Journal: PLoS ONE

Article Title: High Fat Diet Induced Developmental Defects in the Mouse: Oocyte Meiotic Aneuploidy and Fetal Growth Retardation/Brain Defects

doi: 10.1371/journal.pone.0049217

Figure Lengend Snippet: [A] Embryos from mothers on a high fat diet were significantly smaller than those from a control diet [12.1 mm vs 13.2 mm; *p<0.001]. Similarly, placentas from the HFD mothers were significantly smaller than placentas originating from control blastocysts [7.9 mm vs 8.9 mm; *p<0.001]. [B] d14.5 fetuses from HFD [left] and Control [right]; example shown for HFD illustrates the more extreme end of the spectrum of growth retarded fetuses. [C] H & E staining of control fetus and fetal brain vs HFD fetus and HFD fetal brain. All fetuses were from transferred blastocysts. Abnormal development of both the ventricles and the choroid plexus is seen in 40% of the HFD blastocyst-derived fetuses. Scale bars, 500 µm.

Article Snippet: Mice were fed either a high fat diet [HFD] [Diet 58R3; TestDiet, Richmond, IN] containing 36% fat, and 20% protein by nutritional content or an isocaloric control diet [PicoLab Mouse diet 20; LabDiet; Richmond, IN] containing 13% fat and 25% protein for a total of 4 weeks.

Techniques: Control, Staining, Derivative Assay

Journal: bioRxiv

Article Title: Drosophila melanogaster Toll-9 elicits antiviral immunity against Drosophila C virus

doi: 10.1101/2024.06.19.599730

Figure Lengend Snippet: (A) Survival analysis for isogenic control w 1118 flies and Toll-9 0024-G4 flies injected with PBS and DCV. (N=80 flies from three independent experiments) (B) qPCR analysis DCV infected w 1118 flies and Toll-9 0024-G4 flies at 1 dpi and 3 dpi for DCV mRNA. Data are representative of four biological replicates (groups of five flies) each from three independent experiments. Error bars, SEM. Unpaired T test, *p<0.05; ***p<0.001. (C) Western blot analysis to probe DCV capsid protein with anti-DCV antibody from fly lysate of isogenic control w 1118 flies and Toll-9 0024-G4 flies injected with DCV at 1 dpi and 3 dpi. Data are representative of three independent experiments. (D) DCV titer in S2 cells infected with fly lysate from isogenic w 1118 flies and Toll-9 0024-G4 flies injected with DCV calculated as 50% Tissue culture infectious dose (TCID 50 /ml). Data are representative of eight biological replicates (groups of five flies) from four independent experiments. Error bars, SEM. Unpaired T test, *p<0.05; ***p<0.001.

Article Snippet: Six biological replicates of S2 cells or five biological replicates of five flies each per condition were lysed in Trizol Reagent (ThermoFisher 15596), and RNA was isolated by column purification (ZymoResearch R2050) and cDNA was synthesized using the iScript Reverse Transcriptase kit (Bio-Rad, Hercules, CA). qPCR was performed using SYBR green PCR master mix (Bio-Rad).

Techniques: Control, Injection, Infection, Western Blot

Journal: bioRxiv

Article Title: Drosophila melanogaster Toll-9 elicits antiviral immunity against Drosophila C virus

doi: 10.1101/2024.06.19.599730

Figure Lengend Snippet: (A) qPCR for detection of expression of Toll-9 transcripts in mock- and DCV-infected S2 naive and S2 cells stably expressing TOll-9 in presence and absence of CuSO 4 at 1 dpi. (B-C) qPCR analysis of DCV infected S2 naive and S2 cells expressing TOll-9 in presence and absence of CuSO 4 at 1 dpi to detect expression of DCV replicase ORF 1 transcript and Dicer2 mRNA. Data are representative of six biological replicates (well of cells) from three independent experiments. Error bars, SEM. Unpaired T test, **p<0.01; ***p<0.001; ****p<0.0001.

