lentivirus shrna targeting protein kinase amp activated catalytic subunit alpha 1 2  (Santa Cruz Biotechnology)

Journal: iScience

Article Title: Phosphorylated YBX2 is stabilized to promote glycolysis in brown adipocytes

doi: 10.1016/j.isci.2023.108091

Figure Lengend Snippet: AMPK phosphorylates YBX2 at Thr115 and Ser189 (A) Immunoblot analysis (top) of the indicated proteins in BAT. Mice were housed at 22°C or exposed to 4°C for 6 h (n = 3 each group). Bottom panel showed quantification of protein levels. (B) Representative YBX2 protein level (top) in differentiated brown adipocytes treated with 10 μM Cl316,243 for 12 h in the presence of H-89, compound C or Akt inhibitor Ⅷ. Quantification of YBX2 protein level was shown in the bottom panel (n = 3 independent cultures). (C) Representative immunoblot analysis of the indicated proteins in mature brown adipocytes stimulated with A-769662 (10μM) for 12 h. Quantification of protein levels was shown in the bottom panel (n = 4–6 independent cultures). (D) Representative YBX2 protein level in mature brown adipocytes. Brown adipocytes were infected with sh AMPKα1 lentivirus on day 2, and treated with 10 μM Cl316,243 for 12 h on day 6. Quantification of protein levels was shown in right panel (n = 4 independent cultures). (E) Immunoprecipitation of Flag-YBX2 protein followed by western blot to detect AMPKα1 in mature brown adipocytes. The experiment was repeated independently for three times. (F) Representative phosphorylated AMPK substrate levels in in vitro kinase assay. The recombinant GST-YBX2 protein and recombinant AMPKα1β1γ1 kinase were incubated at 30°C for 30 min followed by western blot analysis (top). Three independent experiments were repeated and the quantified phosphorylated substrate levels were shown in bottom panel (n = 3 independent experiments). (G and H) Representative YBX2 protein level in mature brown adipocytes. Preadipocytes were infected with lentiviruses carrying YBX2 WT , T115A , or S189A , and induced for differentiation. Adipocytes were treated with AMPK agonist A-769662 (5 μm) (G) or compound C (10 μm) (H) in the presence of Cl316,243 (10 μM) for 12 h. Quantification of protein levels was shown in the bottom panel (G, n = 4 independent cultures) or in the right panel (H, n = 3 independent cultures). Data are expressed as mean ± SEM of biologically independent samples. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by two-tailed unpaired Student’s t test (for A, C), or by one-way ANOVA (for B, D, F), or by two-way ANOVA (for G, H). See also Figure S2 .

Article Snippet: Ybx1 , Ybx2 , Ybx3 and AMPKα1 short hairpin RNA (shRNA) lentiviral constructs were generated using psp108 vector (Addgene).

Techniques: Western Blot, Infection, Immunoprecipitation, In Vitro, Kinase Assay, Recombinant, Incubation, Two Tailed Test

Journal: iScience

Article Title: Phosphorylated YBX2 is stabilized to promote glycolysis in brown adipocytes

doi: 10.1016/j.isci.2023.108091

Figure Lengend Snippet: Akt phosphorylates YBX2 at Ser 137 (A) Representative YBX2 protein level in mature brown adipocytes treated with 25 μM Akt inhibitor Ⅷ for the indicated time. The experiment was repeated for three times. (B) Immunoblot analysis of the indicated protein in brown adipocytes (left) and quantification of the YBX2 protein level (right, n = 3 individual cultures). The culture medium was supplemented with or without insulin for 24 h from day 5 to day 6 of differentiation. (C) Immunoprecipitation of Flag-YBX2 protein from mature brown adipocytes followed by western blot to detect Akt2. The experiment was repeated for three times. (D) Alignment of YBX1 and YBX2 amino acid sequence around the potential Akt2 substrate motif. (E) Representative phosphorylated Akt motif level in YBX2 protein. In vitro kinase assay used recombinant GST-YBX2 protein and recombinant constitutive active Akt2 kinase followed by western blot analysis (left). The experiment was repeated for three times and quantification of phosphorylated Akt substrate levels was shown in the right panel (n = 3 independent experiments). (F) Immunoblot analysis of YBX2 WT and S137A level in mature brown adipocytes. Cultured adipocytes carrying lentiviral YBX2 WT or S137A were incubated in medium with or without insulin for 24 h before immunoblot analysis. Two independent experiments were repeated and quantification of protein levels was shown in the bottom panel (n = 4 independent cultures). (G) Immunoblot analysis of YBX2 WT and S137A level in mature brown adipocytes. Adipocytes carrying YBX2 WT or S137A were treated with 25 μM Akt inhibitor Ⅷ in the presence of Cl316,243 (10 μm) for 12 h before immunoblot analysis. The experiment was repeated for three times and quantification of protein levels was shown in the bottom panel (n = 3 independent experiments). Data are expressed as mean ± SEM of biologically independent samples. ∗p < 0.05, ∗∗∗p < 0.0001 by two-tailed unpaired Student’s t test (for B), or by one-way ANOVA (for E), or by two-way ANOVA (for F, G). See also Figure S3 .

