Journal: PLoS Biology

Article Title: HIV-1 Tat-mediated astrocytic amyloidosis involves the HIF-1α/lncRNA BACE1-AS axis

doi: 10.1371/journal.pbio.3000660

Figure Lengend Snippet: Representative western blots showing the expression of Aβ mOC64 (A) and APP (B) and p-Tau/Tau (C) in different brain regions—FC, PC, Cer, BS, OC, Thal—of saline and SIV + macaques. (D) RNA-seq analysis showing expression of HIF 1A, APP, BACE1, GFAP, and AIF1 in the FC of saline- and SIV-infected macaques. Data are presented as mean ± SEM; n = 6. Student t test was used to determine the statistical significance: * P < 0.05 versus saline. The data underlying this figure may be found in . AIF1, allograft inflammatory factor 1; APP, amyloid precursor protein; Aβ, amyloid beta; BACE1, β-site cleaving enzyme; BS, brain stem; Cer, cerebellum; FC, frontal cortex; GFAP, glial fibrillary acidic protein; OC, occipital cortex; PC, parietal cortex; RNA-Seq, RNA-sequencing; SIV, simian immunodeficiency virus; Thal, thalamus.

Article Snippet: The following antibodies or reagents used in this study were from the indicated sources: reagents: HIV-1 Tat 101 (Immunodiaognistics, Woburn, MA), cobalt chloride (Sigma-Aldrich, St . Louis , MO , 232696), Actinomycin D (Sigma, St . Louis , MO , A9415), SP6/T7 Transcription Kit (Sigma, St. Louis, MO, 10 999 644 001), Biotin-16-UTP (Sigma, St. Louis, MO, 11 388 908 910), HindIII (NEB, Ipswich, MA,R0104S), EcoRI (NEB, Ipswich, MA, R0101S), Tth111I (NEB, Ipswich, MA, R0185S), Platinum Taq DNA Polymerase High Fidelity (ThermoFisher, Waltham, MA,11304011), 10 mM dNTP mix (ThermoFisher, Waltham, MA, 18427–088), TrackIt 1 Kb Plus DNA Ladder (ThermoFisher, Waltham, MA, 10488–085), QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany, 28704), NEB 5-alpha Competent E. coli (High Efficiency) (NEB, Ipswich, MA, C2987I), T4 DNA Ligase (NEB, Ipswich, MA, M0202S), Light Shift Chemiluminescent RNA EMSA (REMSA) Kit (Thermo, Waltham, MA, 20158), HIF1A purified protein Recombinant Human HIF-1 alpha protein (Abcam, Cambridge, MA, ab154478), DEPC (Sigma, St. Louis, MO, 40718), Positive charged nylon membrane (Ambion, Austin, TX, AM10104), ssRNA Ladder (NEB, Ipswich, MA, N0364S), RNA loading dye (NEB, Ipswich, MA, BO363), 10X MOPS buffer (KD Medical, Columbia, MD, RGF-6170), 10X TBE Electrophoresis buffer (Thermo Scientific, Waltham, MA, B52), Formaldehyde (Sigma, St. Louis, MO, 252549), RNA gel extraction kit (Zymo Research, R1011); antibodies: HIF-1α (Novus Biological Company, Littleton, CO, NB100-449, Millipore Sigma, St. Louis, MO,MAB5382MI), APP (Abcam, Cambridge, MA, ab15272), AβmOC64 (Abcam, Cambridge, MA, ab201060), Aβ 1–42 (Abcam, Cambridge, MA, ab10148), Aβ 1–40 (Abcam, Cambridge, MA, ab76317), BACE1 (Abcam, ab63954), p-Tau (Abcam, Cambridge, MA, ab76128), Tau (Abcam, Cambridge, MA, ab9778), MAP 2 (Abcam, Cambridge, MA, ab5392), GFAP (Sigma-Aldrich, St. Louis, MO, G3893), Lamin B1 (Abcam, Cambridge, MA, ab8982), PHD-2 (Abcam, Cambridge, MA, ab133630), goat anti-rabbit (sc-2004, Santa Cruz Biotechnology, Dallas, TX), goat anti-mouse (sc-2005, Santa Cruz Biotechnology, Dallas, TX), and β-actin (Sigma Aldrich, St. Louis, MO, A5316) were purchased from commercial vendors, as mentioned.