Article Snippet: Six biological replicates of S2 cells or five biological replicates of five flies each per condition were lysed in Trizol Reagent (ThermoFisher 15596), and RNA was isolated by column purification (ZymoResearch R2050) and cDNA was synthesized using the iScript Reverse Transcriptase kit (Bio-Rad, Hercules, CA). qPCR was performed using SYBR green PCR master mix (Bio-Rad).

Techniques: Expressing, Infection, Stable Transfection

Journal: bioRxiv

Article Title: Drosophila melanogaster Toll-9 elicits antiviral immunity against Drosophila C virus

doi: 10.1101/2024.06.19.599730

Figure Lengend Snippet: (A-B) Western blot analysis for indicated antibodies of cell lysate from DCV infected S2 cells and Toll-9 OE cells in presence and absence of CuSO 4 at 1 dpi in presence and absence of Akt inhibitor. Data are representative of three independent experiments. (B-C) Micrograph shows presence of DCV in S2 naive cells and S2 cells expressing Toll-9 in presence and absence of CuSO 4 at 1 dpi in presence and absence of Akt inhibitor. Data are representative of three independent experiments. (E) Viral titer of DCV infected S2 and Toll-9 OE cells culture supernatant in presence and absence of Akt inhibitor calculated as 50% Tissue culture infectious dose (TCID 50 /ml). Data are representative of eight biological replicates (well of cells) from four independent experiments. Error bars, SEM. Unpaired T test, **p<0.01.

Article Snippet: Six biological replicates of S2 cells or five biological replicates of five flies each per condition were lysed in Trizol Reagent (ThermoFisher 15596), and RNA was isolated by column purification (ZymoResearch R2050) and cDNA was synthesized using the iScript Reverse Transcriptase kit (Bio-Rad, Hercules, CA). qPCR was performed using SYBR green PCR master mix (Bio-Rad).

Techniques: Western Blot, Infection, Expressing

Journal: bioRxiv

Article Title: Drosophila melanogaster Toll-9 elicits antiviral immunity against Drosophila C virus

doi: 10.1101/2024.06.19.599730

Figure Lengend Snippet: (A) In silico prediction of signal peptide in Toll-9 protein sequence. Red solid line indicates predicted n-terminal region, orange solid line indicates the predicted center hydrophobic region, and yellow solid line indicates predicted c-terminal region of signal peptide. Black dotted line indicates the cleavage site (CS) of the signal peptide. Sec/SPI: Sec translocon transported secretory signal peptide/Signal Peptidase I Tat/SPI: Tat translocon transported Tat signal peptides/Signal Peptidase I (B) Western blot analysis demonstrating the presence of Toll-9/V5 in endosomes. Endosomal fractions were identified using Rab5 as a microsomal marker, while Actin served as a cytosolic marker. Data are representative of three independent experiments. (C) Micrographs show colocalization of Rab5-early endosome marker (green) and Toll-9 (anti-V5 tag ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (D) Micrographs show colocalization of Rab7-Late endosome marker (green) and Toll-9 (anti-V5 tag ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (E) Micrographs show colocalization of Rab5-early endosome marker (green) and Poly(I:C) (J2 anti-dsRNA ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (F) Toll-9 (anti-V5 tag ab-Green) and Poly(I:C) (J2 anti-dsRNA ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (G) Western blot analysis using the indicated antibodies following immunoprecipitation of V5 tag (Toll-9) using J2 dsRNA antibody from the lysate of Poly (I:C) treated Toll-9 OE and S2 cells in presence and absence of CuSO 4 (500 µM). Data are representative from three independent experiments.

Article Snippet: Six biological replicates of S2 cells or five biological replicates of five flies each per condition were lysed in Trizol Reagent (ThermoFisher 15596), and RNA was isolated by column purification (ZymoResearch R2050) and cDNA was synthesized using the iScript Reverse Transcriptase kit (Bio-Rad, Hercules, CA). qPCR was performed using SYBR green PCR master mix (Bio-Rad).