Article Snippet: Ybx1 , Ybx2 , Ybx3 and AMPKα1 short hairpin RNA (shRNA) lentiviral constructs were generated using psp108 vector (Addgene).

Techniques: Western Blot, Immunoprecipitation, Sequencing, In Vitro, Kinase Assay, Recombinant, Cell Culture, Incubation, Two Tailed Test

Journal: iScience

Article Title: Phosphorylated YBX2 is stabilized to promote glycolysis in brown adipocytes

doi: 10.1016/j.isci.2023.108091

Figure Lengend Snippet: YBX2 regulates glycolysis gene expression in brown adipocytes (A) Immunoblot analysis for the indicated proteins in brown fat cells. Preadipocytes were infected by lentiviral shRNA targeting Ybx2 , followed by differentiation to mature adipocytes. Then cells were treated with or without 10 μM Cl316,243 for 14 h. (B–D) ECAR was measured by Seahorse analyzer with cells treated as in (A) (n = 6). Basal ECAR (C) and maximal ECAR (D) were quantified (n = 6). (E and F) Preadipocytes bearing lentiviral Flag - Ybx2 differentiated to mature adipocytes. Immunoblot analysis for the indicated proteins (E) and the measurement of Ybx2 mRNA level (F) were performed on day 6. (G–I) ECAR was measure by Seahorse analyzer with cells generated as in (E) (n = 6). Basal ECAR (H) and maximal ECAR (I) were quantified (n = 6). (J and K) The WT or mutant YBX2 was introduced into YBX2 knockdown adipocytes by lentiviral transduction. Cl316,243 treated the mature adipocytes for 14 h before harvesting the cells (K). The indicated proteins were detected by western blots. (L) The relative glycolysis metabolite levels were determined by LC-MS/MS in mature brown adipocytes treated by Cl316,243 for 12 h (n = 4 independent samples). Data are expressed as mean ± SEM of biologically independent samples. ∗p < 0.05, ∗∗p < 0.01 by two-way ANOVA (for C, D), by two-tailed unpaired Student’s t test (for L) or by one-way ANOVA (for F, H, I). See also Figure S5 .

Article Snippet: Ybx1 , Ybx2 , Ybx3 and AMPKα1 short hairpin RNA (shRNA) lentiviral constructs were generated using psp108 vector (Addgene).

Techniques: Gene Expression, Western Blot, Infection, shRNA, Generated, Mutagenesis, Knockdown, Transduction, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: iScience

Article Title: Phosphorylated YBX2 is stabilized to promote glycolysis in brown adipocytes

doi: 10.1016/j.isci.2023.108091

Figure Lengend Snippet: YBX2 enhances translation of glycolytic genes in brown adipocytes (A and B) RNA immunoprecipitation followed by real-time PCR to detect YBX2 association with Hk2 (A) and Pkm1 (B) mRNAs. Brown adipocytes expressing HA-YBX2 were subjected to IgG or HA antibody pulldown. The primers used were indicated. n = 3 independent experiments. (C) Immunoblot analysis (left) and protein quantification (right) in brown adipocytes for the indicated proteins. Preadipocytes were infected by lentivirus expressing WT or Y107A/Y109A mutant (Mu) Ybx2 , followed by differentiation induction. n = 3 individual cultures. (D–F) Oxygen consumption rate (D), glucose (E) and lactate levels (F) in culture media of adipocytes in (C) (n = 3 individual cultures). (G and H) Immunoblot analysis (G) and protein quantification (H) for indicated genes in brown adipocytes. The YBX2 protein level in knockdown adipocytes was reconstituted by lentiviral expressing WT or Mu Ybx2 on day 2. n = 3 individual cultures. (I) A Click-iT AHA labeling assay was used to detect newly synthesized glycolytic protein in Ybx2 knockdown brown adipocytes on day 4. The adipocytes were treated with or without Cl316,243 for 14 h before the incubation with AHA for 4 h. The experiment was repeated for three times and the representative images were shown. Data are shown as mean ± SEM of biologically independent samples. ∗p < 0.05, ∗∗p < 0.01 by one-way ANOVA. See also Figure S7 .