Techniques: Western Blot, Expressing, RNA Sequencing Assay, Infection

Journal: PLoS Biology

Article Title: HIV-1 Tat-mediated astrocytic amyloidosis involves the HIF-1α/lncRNA BACE1-AS axis

doi: 10.1371/journal.pbio.3000660

Figure Lengend Snippet: Western blot analysis showing dose- (A) and time-dependent (B) up-regulation of HIF-1α in HPAs exposed to HIV-1 Tat at the indicated dose and time points. Cobalt chloride (CoCl 2 −10 μM) was used as a positive control for HIF-1α expression. (C) Nuclear and cytoplasmic expression of HIF-1α protein 30 minutes following exposure of cells to HIV-1 Tat (3.57 nM). (D) Representative fluorescent photomicrographs showing increased expression of HIF-1α after 30 minutes of HIV-1 Tat (3.57 nM) exposure in HPAs. (E) qPCR analysis demonstrating expression of lncRNA BACE1-AS in HPAs exposed to HIV-1 Tat (3.57 nM) at the indicated time points. (F) Nuclear and cytoplasmic expression of BACE1-AS RNA following exposure to HPAs to HIV-1 Tat (3.57 nM) for 12 hours. (G) Representative FISH and immunofluorescence photomicrographs demonstrating increased expression of BACE1-AS RNA in the GFAP + astrocytes in the FC of SIV + macaques compared with the saline group. Scale bar, 10 μm. Data are presented as mean ± SEM; n = 6. One-way ANOVA followed by Bonferroni post hoc and Student t test was employed to determine the statistical significance: * P < 0.05 versus control. Arrows indicate GFAP-positive astrocytes colocalized with BACE1-AS RNA. The data underlying this figure may be found in . BACE1-AS, BACE1‐antisense transcript; FC, frontal cortex; FISH, fluorescence insitu hybridization; GFAP, glial fibrillary acidic protein; HIF-1α, hypoxia-inducible factor; HPA, human primary astrocyte; lncRNA, long noncoding RNA; qPCR, quantative polymerase chain reaction; SIV, simian immunodeficiency virus; Tat, transactivator of transcription.

Article Snippet: The following antibodies or reagents used in this study were from the indicated sources: reagents: HIV-1 Tat 101 (Immunodiaognistics, Woburn, MA), cobalt chloride (Sigma-Aldrich, St . Louis , MO , 232696), Actinomycin D (Sigma, St . Louis , MO , A9415), SP6/T7 Transcription Kit (Sigma, St. Louis, MO, 10 999 644 001), Biotin-16-UTP (Sigma, St. Louis, MO, 11 388 908 910), HindIII (NEB, Ipswich, MA,R0104S), EcoRI (NEB, Ipswich, MA, R0101S), Tth111I (NEB, Ipswich, MA, R0185S), Platinum Taq DNA Polymerase High Fidelity (ThermoFisher, Waltham, MA,11304011), 10 mM dNTP mix (ThermoFisher, Waltham, MA, 18427–088), TrackIt 1 Kb Plus DNA Ladder (ThermoFisher, Waltham, MA, 10488–085), QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany, 28704), NEB 5-alpha Competent E. coli (High Efficiency) (NEB, Ipswich, MA, C2987I), T4 DNA Ligase (NEB, Ipswich, MA, M0202S), Light Shift Chemiluminescent RNA EMSA (REMSA) Kit (Thermo, Waltham, MA, 20158), HIF1A purified protein Recombinant Human HIF-1 alpha protein (Abcam, Cambridge, MA, ab154478), DEPC (Sigma, St. Louis, MO, 40718), Positive charged nylon membrane (Ambion, Austin, TX, AM10104), ssRNA Ladder (NEB, Ipswich, MA, N0364S), RNA loading dye (NEB, Ipswich, MA, BO363), 10X MOPS buffer (KD Medical, Columbia, MD, RGF-6170), 10X TBE Electrophoresis buffer (Thermo Scientific, Waltham, MA, B52), Formaldehyde (Sigma, St. Louis, MO, 252549), RNA gel extraction kit (Zymo Research, R1011); antibodies: HIF-1α (Novus Biological Company, Littleton, CO, NB100-449, Millipore Sigma, St. Louis, MO,MAB5382MI), APP (Abcam, Cambridge, MA, ab15272), AβmOC64 (Abcam, Cambridge, MA, ab201060), Aβ 1–42 (Abcam, Cambridge, MA, ab10148), Aβ 1–40 (Abcam, Cambridge, MA, ab76317), BACE1 (Abcam, ab63954), p-Tau (Abcam, Cambridge, MA, ab76128), Tau (Abcam, Cambridge, MA, ab9778), MAP 2 (Abcam, Cambridge, MA, ab5392), GFAP (Sigma-Aldrich, St. Louis, MO, G3893), Lamin B1 (Abcam, Cambridge, MA, ab8982), PHD-2 (Abcam, Cambridge, MA, ab133630), goat anti-rabbit (sc-2004, Santa Cruz Biotechnology, Dallas, TX), goat anti-mouse (sc-2005, Santa Cruz Biotechnology, Dallas, TX), and β-actin (Sigma Aldrich, St. Louis, MO, A5316) were purchased from commercial vendors, as mentioned.

