Journal: Oncotarget

Article Title: RNA-dependent protein kinase (PKR) depletes nutrients, inducing phosphorylation of AMP-activated kinase in lung cancer

doi:

Figure Lengend Snippet: (A) and (B) Western blotting of PKR, phosphorylated (p-)PKR, p-eIF2α, AMPK, p-AMPK T172 , p-AMPK T485 , tuberous sclerosis complex 2 (TSC2), p-TSC2, acetyl-CoA carboxylase (ACC), p-ACC, mammalian target of rapamycin (mTOR), and p-mTOR protein expression in human lung cancer cell lines (H1299, A549, and H322) 72 hours after transfection with PBS (control [con]), Ad-PKR (2,500 viral particles/cell), Ad-PKRΔ6 (2,500 viral particles/cell), or Ad-Luc (2,500 viral particles/cell). The expression of actin was used as a loading control.

Article Snippet: We obtained the following antibodies from Cell Signaling Technology: AMPK (2532), phospho-AMPK α (Thr172) (40H9) (2535), p-TSC2 (Ser1387) (5584), acetyl-CoA carboxylase (C83B10) (3676), phospho-acetyl-CoA carboxylase (Ser79) (3661), phospho-mTOR (Ser2448) (5536), phospho-LKB1 (Ser428) (C67A3) (3482), TAK1 (4505), and phospho-TAK1 (Thr184/187) (90C7) (4508).

Techniques: Western Blot, Expressing, Transfection, Control

Journal: International Journal of Nanomedicine

Article Title: Peripheral Serum Exosomes Isolated from Patients with Acute Myocardial Infarction Promote Endothelial Cell Angiogenesis via the miR-126-3p/TSC1/mTORC1/HIF-1α Pathway

doi: 10.2147/IJN.S338937

Figure Lengend Snippet: miR-126-3p promotes angiogenesis by directly targeting TSC1. ( A ) Screening miR-126-3p-targeted genes ( B ) Relative mRNA expression of TSC1 in HUVECs after treatment with mimic miR-126-3p, mimic NC, inhibitor miR-126-3p, or inhibitor NC. ( C ) Matched base pairs of the miR-126-3p binding area and reporter plasmids including wild- or mutant-type TSC1. ( D ) Relative luciferase reporter activity of vectors and a fragment of TSC1 UTR with wild- or mutant-type miR-126-3p binding sites after co-transfection with the four groups. ( E ) Western blotting showed the relative protein expression of TSC1, pS6, S6, HIF-1α, and VEGFA in HUVECs treated with the mimic miR-126-3p, mimic NC, inhibitor miR-126-3p, or inhibitor NC. ( F ) Protein expression quantification. ( G ) Western blotting showed the relative protein expression of TSC1, pS6, S6, HIF-1α, and VEGFA in HUVECs treated with the mimic NC, mimic miR-126-3p, mimic miR-126-3p + oe.NC, mimic miR-126-3p + oe.TSC1. ( H ) Protein expression quantification. * p < 0.05, ** p < 0.01.

Article Snippet: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins; these were then transferred onto polyvinylidene difluoride membranes (Millipore, Massachusetts, USA), and incubated at 4 °C overnight together with primary antibodies against Alix (Abcam, ab186429, UK), CD63 (Abcam, ab217345), TSG101 (Abcam, ab125011), TSC1 (CST, 6935, China), HIF-1α (Abcam, ab179483), phospho-S6 S240/244 (CST, 5364), S6 (CST, 2217), VEGFA (Abcam, ab214424), and GAPDH (Bioss, Beijing, China).

Techniques: Expressing, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Cotransfection, Western Blot

Journal: International Journal of Nanomedicine

Article Title: Peripheral Serum Exosomes Isolated from Patients with Acute Myocardial Infarction Promote Endothelial Cell Angiogenesis via the miR-126-3p/TSC1/mTORC1/HIF-1α Pathway

doi: 10.2147/IJN.S338937

Figure Lengend Snippet: miR-126-3p-targeted TSC1 promotes angiogenesis in vitro. The mimic NC, mimic miR-126-3p, mimic miR-126-3p + oe. NC, and mimic miR-126-3p + oe. TSC1 were transfected into HUVECs. ( A ) HUVEC proliferation was measured by manual cell counting, CCK8 absorbance ( B ), and EdU staining ( C and D ). Scale bar, 50 μm. Cell migration was assessed by scratch wound assays ( E ) and analyzed as the ratio of non-migrated area divided by the baseline wound area ( F ). Scale bar, 100 μm. ( G ) Microvessels were quantified using aortic sprouting assays ( H ). Scale bar, 200 μm. ** p < 0.01, *** p < 0.001.

