat. no. #4691), total PDK1 (cat. no. #3062), total GSK-3β (cat. no. #9832) and Myc-tag (cat. no. #2278) were obtained from Cell Signaling Technology, Inc. Antibodies against β-tubulin (cat. no. sc-9104) and GAPDH (cat. no. sc-25778) were purchased from Santa Cruz Biotechnology, Inc.

Techniques: Western Blot, Transfection, Plasmid Preparation, Construct, Small Interfering RNA

Journal: Neural Regeneration Research

Article Title: Poly-L-ornithine blocks the inhibitory effects of fibronectin on oligodendrocyte differentiation and promotes myelin repair

doi: 10.4103/1673-5374.353493

Figure Lengend Snippet: PLO and FN function through ERK and PI3K/Akt signaling pathways. (A) The diagram shows the integrins (αV, β1, β3, β5) expressed in oligodendrocytes that specifically bind to FN. Data are collected from published literature. (B) Quantitative analysis of the expression levels (normalized by the PDL group) of integrins (αV, β1, β3, β5) during the proliferation and differentiation stages of oligodendrocytes by real-time quantitative polymerase chain reaction. (C) Representative western blots of Akt, P-Akt (Thr308), S6K, P-S6K, Erk1/2, and P-Erk1/2 proteins from OPCs cultured with different coating substances for 2 days in proliferation medium (2 d PM). (D) Quantification of the western blot results (normalized by the PDL group) showed in C. (E) Representative western blots of GSK3β, P-GSK3β, CREB, P-CREB, Akt, P-Akt, Erk, and P-Erk proteins from cells cultured for 1 day in differentiation medium (1d DM). (F) Quantification (normalized by the PDL group) of the results in E. (G) Representative western blots of Akt, P-Akt, Erk, P-Erk, GSK3β, P-GSK3β, CREB, and P-CREB proteins from cells cultured for 3 days in differentiation medium (3d DM). (H) Quantification (normalized by the PDL group) of the results in G. All data are expressed as mean ± SD from at least three independent experiments. * P < 0.05, ** P < 0.01 (one-way analysis of variance followed by Tukey’s multiple comparison test). Akt: Protein kinase B; CREB: cyclic adenosine monophosphate response element binding protein; DM: differentiation medium; Erk1/2: extracellular signal-regulated kinases 1 and 2; FN: fibronectin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSK3β: glycogen synthase kinase 3 beta; P-Akt; phospho-protein kinase B; P-CREB: phospho-cyclic adenosine monophosphate response element binding protein; PDL: poly-D-lysine; P-Erk1/2: phospho-extracellular signal-regulated kinases 1 and 2; P-GSK3β: phospho-glycogen synthase kinase 3 beta; PI3K: phosphoinositide 3-kinase; PLO: poly-L-ornithine; PM: proliferation medium; P-S6k: phospho-ribosomal S6 kinase; S6k: ribosomal S6 kinase.

Article Snippet: The primary antibodies were as follows: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:10,000, rabbit, Proteintech, Cat# 10494-1-AP, RRID: AB_2263076), protein kinase B (PKB, known as Akt; 1:1000, rabbit, Cell Signaling Technology, Cat# 9272, RRID: AB_329827), phospho-protein kinase B (Ser473) (P-PKB, known as P-AKT; 1:1000, rabbit, Cell Signaling Technology, Cat# 9271, RRID: AB_329825), cyclic adenosine monophosphate response element binding protein (CREB; 1:1000, rabbit, Cell Signaling Technology, Cat# 9197, RRID: AB_331277), phospho-cyclic adenosine monophosphate response element binding protein (P-CREB; 1:1000, rabbit, Cell Signaling Technology, Cat# 9198, RRID: AB_2561044), extracellular signal-regulated kinases 1 and 2 (Erk1/2; 1:1000, rabbit, Cell Signaling Technology, Cat# 4695, RRID: AB_390779), phospho-extracellular signal-regulated kinases 1 and 2 (Thr202/Tyr204) (P-Erk1/2; 1:1000, rabbit, Cell Signaling Technology, Cat# 4370, RRID: AB_2315112); glycogen synthase kinase 3 beta (GSK3β; 1:1000, rabbit, Affinity, Cincinnati, OH, USA, Cat# AF5016, RRID: AB_2834935), phospho-glycogen synthase kinase 3 beta (P-GSK3β; 1:1000, rabbit, Affinity, Cat# AF2016, RRID: AB_2834439), ribosomal S6 kinase (S6Κ; 1:1000, rabbit, Affinity, Cat# AF6226, RRID: AB_2835100), phospho-ribosomal S6 kinase (P-S6K; 1:1000, rabbit, Affinity, Cat# AF3228, RRID: AB_2834654), CyclinD1 (1:1000, rabbit, Abcam, Cat# AB134175, RRID: AB_2750906), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP; 1:1000, mouse, Calbiochem, Darmstadt, Germany, Cat# NE1020, RRID: AB_2043400), and myelin basic protein (MBP; 1:1000, mouse, Calbiochem, Cat# NE1018, RRID: AB_2140494).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, Binding Assay

Journal: International Journal of Oncology

Article Title: A disintegrin and metalloprotease 12 contributes to colorectal cancer metastasis by regulating epithelial-mesenchymal transition

doi: 10.3892/ijo.2023.5498

Figure Lengend Snippet: Effects of ADAM12 on oncogenic signaling pathways in the human colorectal cancer cells. Representative western blot images and semi-quantification graphs for the phosphorylation levels of PDK1, GSK-3β and AKT in AV-transfected or AS-transfected DLD1 or SW480 cells. Data are presented as the mean ± SD (n=3). * P<0.05. ADAM12, a disintegrin and metalloprotease 12; PDK1, phosphoinositide-dependent protein kinase 1; GSK-3β, glycogen synthase kinase-3β; EV, empty pcDNA6-myc vector; AV, ADAM12-pcDNA6-myc construct; SS, scrambled siRNA; AS, ADAM12 siRNA; siRNA, small interfering RNA; p-, phosphorylated; T-, total.

Article Snippet: Antibodies against E-cadherin (cat. no. #14472), Snail (cat. no. #3879), vimentin (cat. no. #5741), claudin-1 (cat. no. #4933), integrin α5 (cat. no. #4705), integrin β1 (cat. no. #9699), integrin β3 (cat. no. #13166), phosphorylated (p)-AKT (S473) (cat. no. #4060), p-phosphoinositide-dependent protein kinase 1 (PDK1) (S241) (cat. no. #3438), p-glycogen synthase kinase-3β (GSK-3β) (S9) (cat. no. #9323), total AKT (cat. no. #4691), total PDK1 (cat. no. #3062), total GSK-3β (cat. no. #9832) and Myc-tag (cat. no. #2278) were obtained from Cell Signaling Technology, Inc. Antibodies against β-tubulin (cat. no. sc-9104) and GAPDH (cat. no. sc-25778) were purchased from Santa Cruz Biotechnology, Inc.

Techniques: Western Blot, Transfection, Plasmid Preparation, Construct, Small Interfering RNA