Techniques: Western Blot, Positive Control, Expressing, Immunofluorescence, Fluorescence, Hybridization, Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: HIV-1 Tat-mediated astrocytic amyloidosis involves the HIF-1α/lncRNA BACE1-AS axis

doi: 10.1371/journal.pbio.3000660

Figure Lengend Snippet: (A) qPCR analysis demonstrating expression of HIF-1α, BACE1-AS, and BACE1 mRNA in HPAs transfected with either HIF-1α or scrambled siRNA in the presence or absence of HIV-1 Tat (3.57 nM; 24 hours). (B) Representative western blots showing the expression of HIF-1α, BACE1, and Aβ mOC64 proteins in HPAs transfected with either HIF-1α or scrambled siRNA. (C) ELISA showing the protein levels of Aβ 42 in the supernatant fluids of HPAs transfected with either HIF-1α or scrambled siRNA. (D) qPCR demonstrating expression of BACE1-AS, BACE1, and HIF-1α RNA in HPAs transfected with either BACE1-AS or scrambled siRNA. (E) Representative western blots showing the expressions of BACE1, Aβ mOC64, and HIF-1α in HPA transfected with either BACE1-AS or scrambled siRNA. (F) ELISA showing the protein levels of Aβ42 in the supernatant of HPA transfected with either BACE1-AS or scrambled siRNA. Data are presented as mean ± SEM; n = 6. One-way ANOVA followed by Bonferroni post hoc test was used to determine the statistical significance: * P < 0.05 versus control, # P < 0.05 versus Tat. The data underlying this figure may be found in . Aβ, amyloid beta; BACE1, β-site cleaving enzyme; BACE1-AS, BACE1‐antisense transcript; HIF-1α, hypoxia-inducible factor; HPA, human primary astrocyte; qPCR, quantative polymerase chain reaction; siRNA, small interfering RNA; Tat, transactivator of transcription.

Article Snippet: The following antibodies or reagents used in this study were from the indicated sources: reagents: HIV-1 Tat 101 (Immunodiaognistics, Woburn, MA), cobalt chloride (Sigma-Aldrich, St . Louis , MO , 232696), Actinomycin D (Sigma, St . Louis , MO , A9415), SP6/T7 Transcription Kit (Sigma, St. Louis, MO, 10 999 644 001), Biotin-16-UTP (Sigma, St. Louis, MO, 11 388 908 910), HindIII (NEB, Ipswich, MA,R0104S), EcoRI (NEB, Ipswich, MA, R0101S), Tth111I (NEB, Ipswich, MA, R0185S), Platinum Taq DNA Polymerase High Fidelity (ThermoFisher, Waltham, MA,11304011), 10 mM dNTP mix (ThermoFisher, Waltham, MA, 18427–088), TrackIt 1 Kb Plus DNA Ladder (ThermoFisher, Waltham, MA, 10488–085), QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany, 28704), NEB 5-alpha Competent E. coli (High Efficiency) (NEB, Ipswich, MA, C2987I), T4 DNA Ligase (NEB, Ipswich, MA, M0202S), Light Shift Chemiluminescent RNA EMSA (REMSA) Kit (Thermo, Waltham, MA, 20158), HIF1A purified protein Recombinant Human HIF-1 alpha protein (Abcam, Cambridge, MA, ab154478), DEPC (Sigma, St. Louis, MO, 40718), Positive charged nylon membrane (Ambion, Austin, TX, AM10104), ssRNA Ladder (NEB, Ipswich, MA, N0364S), RNA loading dye (NEB, Ipswich, MA, BO363), 10X MOPS buffer (KD Medical, Columbia, MD, RGF-6170), 10X TBE Electrophoresis buffer (Thermo Scientific, Waltham, MA, B52), Formaldehyde (Sigma, St. Louis, MO, 252549), RNA gel extraction kit (Zymo Research, R1011); antibodies: HIF-1α (Novus Biological Company, Littleton, CO, NB100-449, Millipore Sigma, St. Louis, MO,MAB5382MI), APP (Abcam, Cambridge, MA, ab15272), AβmOC64 (Abcam, Cambridge, MA, ab201060), Aβ 1–42 (Abcam, Cambridge, MA, ab10148), Aβ 1–40 (Abcam, Cambridge, MA, ab76317), BACE1 (Abcam, ab63954), p-Tau (Abcam, Cambridge, MA, ab76128), Tau (Abcam, Cambridge, MA, ab9778), MAP 2 (Abcam, Cambridge, MA, ab5392), GFAP (Sigma-Aldrich, St. Louis, MO, G3893), Lamin B1 (Abcam, Cambridge, MA, ab8982), PHD-2 (Abcam, Cambridge, MA, ab133630), goat anti-rabbit (sc-2004, Santa Cruz Biotechnology, Dallas, TX), goat anti-mouse (sc-2005, Santa Cruz Biotechnology, Dallas, TX), and β-actin (Sigma Aldrich, St. Louis, MO, A5316) were purchased from commercial vendors, as mentioned.