Techniques: In Silico, Sequencing, Western Blot, Marker, Immunoprecipitation

Journal: bioRxiv

Article Title: Drosophila melanogaster Toll-9 elicits antiviral immunity against Drosophila C virus

doi: 10.1101/2024.06.19.599730

Figure Lengend Snippet: (A-B) qPCR analysis DCV infected S2 naive and S2 cells expressing TOll-9 in presence and absence of CuSO 4 at 1 dpi to detect proapoptotic genes Reaper and Hid . (C) Percentage of Annexin V positive cells from DCV infected S2 naive and S2 cells expressing TOll-9 in presence and absence of CuSO 4 at 1 dpi. Data are representative of six biological replicates (well of cells) from three independent experiments. Error bars, SEM. Unpaired T test, **p<0.01; ***p<0.001.

Article Snippet: Six biological replicates of S2 cells or five biological replicates of five flies each per condition were lysed in Trizol Reagent (ThermoFisher 15596), and RNA was isolated by column purification (ZymoResearch R2050) and cDNA was synthesized using the iScript Reverse Transcriptase kit (Bio-Rad, Hercules, CA). qPCR was performed using SYBR green PCR master mix (Bio-Rad).

Techniques: Infection, Expressing

Journal: The Journal of biological chemistry

Article Title: Mitochondrial protein import. Tom40 plays a major role in targeting and translocation of preproteins by forming a specific binding site for the presequence.

doi: 10.1074/jbc.272.30.18725

Figure Lengend Snippet: FIG. 1. A preprotein bound to OMV under cis and trans condi- tions can be cross-linked to various TOM components. A, a pre- protein can be co-immunoprecipitated with antibodies against Tom40, only when bound to the trans site. Radioactively labeled pSu9-DHFR was bound to OMV for 20 min at 0 or 25 °C in the presence or absence of MTX/NADPH, respectively. The reaction mixtures were chilled on ice and diluted with LSW or HSW buffers (containing 20 or 120 mM KCl, respectively). OMV together with cis or trans site-bound material were reisolated (20 min, 125,000 3 g) and resuspended in SEM buffer with or without MTX/NADPH. Co-immunoprecipitation with antibodies against Tom40 or with antibodies derived from preimmune serum was performed. As a control for the input, an aliquot (Total) representing 25% of the material used for co-immunoprecipitation is shown. B, pSu9- DHFR was bound to OMV under cis and trans conditions and the OMV were washed as described in A. To the indicated samples, 13 mM of F1b

Article Snippet: For cross-linking experiments, OMV or mitochondrial pellets were resuspended in SEM buffer (220 mM sucrose, 1 mM EDTA, and 10 mM MOPS, pH 7.2 containing CCCP in the case of mitochondria), and either 117 mM disuccinimidyl glutarate (DSG) or 1 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (both from Pierce) was added.

Techniques: Immunoprecipitation, Labeling, Derivative Assay, Control

Journal: The Journal of biological chemistry

Article Title: Mitochondrial protein import. Tom40 plays a major role in targeting and translocation of preproteins by forming a specific binding site for the presequence.

doi: 10.1074/jbc.272.30.18725

Figure Lengend Snippet: FIG. 2. A presequence polypeptide bound to OMV under cis and trans conditions can be cross-linked to various TOM com- ponents. A, OMV were incubated with radioactively labeled pSu9(17) for 15 min at 0 °C or 25 °C and diluted with LSW or HSW buffers, respectively. After reisolation (20 min, 125,000 3 g), the OMV were resuspended in SEM buffer, and the cross-linking reagents DSG (117 mM) and EDC (1 mM) were added as indicated. After incubation for 40 min at 0 °C, the cross-linkers were quenched. Further analysis was as in Fig. 1B. B, identification of the cross-linking products by immuno- precipitation. OMV were incubated with pSu9(17) for 15 min at 0 °C, diluted with LSW buffer, and reisolated (20 min, 125,000 3 g). The pellets were resuspended in SEM buffer, and DSG (117 mM) was added. Aliquots were removed before (2DSG) and after the cross-linking reac- tion (40 min at 0 °C; 1DSG). Further analysis and immunoprecipita- tion with antibodies directed against Tom20, Tom22, Tom40, and Tom70 or antibodies derived from preimmune serum were as in Fig. 1C. Apparent molecular masses are given on the left.