Article Snippet: Ybx1 , Ybx2 , Ybx3 and AMPKα1 short hairpin RNA (shRNA) lentiviral constructs were generated using psp108 vector (Addgene).

Techniques: RNA Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Infection, Mutagenesis, Knockdown, Labeling, Synthesized, Incubation

Journal: Journal of Korean Neurosurgical Society

Article Title: EID3 Promotes Glioma Cell Proliferation and Survival by Inactivating AMPKα1

doi: 10.3340/jkns.2021.0298

Figure Lengend Snippet: EID3 preferentially expressed in glioma cells and correlated with AMP-activated protein kinase α1 (AMPKα1) downregulation. EID3 preferentially expressed in glioma cells and correlated with AMPKα1 downregulation. A : mRNa expression of EID3 in primary astrocyte, neuronal cell (HCN-1a), and A172 glioma cells assessed by real-time polymerase chain reaction assays. B : Protein expression of EID3, AMPKα1, pS6K1, and S6K1 in primary astrocyte, neuronal cell (HCN-1a), and A172 glioma cells assessed by WB assays. Tubulin was used as an internal control. C : EID3 and AMPKα1 protein expression in human glioma tissues and paired surrounding normal brain tissues assessed by WB assays. EID3 : EP300-interacting inhibitor of differentiation, WB : Western blot.

Article Snippet: Two lentiviral AMPKα1 shRNAs were purchased from Santa Cruz Biotech (shAMPKα1-1; Santa Cruz, CA, USA) and GeneChem (shAMPKα1-2; Montreal, Canada).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Journal of Korean Neurosurgical Society

Article Title: EID3 Promotes Glioma Cell Proliferation and Survival by Inactivating AMPKα1

doi: 10.3340/jkns.2021.0298

Figure Lengend Snippet: Biological functions of EID3 in glioma. A : Stable A172 cells expressing EID3 shRNas (sh-1, sh-2), and scramble control shRNa were subjected to real-time polymerase chain reaction and WB assays. EID3 expression was knocking down efficiently with two shRNas targeting different sites of EID3 cdNa. EID3 knocking down could restore AMPKα1 and inhibit S6K1 expression. B : Colony formation assays were used to evaluate the effects of EID3 knocking down on glioma cell proliferation. C : Transwell assays were conducted to evaluate the effects of EID3 knocking down on glioma cell migration and invasion. D : Effects of EID3 knocking down on glioma cell apoptosis (left), survival (middle), and death (right) were evaluated by certain function assays. EID3 shRNa could induce apoptosis in glioma cells compared to the scramble non-sense control shRNa. E : Expressions of listed autophagy-associated genes were detected by WB assays after EID3 knocking down. F : In vivo growth curves (left) and weights (right) of glioma tumors in SCID mice. The tumors generated from EID3 knocking down A172 cells grew significantly slower than control tumors. * p <0.05 vs. sh-C. EID3 : EP300-interacting inhibitor of differentiation, AMPKα1 : AMP-activated protein kinase α1, ELISA : enzyme linked immunosorbent assay, OD : optical density, MTT : methyl thiazolyl tetrazolium, WB : Western blot, SCID : severe combined immune deficiency.

Article Snippet: Two lentiviral AMPKα1 shRNAs were purchased from Santa Cruz Biotech (shAMPKα1-1; Santa Cruz, CA, USA) and GeneChem (shAMPKα1-2; Montreal, Canada).