Techniques: Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Small Interfering RNA

Journal: PLoS Biology

Article Title: HIV-1 Tat-mediated astrocytic amyloidosis involves the HIF-1α/lncRNA BACE1-AS axis

doi: 10.1371/journal.pbio.3000660

Figure Lengend Snippet: (A) qPCR showing expression of BACE1, BACE1-AS, and HIF-1α RNA in HPAs transfected with either BACE1 or scrambled siRNA. (B) Representative western blots showing the expressions of BACE1, Aβ mOC64, and HIF-1α in HPA transfected with either BACE1 or scrambled siRNA. (C) ELISA showing the protein levels of Aβ 42 in the supernatant of HPA transfected with either BACE1 or scrambled siRNA. (D) qPCR showing mRNA expressions of BACE1, BACE1-AS, and HIF-1α in HPA transfected with either BACE1–BACE1-AS or scrambled siRNA. (E) Representative western blots showing the expressions of BACE1, Aβ mOC64, and HIF-1α in HPA transfected with either BACE1–BACE1-AS or scrambled siRNA. (F) ELISA showing the protein levels of Aβ42 in the supernatant of HPA transfected with either BACE1–BACE1-AS or scrambled siRNA. Data are presented as mean ± SEM; n = 6. One-way ANOVA followed by Bonferroni post hoc test was used to determine the statistical significance: * P < 0.05 versus control, # P < 0.05 versus Tat. The data underlying this figure may be found in . Aβ, amyloid beta; BACE1, β-site cleaving enzyme; BACE1-AS, BACE1‐antisense transcript; HIF-1α, hypoxia-inducible factor; HPA, human primary astrocyte; qPCR, quantative polymerase chain reaction; siRNA, small interfering RNA; Tat, transactivator of transcription.

Article Snippet: The following antibodies or reagents used in this study were from the indicated sources: reagents: HIV-1 Tat 101 (Immunodiaognistics, Woburn, MA), cobalt chloride (Sigma-Aldrich, St . Louis , MO , 232696), Actinomycin D (Sigma, St . Louis , MO , A9415), SP6/T7 Transcription Kit (Sigma, St. Louis, MO, 10 999 644 001), Biotin-16-UTP (Sigma, St. Louis, MO, 11 388 908 910), HindIII (NEB, Ipswich, MA,R0104S), EcoRI (NEB, Ipswich, MA, R0101S), Tth111I (NEB, Ipswich, MA, R0185S), Platinum Taq DNA Polymerase High Fidelity (ThermoFisher, Waltham, MA,11304011), 10 mM dNTP mix (ThermoFisher, Waltham, MA, 18427–088), TrackIt 1 Kb Plus DNA Ladder (ThermoFisher, Waltham, MA, 10488–085), QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany, 28704), NEB 5-alpha Competent E. coli (High Efficiency) (NEB, Ipswich, MA, C2987I), T4 DNA Ligase (NEB, Ipswich, MA, M0202S), Light Shift Chemiluminescent RNA EMSA (REMSA) Kit (Thermo, Waltham, MA, 20158), HIF1A purified protein Recombinant Human HIF-1 alpha protein (Abcam, Cambridge, MA, ab154478), DEPC (Sigma, St. Louis, MO, 40718), Positive charged nylon membrane (Ambion, Austin, TX, AM10104), ssRNA Ladder (NEB, Ipswich, MA, N0364S), RNA loading dye (NEB, Ipswich, MA, BO363), 10X MOPS buffer (KD Medical, Columbia, MD, RGF-6170), 10X TBE Electrophoresis buffer (Thermo Scientific, Waltham, MA, B52), Formaldehyde (Sigma, St. Louis, MO, 252549), RNA gel extraction kit (Zymo Research, R1011); antibodies: HIF-1α (Novus Biological Company, Littleton, CO, NB100-449, Millipore Sigma, St. Louis, MO,MAB5382MI), APP (Abcam, Cambridge, MA, ab15272), AβmOC64 (Abcam, Cambridge, MA, ab201060), Aβ 1–42 (Abcam, Cambridge, MA, ab10148), Aβ 1–40 (Abcam, Cambridge, MA, ab76317), BACE1 (Abcam, ab63954), p-Tau (Abcam, Cambridge, MA, ab76128), Tau (Abcam, Cambridge, MA, ab9778), MAP 2 (Abcam, Cambridge, MA, ab5392), GFAP (Sigma-Aldrich, St. Louis, MO, G3893), Lamin B1 (Abcam, Cambridge, MA, ab8982), PHD-2 (Abcam, Cambridge, MA, ab133630), goat anti-rabbit (sc-2004, Santa Cruz Biotechnology, Dallas, TX), goat anti-mouse (sc-2005, Santa Cruz Biotechnology, Dallas, TX), and β-actin (Sigma Aldrich, St. Louis, MO, A5316) were purchased from commercial vendors, as mentioned.