Article Snippet: For cross-linking experiments, OMV or mitochondrial pellets were resuspended in SEM buffer (220 mM sucrose, 1 mM EDTA, and 10 mM MOPS, pH 7.2 containing CCCP in the case of mitochondria), and either 117 mM disuccinimidyl glutarate (DSG) or 1 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (both from Pierce) was added.

Techniques: Incubation, Labeling, Immunoprecipitation, Derivative Assay

Journal: The Journal of biological chemistry

Article Title: Mitochondrial protein import. Tom40 plays a major role in targeting and translocation of preproteins by forming a specific binding site for the presequence.

doi: 10.1074/jbc.272.30.18725

Figure Lengend Snippet: FIG. 3. Cross-linking of preproteins to Tom40 occurs inde- pendently of surface receptors. A, pSu9-DHFR or B, pSu9(17) were incubated with OMV that were pretreated with trypsin (30 mg/ml) or were mock-treated. The binding incubation was for 20 min at 25 °C (pSu9-DHFR) or 0 °C (pSu9(17)). The reaction mixture was chilled on ice and diluted with HSW (pSu9-DHFR) or LSW (pSu9(17)) buffers before reisolation of the OMV. The pellets were resuspended in SEM buffer, and the cross-linker EDC was added as indicated for 40 min at 0 °C. Further analysis was as in Fig. 1. Apparent molecular masses are given on the left. FIG. 4. A purified presequence peptide can be cross-linked to Tom22 and Tom40. The F1b presequence peptide or the control pep- tide CH4 (35 mM) were incubated with OMV that were pretreated with or without trypsin for 20 min at 0 °C as indicated. As a control, an aliquot was removed before the addition of the peptides. The reaction mixture was diluted with LSW and HSW buffers before reisolation of the OMV. The pellets were resuspended in SEM buffer, and 1 mM EDC was added as indicated. After quenching of the cross-linker the proteins were precipitated with trichloroacetic acid, solubilized with sample buffer, and separated by SDS-PAGE. After blotting onto nitrocellulose membranes, immunostaining analysis was performed for A, Tom22 or B, Tom40. In both panels the cross-linking product with a small TOM component is marked with a and the one with the presequence peptide with b.

Article Snippet: For cross-linking experiments, OMV or mitochondrial pellets were resuspended in SEM buffer (220 mM sucrose, 1 mM EDTA, and 10 mM MOPS, pH 7.2 containing CCCP in the case of mitochondria), and either 117 mM disuccinimidyl glutarate (DSG) or 1 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (both from Pierce) was added.

Techniques: Incubation, Binding Assay, Purification, Control, SDS Page, Immunostaining

Journal: The Journal of biological chemistry

Article Title: Mitochondrial protein import. Tom40 plays a major role in targeting and translocation of preproteins by forming a specific binding site for the presequence.

doi: 10.1074/jbc.272.30.18725

Figure Lengend Snippet: FIG. 5. Preproteins accumulated at the outer membrane of intact mitochondria can be cross-linked to the TOM components in a similar fashion as in OMV. A, pSu9-DHFR or B, pSu9(17) were incubated with freshly isolated mitochondria that had been treated with CCCP (38 mM) and apyrase (10 U/ml) to deplete the membrane potential and matrix ATP, respectively. After incubation for 20 min at 0 or 25 °C, the reaction mixture was chilled on ice and diluted with LSW and HSW buffers as indicated. Mitochondria were reisolated (10 min, 12,000 3 g), the pellets were resuspended in SEM buffer containing 38 mM CCCP. DSG or EDC were added as indicated for 40 min at 0 °C. Further analysis was as in Fig. 1B. Apparent molecular masses are given on the left. The position of the cross-linking products of the preproteins with Tom20, Tom22, and Tom40 are marked on the right.

Article Snippet: For cross-linking experiments, OMV or mitochondrial pellets were resuspended in SEM buffer (220 mM sucrose, 1 mM EDTA, and 10 mM MOPS, pH 7.2 containing CCCP in the case of mitochondria), and either 117 mM disuccinimidyl glutarate (DSG) or 1 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (both from Pierce) was added.

Techniques: Membrane, Incubation, Isolation