Techniques: Expressing, Control, shRNA, Real-time Polymerase Chain Reaction, Migration, In Vivo, Generated, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Journal of Korean Neurosurgical Society

Article Title: EID3 Promotes Glioma Cell Proliferation and Survival by Inactivating AMPKα1

doi: 10.3340/jkns.2021.0298

Figure Lengend Snippet: AMP-activated protein kinase (AMPK) signaling activation mediated cell death and apoptosis caused by EID3 knocking down. A : Validation of the transfection efficiencies of AMPKα1 silence in stable A172 cells that expressing EID3 shRNas. B : Effects of AMPKα1 knocking down on apoptosis (left), survival (middle), and death (right) of glioma cell expressing EID3 shRNas. The cell viability reduction and cell apoptosis could be largely attenuated by AMPKα1 knocking down. C : Validation of the transfection efficiencies of dN-AMPKα1. D : Effects of dN-AMPKα1 transfection on the activation status of AMPK and ACC were evaluated by WB assays. E and F : Effects of dN-AMPKα1 transfection on glioma survival and death in A172 cells expressing control shRNa or EID3 shRNas. The forced expression of dN-AMPKα1 did not rescue the inhibition effects of EID3 knocking down on cell survival and death. G : mRNa expression of AMPKα1 assessed by real-time polymerase chain reaction (RT-PcR) after EID3 knocking down. Neither EID3 shRNa could decrease AMPKα1 mRNa expression. H : Protein expression of AMPKα1 assessed by WB after MG132 treatment. I : mRNa expression of AMPKα1 assessed by RTPcR after MG132 treatment. MG132 showed no significant influence on the mRNa expression of AMPKα1 ( p =0.677). J : Protein expression of AMPKα1 assessed by WB after cycloheeximide treatment in EID3 knocking down A172 cells. * p <0.05 vs. sh-C. † p <0.05 vs. no shRNa. ‡ p <0.05 vs. parental or vector in shEID3. EID3 : EP300-interacting inhibitor of differentiation, DN : dominant negative, ACC : acetyl-coa carboxylase, MTT : methyl thiazolyl tetrazolium, OD : optical density, WB : Western blot.

Article Snippet: Two lentiviral AMPKα1 shRNAs were purchased from Santa Cruz Biotech (shAMPKα1-1; Santa Cruz, CA, USA) and GeneChem (shAMPKα1-2; Montreal, Canada).

Techniques: Activation Assay, Biomarker Discovery, Transfection, Expressing, Control, shRNA, Inhibition, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Dominant Negative Mutation, Western Blot

Journal: Journal of Korean Neurosurgical Society

Article Title: EID3 Promotes Glioma Cell Proliferation and Survival by Inactivating AMPKα1

doi: 10.3340/jkns.2021.0298

Figure Lengend Snippet: AMP-activated protein kinase (AMPK) mediated viability reduction and cell death in EID3 knocking down primary glioma cells. A : Protein expression of EID3, AMPKα1, pS6K1, and S6K1 in three tumor with descending EID3 level from PDX models. B : Growth curves indicated that EID3 expression level correlated with glioma growth speed in PDX models. T1 (high EID3) grew significantly faster than T2 and T3. T2 also showed significantly faster growth than T3 did. C : Effects of EID3 knocking down on the expression level of AMPKα1, pS6K1, and S6K1 in primary glioma cells generated from PDX model T1 (high EID3 expression). D : Expressions of listed autophagy-associated genes were detected by Western blot assays after EID3 knocking down in in primary glioma cells generated from PDX model T1. E : Effects of AMPKα1 knocking down on S6K1 activation status in primary glioma cells expressing EID3 shRNa3. F : Effects of AMPKα1 knocking down on cell survival (left) and death (right) in primary glioma cells expressing EID3 shRNa3. AMPKα1 knocking down largely attenuated the viability reduction and cell death caused by EID3 inhibition ( p <0.05). * p <0.05. EID3 : EP300-interacting inhibitor of differentiation, MTT : methyl thiazolyl tetrazolium, OD : optical density, PDX : patient-derived xenograft.

Article Snippet: Two lentiviral AMPKα1 shRNAs were purchased from Santa Cruz Biotech (shAMPKα1-1; Santa Cruz, CA, USA) and GeneChem (shAMPKα1-2; Montreal, Canada).

Techniques: Expressing, Generated, Western Blot, Activation Assay, Inhibition, Derivative Assay