Techniques: Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Small Interfering RNA

Journal: PLoS Biology

Article Title: HIV-1 Tat-mediated astrocytic amyloidosis involves the HIF-1α/lncRNA BACE1-AS axis

doi: 10.1371/journal.pbio.3000660

Figure Lengend Snippet: (A) RIP assay demonstrating enrichment of BACE1-AS RNA (qPCR) in HIF-1α bound RNA complexes. (B) RNA-seq data from HIF-1α pulled down RNA-enriched product from RIP assay. (C) ChIP assay showing enrichment of BACE1 promoter in the HIF-1α–bound DNA complexes. ChIP was also done in the presence of BACE1-AS siRNA-transfected cells. PCR products at the end of ChIP assay were subjected to agarose gel electrophoresis, demonstrating enrichment of the BACE1 promoter. (D) Representative EMSA image showing dose-dependent binding of HIF-1α to BACE1-AS RNA. (E) Simulated structure of HIF-1α binding to lncRNA BACE-1AS. Data are presented as mean ± SEM; n = 6. Student t test/one-way ANOVA was employed to determine the statistical significance between two groups: * P < 0.05 versus control. The data underlying this figure may be found in . BACE1, β-site cleaving enzyme; BACE1-AS, BACE1‐antisense transcript; BL, biotin-labeled; ChIP, chromatin immunoprecipitation; EMSA, electrophorectic mobility shift assay; HIF-1α, hypoxia-inducible factor; HPA, human primary astrocyte; IgG, immunoglobulin G; lncRNA, long noncoding RNA; PS, positive control; qPCR, quantative polymerase chain reaction; RIP, RNA immunoprecipitation; RNA-seq, RNA-sequencing; siRNA, small interfering RNA.