Article Snippet: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins; these were then transferred onto polyvinylidene difluoride membranes (Millipore, Massachusetts, USA), and incubated at 4 °C overnight together with primary antibodies against Alix (Abcam, ab186429, UK), CD63 (Abcam, ab217345), TSG101 (Abcam, ab125011), TSC1 (CST, 6935, China), HIF-1α (Abcam, ab179483), phospho-S6 S240/244 (CST, 5364), S6 (CST, 2217), VEGFA (Abcam, ab214424), and GAPDH (Bioss, Beijing, China).

Techniques: In Vitro, Transfection, Cell Counting, Staining, Migration

Journal: Oncotarget

Article Title: Sporadic renal angiomyolipoma in a patient with Birt-Hogg-Dubé: chaperones in pathogenesis

doi: 10.18632/oncotarget.25164

Figure Lengend Snippet: ( A ) HEK293 cells were transiently transfected with 2 µg of either WT or L460QsX25 mutated FLCN-FLAG. Expression was assessed by immunoblotting. Empty vector (EV) was used as a control. ( B ) HEK293 cells were transiently transfected with 2 µg WT or 6 µg L460QsX25 mutated FLCN-FLAG. Expression of FLCN-FLAG and Tsc2 was assessed by immunoblotting. EV was used as a control. ( C ) HEK293 cells were transiently transfected with 1 µg of EV or FLCN-L460QsX25-FLAG for 24 hr and then treated with 200 nM proteasome inhibitor bortezomib (BZ) for 4 hr prior to protein extraction. Stability of FLCN-FLAG and Tsc2 was assessed by immunoblotting. ( D ) WT and L460QsX25 mutated FLCN-FLAG were transiently expressed and immunoprecipitated from HEK293 cells. Co-immunoprecipitation (Co-IP) of endogenous FNIP1, Tsc1, Hsp90, and Hsp70 was assessed by immunoblotting. EV was used as a control. ( E ) HEK293 cells were transiently transfected with either EV or FLCN-L460QsX25-FLAG with or without co-transfection of Tsc1-HA. Those cells without co-expression of Tsc1-HA instead had additional EV co-transfected. Stability of FLCN-FLAG was assessed by immunoblotting. Overexpression of Tsc1 was demonstrated by probing the blot with an anti-Tsc1 antibody. GAPDH was used as a loading control. ( F ) HEK293 cells were transiently transfected with either EV or FLCN-L460QsX25-FLAG with or without co-transfection of FNIP1-HA. Those cells without co-expression of FNIP1-HA instead had additional EV co-transfected. Stability of FLCN-FLAG was assessed by immunoblotting. Overexpression of FNIP1 was demonstrated by probing the blot with an anti-FNIP1 antibody. GAPDH was used as a loading control.

Article Snippet: Primary antibodies recognizing FLAG 1:8000 (ThermoFisher Scientific; PA1-984B), Hsp90-835-16F1 1:8000 (ENZO Life Sciences; ADI-SPA-835), GAPDH 1:10,000 (ENZO Life Sciences; ADI-CSA-335), Hsp70 1:40,000 (StressMarq; SPC-103), FLCN 1:4000 (Cell Signaling Technologies (CST); 3697), Tsc1 1:1000 (CST; 4906), Tsc2 1:1000 (CST; 3990), phos-mTOR 1:1000 (S2448; CST; 5536), mTOR 1:1000 (CST; 2983), phos-S6K 1:6000 (T389; CST; 9234), phos-4EBP1 1:2000 (T37/46; CST; 2855), 4EBP1 1:2000 (CST; 9644), phos-Akt 1:2000 (S473; CST; 4060); Akt 1:2000 (CST; 9727), S6K 1:6000 (SantaCruz Biotechnology; sc-8418) were used for immunoblotting.

Techniques: Transfection, Expressing, Western Blot, Plasmid Preparation, Control, Protein Extraction, Immunoprecipitation, Co-Immunoprecipitation Assay, Cotransfection, Over Expression