Article Snippet: The following antibodies or reagents used in this study were from the indicated sources: reagents: HIV-1 Tat 101 (Immunodiaognistics, Woburn, MA), cobalt chloride (Sigma-Aldrich, St . Louis , MO , 232696), Actinomycin D (Sigma, St . Louis , MO , A9415), SP6/T7 Transcription Kit (Sigma, St. Louis, MO, 10 999 644 001), Biotin-16-UTP (Sigma, St. Louis, MO, 11 388 908 910), HindIII (NEB, Ipswich, MA,R0104S), EcoRI (NEB, Ipswich, MA, R0101S), Tth111I (NEB, Ipswich, MA, R0185S), Platinum Taq DNA Polymerase High Fidelity (ThermoFisher, Waltham, MA,11304011), 10 mM dNTP mix (ThermoFisher, Waltham, MA, 18427–088), TrackIt 1 Kb Plus DNA Ladder (ThermoFisher, Waltham, MA, 10488–085), QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany, 28704), NEB 5-alpha Competent E. coli (High Efficiency) (NEB, Ipswich, MA, C2987I), T4 DNA Ligase (NEB, Ipswich, MA, M0202S), Light Shift Chemiluminescent RNA EMSA (REMSA) Kit (Thermo, Waltham, MA, 20158), HIF1A purified protein Recombinant Human HIF-1 alpha protein (Abcam, Cambridge, MA, ab154478), DEPC (Sigma, St. Louis, MO, 40718), Positive charged nylon membrane (Ambion, Austin, TX, AM10104), ssRNA Ladder (NEB, Ipswich, MA, N0364S), RNA loading dye (NEB, Ipswich, MA, BO363), 10X MOPS buffer (KD Medical, Columbia, MD, RGF-6170), 10X TBE Electrophoresis buffer (Thermo Scientific, Waltham, MA, B52), Formaldehyde (Sigma, St. Louis, MO, 252549), RNA gel extraction kit (Zymo Research, R1011); antibodies: HIF-1α (Novus Biological Company, Littleton, CO, NB100-449, Millipore Sigma, St. Louis, MO,MAB5382MI), APP (Abcam, Cambridge, MA, ab15272), AβmOC64 (Abcam, Cambridge, MA, ab201060), Aβ 1–42 (Abcam, Cambridge, MA, ab10148), Aβ 1–40 (Abcam, Cambridge, MA, ab76317), BACE1 (Abcam, ab63954), p-Tau (Abcam, Cambridge, MA, ab76128), Tau (Abcam, Cambridge, MA, ab9778), MAP 2 (Abcam, Cambridge, MA, ab5392), GFAP (Sigma-Aldrich, St. Louis, MO, G3893), Lamin B1 (Abcam, Cambridge, MA, ab8982), PHD-2 (Abcam, Cambridge, MA, ab133630), goat anti-rabbit (sc-2004, Santa Cruz Biotechnology, Dallas, TX), goat anti-mouse (sc-2005, Santa Cruz Biotechnology, Dallas, TX), and β-actin (Sigma Aldrich, St. Louis, MO, A5316) were purchased from commercial vendors, as mentioned.

Techniques: RNA Sequencing Assay, Transfection, Agarose Gel Electrophoresis, Binding Assay, Labeling, Chromatin Immunoprecipitation, Mobility Shift, Positive Control, Polymerase Chain Reaction, Immunoprecipitation, Small Interfering RNA

Journal: PLoS Biology

Article Title: HIV-1 Tat-mediated astrocytic amyloidosis involves the HIF-1α/lncRNA BACE1-AS axis

doi: 10.1371/journal.pbio.3000660

Figure Lengend Snippet: (A) qPCR showing expression of HIF-1α mRNA in HPAs exposed to HIV-1 Tat (3.57 nM) at the indicated time points. (B) qPCR showing expression of PHD-2 in HPAs exposed to HIV-1 Tat (3.57 nM) at the indicated time points. GAPDH was used as an internal control for mRNA expression. (C) Representative western blot showing time-dependent down-regulation of PHD-2 in HPAs exposed to HIV-1 Tat at the indicated time points. β-actin was used as an internal control. (D) Regression analysis showing stabilization of HIF-1α mRNA in HPAs transfected with either scrambled or HIF-1α siRNA in the presence or absence of HIV-1 Tat. (E) Regression lines showing mRNA stabilization and indicating the half-lives of BACE1 in HPA transfected with scrambled/HIF-1α siRNA, with/without HIV-1 Tat exposure. (F) Regression lines showing mRNA stabilization and indicating the half-lives of BACE1 in HPA transfected with scrambled/BACE1-AS siRNA, with/without HIV-1 Tat exposure. (G) Regression lines showing mRNA stabilization and indicating the half-lives of BACE1-AS in HPA with/without HIV-1 Tat exposure. Data are presented as mean ± SEM; n = 6. One-way ANOVA followed by Bonferroni post hoc test was used to determine the statistical significance between multiple groups: * P < 0.05 versus control, # P < 0.05 versus Tat. The data underlying this figure may be found in . BACE1, β-site cleaving enzyme; BACE1-AS, BACE1‐antisense transcript; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HIF-1α, hypoxia-inducible factor; HPA, human primary astrocyte; PHD-2, prolyl hydroxylase 2; qPCR, quantitative polymerase chain reaction; siRNA, small interfering RNA; Tat, transactivator of transcription.

Article Snippet: The following antibodies or reagents used in this study were from the indicated sources: reagents: HIV-1 Tat 101 (Immunodiaognistics, Woburn, MA), cobalt chloride (Sigma-Aldrich, St . Louis , MO , 232696), Actinomycin D (Sigma, St . Louis , MO , A9415), SP6/T7 Transcription Kit (Sigma, St. Louis, MO, 10 999 644 001), Biotin-16-UTP (Sigma, St. Louis, MO, 11 388 908 910), HindIII (NEB, Ipswich, MA,R0104S), EcoRI (NEB, Ipswich, MA, R0101S), Tth111I (NEB, Ipswich, MA, R0185S), Platinum Taq DNA Polymerase High Fidelity (ThermoFisher, Waltham, MA,11304011), 10 mM dNTP mix (ThermoFisher, Waltham, MA, 18427–088), TrackIt 1 Kb Plus DNA Ladder (ThermoFisher, Waltham, MA, 10488–085), QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany, 28704), NEB 5-alpha Competent E. coli (High Efficiency) (NEB, Ipswich, MA, C2987I), T4 DNA Ligase (NEB, Ipswich, MA, M0202S), Light Shift Chemiluminescent RNA EMSA (REMSA) Kit (Thermo, Waltham, MA, 20158), HIF1A purified protein Recombinant Human HIF-1 alpha protein (Abcam, Cambridge, MA, ab154478), DEPC (Sigma, St. Louis, MO, 40718), Positive charged nylon membrane (Ambion, Austin, TX, AM10104), ssRNA Ladder (NEB, Ipswich, MA, N0364S), RNA loading dye (NEB, Ipswich, MA, BO363), 10X MOPS buffer (KD Medical, Columbia, MD, RGF-6170), 10X TBE Electrophoresis buffer (Thermo Scientific, Waltham, MA, B52), Formaldehyde (Sigma, St. Louis, MO, 252549), RNA gel extraction kit (Zymo Research, R1011); antibodies: HIF-1α (Novus Biological Company, Littleton, CO, NB100-449, Millipore Sigma, St. Louis, MO,MAB5382MI), APP (Abcam, Cambridge, MA, ab15272), AβmOC64 (Abcam, Cambridge, MA, ab201060), Aβ 1–42 (Abcam, Cambridge, MA, ab10148), Aβ 1–40 (Abcam, Cambridge, MA, ab76317), BACE1 (Abcam, ab63954), p-Tau (Abcam, Cambridge, MA, ab76128), Tau (Abcam, Cambridge, MA, ab9778), MAP 2 (Abcam, Cambridge, MA, ab5392), GFAP (Sigma-Aldrich, St. Louis, MO, G3893), Lamin B1 (Abcam, Cambridge, MA, ab8982), PHD-2 (Abcam, Cambridge, MA, ab133630), goat anti-rabbit (sc-2004, Santa Cruz Biotechnology, Dallas, TX), goat anti-mouse (sc-2005, Santa Cruz Biotechnology, Dallas, TX), and β-actin (Sigma Aldrich, St. Louis, MO, A5316) were purchased from commercial vendors, as mentioned.

Techniques: Expressing, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: PLoS Biology

Article Title: HIV-1 Tat-mediated astrocytic amyloidosis involves the HIF-1α/lncRNA BACE1-AS axis

doi: 10.1371/journal.pbio.3000660

Figure Lengend Snippet: HPAs exposed to HIV-1 Tat showed increased expression and accumulation of HIF-1α involving differential regulatory mechanisms—transcription, RNA stabilization (posttranscription), translation, and posttranslational stabilization (decreased expression of PHD-2) (1). Upon accumulation, HIF-1α is translocated into the nucleus, where it forms a complex with the lncRNA BACE1-AS (which is also up-regulated following HIV-1 Tat exposure).The HIF-1α–lncRNA BACE1-AS complex, in turn, binds to the BACE1 promoter to induce its transcription (2). Following transcriptional induction of BACE1 mRNA, it subsequently binds to the BACE1-AS, resulting, in turn, in stabilization of BACE1 mRNA (3). This then leads to increased translation and activity of BACE1 (4). Additionally, HIV-1 Tat has also been shown to induce the expression of APP mRNA, resulting in up-regulated expression of APP protein, which then is subject to cleavage by BACE1, leading to the release of various Aβ forms into the extracellular space. Oligomerization of the toxic Aβ forms leads to deposition of insoluble amyloid plaques in select brain regions, which are neurotoxic and can also independently interact with HIV-1 Tat to further aggravate HAND-associated toxicity. APP, amyloid precursor protein; Aβ, amyloid beta; BACE1, β-site cleaving enzyme; BACE1-AS, BACE1‐antisense transcript; HAND, HIV-associated neurocognitive disorder; HIF-1α, hypoxia-inducible factor; HPA, human primary astrocyte; lncRNA, long noncoding RNA; PHD-2, prolyl hydroxylase 2; Tat, transactivator of transcription. Original creation .

Article Snippet: The following antibodies or reagents used in this study were from the indicated sources: reagents: HIV-1 Tat 101 (Immunodiaognistics, Woburn, MA), cobalt chloride (Sigma-Aldrich, St . Louis , MO , 232696), Actinomycin D (Sigma, St . Louis , MO , A9415), SP6/T7 Transcription Kit (Sigma, St. Louis, MO, 10 999 644 001), Biotin-16-UTP (Sigma, St. Louis, MO, 11 388 908 910), HindIII (NEB, Ipswich, MA,R0104S), EcoRI (NEB, Ipswich, MA, R0101S), Tth111I (NEB, Ipswich, MA, R0185S), Platinum Taq DNA Polymerase High Fidelity (ThermoFisher, Waltham, MA,11304011), 10 mM dNTP mix (ThermoFisher, Waltham, MA, 18427–088), TrackIt 1 Kb Plus DNA Ladder (ThermoFisher, Waltham, MA, 10488–085), QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany, 28704), NEB 5-alpha Competent E. coli (High Efficiency) (NEB, Ipswich, MA, C2987I), T4 DNA Ligase (NEB, Ipswich, MA, M0202S), Light Shift Chemiluminescent RNA EMSA (REMSA) Kit (Thermo, Waltham, MA, 20158), HIF1A purified protein Recombinant Human HIF-1 alpha protein (Abcam, Cambridge, MA, ab154478), DEPC (Sigma, St. Louis, MO, 40718), Positive charged nylon membrane (Ambion, Austin, TX, AM10104), ssRNA Ladder (NEB, Ipswich, MA, N0364S), RNA loading dye (NEB, Ipswich, MA, BO363), 10X MOPS buffer (KD Medical, Columbia, MD, RGF-6170), 10X TBE Electrophoresis buffer (Thermo Scientific, Waltham, MA, B52), Formaldehyde (Sigma, St. Louis, MO, 252549), RNA gel extraction kit (Zymo Research, R1011); antibodies: HIF-1α (Novus Biological Company, Littleton, CO, NB100-449, Millipore Sigma, St. Louis, MO,MAB5382MI), APP (Abcam, Cambridge, MA, ab15272), AβmOC64 (Abcam, Cambridge, MA, ab201060), Aβ 1–42 (Abcam, Cambridge, MA, ab10148), Aβ 1–40 (Abcam, Cambridge, MA, ab76317), BACE1 (Abcam, ab63954), p-Tau (Abcam, Cambridge, MA, ab76128), Tau (Abcam, Cambridge, MA, ab9778), MAP 2 (Abcam, Cambridge, MA, ab5392), GFAP (Sigma-Aldrich, St. Louis, MO, G3893), Lamin B1 (Abcam, Cambridge, MA, ab8982), PHD-2 (Abcam, Cambridge, MA, ab133630), goat anti-rabbit (sc-2004, Santa Cruz Biotechnology, Dallas, TX), goat anti-mouse (sc-2005, Santa Cruz Biotechnology, Dallas, TX), and β-actin (Sigma Aldrich, St. Louis, MO, A5316) were purchased from commercial vendors, as mentioned.

Techniques: Expressing, Activity Assay

Journal: Biochemical pharmacology

Article Title: A systematic evaluation of microRNAs in regulating human hepatic CYP2E1

doi: 10.1016/j.bcp.2017.04.020

Figure Lengend Snippet: Hsa-miR-214-3p oligonucleotides interact directly with CYP2E1 mRNA oligonucleotides. RNA EMSA showed that hsa-miR-214-3p interacts with CYP2E1 mRNA oligonucleotides (three target sequences) to form stable complexes (Arrows) (lane 3 in A, B and C). The 50 × cold probe reduces the density associated with the hsa-miR-214-3p/CYP2E1 mRNA complex (lane 5 in A, B and C) but not with hsa-miR-942-5p (lane 5 in D). Hsa-miR-214-3p/CYP2E1 mRNA/protein complexes were observed by adding HepaRG protein extract in B and C (lane 6 and 7, Upper arrows in B and C), but similar complexes were not detected in A and D.

Article Snippet: RNA EMSAs were performed using LightShift Chemiluminescent RNA EMSA Kits (Thermo Scientific) according to the manufacturer’s protocol, except that the 1 × REMSA Binding Buffer was adjusted to contain an additional 5% glycerol, 200 mM KCl, 100 mM MgCl 2 , and 200 nmol synthetic miRNA or/and cognate mRNA oligonucleotides for 20 μL basic reaction mixtures.

Techniques: