Journal: Frontiers in Physiology

Article Title: β-Cyclodextrins Decrease Cholesterol Release and ABC-Associated Transporter Expression in Smooth Muscle Cells and Aortic Endothelial Cells

doi: 10.3389/fphys.2016.00185

Figure Lengend Snippet: Cholesterol efflux from ABAE and SMCs . Cells were first labeled with [ 3 H]-cholesterol (0.5 μCi/mL) for 36 h at 37°C and then equilibrated for 24 h in DMEM/0.05% BSA at 37°C. Cellular [ 3 H]-cholesterol loads in DPM per μg of proteins were compared between ABAE and SMCs (A) . Data represent the mean ± SEM (in DPM/μg of proteins), with n = 6 from two sets of experiments. Paired- t -test was performed, *** p < 0.001 when compared with ABAE condition. [ 3 H]-cholesterol released from both cell types was then measured 6 h after medium supplementation with either BSA (control condition) or cholesterol acceptors [ApoA-I (20 μg/mL) or HDL (50 μg/mL)] at 37°C (B) . Data represent the mean ± SEM (in DPM/μg of proteins), with n = 6 from two sets of experiments. One-way ANOVA test followed by Bonferroni correction was performed in which *** p < 0.001 refers to the control condition and ### p < 0.001 refers to the matched HDL condition in ABAE.

Article Snippet: ApoA-I and HDL were purchased from PROSCI Incorporated (Poway, CA, USA).

Techniques: Labeling, Control

Journal: Frontiers in Physiology

Article Title: β-Cyclodextrins Decrease Cholesterol Release and ABC-Associated Transporter Expression in Smooth Muscle Cells and Aortic Endothelial Cells

doi: 10.3389/fphys.2016.00185

Figure Lengend Snippet: Cholesterol release from ABAE (A,C) and from SMCs (B,D) upon CD-treatment . Cells were first labeled with [ 3 H]-cholesterol (0.5 μCi/mL) for 36 h at 37°C and then equilibrated in DMEM/0.05% BSA for 24 h at 37°C. [ 3 H]-cholesterol released in the medium from both cell types was then measured 6 h after medium supplementation with cholesterol acceptors [ApoA-I (20 μg/mL) or HDL (50 μg/mL)] at 37°C. The radioactivity contained in the culture medium, that corresponds to the cholesterol released from cells was measured. Data are expressed as the mean ± SEM (in DPM/μg of proteins, n = 6 from two sets of experiments). Statistical analysis: a one-way ANOVA followed by Dunnett's test for multiple comparisons in which * p < 0.05; *** p < 0.001 compared with the control condition (open bars) without any CD treatment.

Article Snippet: ApoA-I and HDL were purchased from PROSCI Incorporated (Poway, CA, USA).

Techniques: Labeling, Radioactivity, Control

Journal: Frontiers in Physiology

Article Title: β-Cyclodextrins Decrease Cholesterol Release and ABC-Associated Transporter Expression in Smooth Muscle Cells and Aortic Endothelial Cells

doi: 10.3389/fphys.2016.00185

Figure Lengend Snippet: Effect of 10 μM T0901317 on cholesterol efflux to ApoA-I and HDL in ABAE (A) and SMCs (B) treated or nor with Rameβ . Cells were first labeled with [ 3 H]-cholesterol as previously described and incubated in the presence of 1 mM Rameβ, 10 μM T0901317 or both during 24 h. Then, [ 3 H]-cholesterol released in the medium from both cell types was measured 6 h after medium supplementation with either ApoA-I (20 μg/mL) or HDL (50 μg/mL). Data are expressed as the mean ± SEM (in DPM/μg of proteins, n = 6 from two sets of experiments). Control condition (without any treatment) is used as the reference level and set to 100%. Statistical analysis: a one-way ANOVA followed by Dunnett's test for multiple comparisons in which * p < 0.05; ** p < 0.01; *** p < 0.001 compared with the untreated condition (Control condition, open bars) and in which ## p < 0.01 when Rameβ + T0901317 condition was compared with Rameβ condition.

Article Snippet: ApoA-I and HDL were purchased from PROSCI Incorporated (Poway, CA, USA).

Techniques: Labeling, Incubation, Control

Journal: Scientific Reports

Article Title: Comparison of the sensitivity of Western blotting between PVDF and NC membranes

doi: 10.1038/s41598-021-91521-8

Figure Lengend Snippet: Antibodies, lectins and proteins, as well as their molecular weights, transfer time and incubate time.

Article Snippet: Rabbit anti-ceruloplasmin (CerP) antibody, rabbit anti-transferrin (TF) antibody and rabbit anti-Apolipoprotein A1 (ApoA1) antibody were purchased from Boster Corporation (Wuhan, China).

Techniques: Molecular Weight, High Molecular Weight

Journal: Scientific Reports

Article Title: Comparison of the sensitivity of Western blotting between PVDF and NC membranes

doi: 10.1038/s41598-021-91521-8

Figure Lengend Snippet: Comparison of the re-probed ability of PVDF membrane and NC membrane. The pooled sera proteins (3.0, 1.5, 0.7, 0.3 and 0.1 μg) were separated by 8% SDS-PAGE, and transferred to PVDF membranes (up) and NC membrane (down), respectively. ( a ) Staining with AAL and then re-probed with PHA-E; ( b ) staining with ApoA1 and then re-probing with IgG; ( c ) Staining with PHA-E and then re-probing with A2M; ( d ) staining with A2M and then re-probing with PHA-E. Band intensities were statistically analyzed (n = 3 individual experiments) and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. Pink bar, PVDF membrane; Blue bar, NC membrane. Band intensities were analyzed *Significantly different p < 0.05, ** p < 0.01. N.S., not significant. All values are means ± S.E. (error bars).

Article Snippet: Rabbit anti-ceruloplasmin (CerP) antibody, rabbit anti-transferrin (TF) antibody and rabbit anti-Apolipoprotein A1 (ApoA1) antibody were purchased from Boster Corporation (Wuhan, China).

Techniques: Comparison, Membrane, SDS Page, Staining, Software

Journal: Cells

Article Title: Geniposide Ameliorated Dexamethasone-Induced Cholesterol Accumulation in Osteoblasts by Mediating the GLP-1R/ABCA1 Axis

doi: 10.3390/cells10123424

Figure Lengend Snippet: GEN ameliorated DEX-induced cholesterol accumulation by increasing ABCA1 expression. ( A , B ) The in vivo immunohistochemical examination of ABCA1 expression was conducted. ( C – E ) The protein expression of ABCA1 and ApoA-I was detected by western blot in DEX-treated MC3T3-E1 cells. ( F ) The level of the total intracellular cholesterol was measured using the ELISA kit in MC3T3-E1 cells co-treated with DEX and DIDS (200 μM). ( G – I ) The protein expression of ABCA1 and ApoA-I was detected by western blot in DEX/DIDS-treated MC3T3-E1 cells. ( J ) The ALP staining and the Alizarin Red S staining assays were conducted (×100 magnification). ( K – M ) The protein expression of RUNX2 and OPN was detected by western blot in DEX/DIDS-treated MC3T3-E1 cells. All experiments were implemented separately in triplicate. * p < 0.05; ** p < 0.01. OIM, osteogenic induction medium. NC, negative control; 50 mg/kg, Dex + 50 mg/kg GEN; 100 mg/kg, Dex + 100 mg/kg GEN.

Article Snippet: After being blocked in TBS containing 5% skimmed milk for 1 h, the membranes were co-incubated with the primary antibodies at 4 °C overnight against RUNX2 (1:1000 dilution; MyBioSource, cat.no.127554, San Diego, CA, USA), OPN (1:1000 dilution; Proteintech, cat.no.22952-1-AP, Rosemont, IL, USA), GLP-1R (1:1000 dilution; ABGENT, cat.no.AP52040), ABCA1 (1:1000 dilution; Affinity, cat.no.DF8233), apoA-I (1:1000 dilution; Affinity, cat.no.DF6264), and GAPDH (1:1000 dilution; Affinity, cat.no.AF7021), and then with HRP-labeled goat anti-rabbit secondary antibody (1:5000 dilution; Boster Biological Technology, Wuhan, China).

Techniques: Expressing, In Vivo, Immunohistochemical staining, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Negative Control

Journal: Cells

Article Title: Geniposide Ameliorated Dexamethasone-Induced Cholesterol Accumulation in Osteoblasts by Mediating the GLP-1R/ABCA1 Axis

doi: 10.3390/cells10123424

Figure Lengend Snippet: GEN promoted ABCA1-mediated cholesterol metabolism in a GLP-1R-dependent manner. ( A , B ) The in vivo immunohistochemical examination of GLP-1R expression was conducted. ( C , D ) The protein expression of GLP-1R was determined by western blot in DEX-treated MC3T3-E1 cells. ( E ) The ALP staining and the Alizarin Red S staining assay were performed in EX-treated MC3T3-E1 cells (×100 magnification). ( F ) The level of the total intracellular cholesterol was measured using the ELISA kit. ( G – K ) The protein expression of GLP-1R, ABCA1, RUNX2, and OPN was detected by western blot. All experiments were implemented separately in triplicate. * p < 0.05; ** p < 0.01. NC, negative control; 50 mg/kg, Dex + 50 mg/kg GEN; 100 mg/kg, Dex + 100 mg/kg GEN; EX, Exendin9-39.

Article Snippet: After being blocked in TBS containing 5% skimmed milk for 1 h, the membranes were co-incubated with the primary antibodies at 4 °C overnight against RUNX2 (1:1000 dilution; MyBioSource, cat.no.127554, San Diego, CA, USA), OPN (1:1000 dilution; Proteintech, cat.no.22952-1-AP, Rosemont, IL, USA), GLP-1R (1:1000 dilution; ABGENT, cat.no.AP52040), ABCA1 (1:1000 dilution; Affinity, cat.no.DF8233), apoA-I (1:1000 dilution; Affinity, cat.no.DF6264), and GAPDH (1:1000 dilution; Affinity, cat.no.AF7021), and then with HRP-labeled goat anti-rabbit secondary antibody (1:5000 dilution; Boster Biological Technology, Wuhan, China).

Techniques: In Vivo, Immunohistochemical staining, Expressing, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: bioRxiv

Article Title: Engineered high-density lipoprotein particles that chaperone bioactive lipid mediators to combat endothelial dysfunction and thromboinflammation

doi: 10.1101/2022.02.14.480375

Figure Lengend Snippet: (A) Schematic model of ApoA1-ApoM (A1M). (B) Purified A1M (4 μg) was separated by reducing 10% SDS-PAGE and stained with Coomassie brilliant blue. (C) Purified A1M (100 μg) was incubated or not with S1P for 24 hours, purified by gel filtration chromatography and analyzed for S1P content by electrospray ionization-MS/MS. The resulting data are the mean + S.D.; N=5. (D, E, F) Representative FPLC elution profiles (OD 280) of 200μl of mouse plasma, purified recombinant A1M, and A1M after lipidation and S1P loading. (D) FPLC elution of plasma standards: LDL (22-26ml) HDL (28-32ml), Soluble protein fraction (32-38ml). (E) nascent A1M protein (100μg) (F) lipidated A1M/S1P (1 mg). G) Representative negative-stain EM image of A1M with some particles highlighted in white circle (left) and 2D averages with potential ApoM densities indicated by arrows (right). Box dimension of each 2D average is 215 Å.

Article Snippet: Plasmids for murine ApoA1 (Cat# MR203500) and murine ApoM (Cat# MR201811) were obtained from OriGene.

Techniques: Purification, SDS Page, Staining, Incubation, Filtration, Chromatography, Tandem Mass Spectroscopy, Recombinant

Journal: bioRxiv

Article Title: Engineered high-density lipoprotein particles that chaperone bioactive lipid mediators to combat endothelial dysfunction and thromboinflammation

doi: 10.1101/2022.02.14.480375

Figure Lengend Snippet: (A) A1M/S1P-dependent enhancement of barrier function in HUVEC (24 μg/ml A1M contains ~ 300 nM S1P). (B) Comparison of A1M/S1P and ApoM-Fc/S1P by TEER using ApoA1-ApoM/S1P (16μg/ml of A1M or ApoM-Fc ~ 200nM S1P). Unloaded chaperones were used as controls. Data are presented as area under the curve (N = 3; mean ± SD. P < 0.0001, two-way ANOVA followed by paired student t-test t test). (C) HUVECs were analyzed for barrier protection by TEER analysis using ApoM-Fc/S1P (30 nM) and Ang-1 (300 ng/ml) individually or in combination. (D) HUVECs were analyzed for barrier protection by TEER analysis in response to thrombin (1U/ml) treatment using ApoM-Fc/S1P (200nM), Ang-1 (300ng/ml) or in combination. For C and D, data were analyzed by non-parametric t-test (Mann-Whitney) (****P<0.0001). (E-H) HUVECs were analyzed for barrier protection by TEER analysis using either A1M/S1P (30nM) (E) or ApoM-Fc/S1P (30nM) (G) in conjunction with APC (5μg/ml). After 1 hour pretreatment, thrombin was added for an additional 2 hours. Area Under the Curve was analyzed (n=3) followed by non-parametric t-test (Mann-Whitney) of control or individual treatments vs combined treatments (F,G). ****P<0.0001 for (E,G) and P<0.01 for (F,G).

Article Snippet: Plasmids for murine ApoA1 (Cat# MR203500) and murine ApoM (Cat# MR201811) were obtained from OriGene.

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: Engineered high-density lipoprotein particles that chaperone bioactive lipid mediators to combat endothelial dysfunction and thromboinflammation

doi: 10.1101/2022.02.14.480375

Figure Lengend Snippet: (A) HMEC NF-κB-luciferase reporter cells were assayed for TNFα-induced NF-κB luciferase reporter activity in the presence of ApoA1, A1M and A1M/S1P. (N=3; Mean + S.D.; **P< 0.01 *P<0.05 Student t-test). (B) Effect of ApoM-Fc and Albumin-S1P on TNFα-induced NF-κB luciferase reporter activity (N= 3 Mean + S.D.). (C) HUVECs were assayed for TNFα induction of ICAM-1 expression by immunoblot analysis. Cultures were pre-treated for 10 minutes with ApomFc/S1P (100nM), Iloprost (200nM), or both in combination, as well as A1M, A1M/S1P, A1M/Iloprosst (all 200μg/ml) and induced with TNFα (10ng/ml) for 5 hours. Image J was used to obtain semi-quantitative values from scans of immunoblots (N=3).

Article Snippet: Plasmids for murine ApoA1 (Cat# MR203500) and murine ApoM (Cat# MR201811) were obtained from OriGene.

Techniques: Luciferase, Activity Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Engineered high-density lipoprotein particles that chaperone bioactive lipid mediators to combat endothelial dysfunction and thromboinflammation

doi: 10.1101/2022.02.14.480375

Figure Lengend Snippet: (A) Isolated peritoneal Mouse Neutrophils were assayed for oxidative burst after fMLF stimulation. Cells were pre-incubated for 10 minutes with vehicle control, ApoM-Fc/S1P (S1P 100nM), ApoA1/Iloprost (160nM), or in combination. Data are presented as normalized luminol fluorescence (B) Composite data for N=4 independent experiments. Area under the Curve (AUC) data were analyzed by non-parametric t-test (Mann-Whitney) and P values <0.0001. (C) Mice were treated with with thioglycolate and either PBS, A1M (200μg) or A1M/S1P (200μg). Peritoneal cells (4 h) were counted and analyzed by flow cytometry. Data were analyzed by one-way ANOVA followed by student t-test P=0.0317. (D) Peritoneal lavage supernatant from (C) was assayed by cytokine array analysis. Resulting blots were analyzed on IMAGEJ and differentially expressed analytes were quantified and data are expressed after normalization (Control = 100%). Data was analyzed by 2-way ANOVA followed by multiple paired student t-test (C5/C5a P= 0.028, G-CSF P= 0.004, sICAM-1 P= 0.025, IL-6 P= 0.001, IL-16 P= 0.0007, M-CSF P= 0.049, CCL2 P=0.009).

Article Snippet: Plasmids for murine ApoA1 (Cat# MR203500) and murine ApoM (Cat# MR201811) were obtained from OriGene.

Techniques: Isolation, Incubation, Fluorescence, MANN-WHITNEY, Flow Cytometry

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Targeting the Apoa1 locus for liver-directed gene therapy

doi: 10.1016/j.omtm.2021.04.011

Figure Lengend Snippet: Strategy for therapeutic targeting of the Apoa1 locus for liver gene therapy AAV-CRISPR encodes a gRNA for targeting the Apoa1 3′ UTR, and SaCas9 driven by a liver-specific promoter (HLP). The gRNA target site (red) and protospacer adjacent motif (PAM) sequence (blue) in the Apoa1 3′ UTR are shown. AAV-Donor contains the final coding exon of Apoa1 (exon 4) fused to a 2A ribosomal skipping sequence upstream of a therapeutic gene and synthetic polyadenylation signal (pA), flanked by homology arms (HR) to the Apoa1 locus. Following correct integration by HDR, the Apoa1 promoter drives the expression of a bicistronic mRNA consisting of Apoa1 and therapeutic transgene. Translation results in expression of apoA1 with a C-terminal 2A epitope tag, as well as the therapeutic protein including an N-terminal proline. Created with BioRender.

Article Snippet: Primary antibodies to the FLAG tag (1:5,000, rabbit, 600-401-383, Rockland), 2A peptide (1:5,000, rabbit, ABS31, Sigma-Aldrich), APOE (1:1,000, rabbit, ab52607, Abcam), FAH (1:1,000, rabbit, SAB2100745, Sigma-Aldrich), apoB (1:5,000, rabbit, K23300R, Meridian), apoA1 (1:5,000, rabbit, K23500R, Meridian), alpha-1 antitrypsin (Aat; 1:1,000, rabbit, 16382-1-AP, Proteintech), and beta tubulin (1:500, mouse, University of Iowa Developmental Studies Hybridoma Bank E7) were diluted in 1% BSA in PBS-T and membranes were incubated overnight at 4 degrees.

Techniques: CRISPR, Sequencing, Expressing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Targeting the Apoa1 locus for liver-directed gene therapy

doi: 10.1016/j.omtm.2021.04.011

Figure Lengend Snippet: Targeted integration and expression from the Apoa1 locus in vivo (A) Adult C57BL/6J mice were intraperitoneally injected with AAV-CRISPR (5 × 10 11 GC) and/or an AAV-Donor template (5 × 10 11 GC) encoding a far-red fluorescent protein (mKate2). Control mice were injected with AAV-GFP (5 × 10 11 GC). Livers and plasma were harvested for analysis at 12 weeks post-injection. (B) PCR from liver showing integration of AAV-Donor into the Apoa1 locus. Two main products were observed: correct HDR (1,139 bp) and NHEJ insertion (2,031 bp). The forward primer binds to the Apoa1 locus upstream of the 5′ HR and reverse primer binds to the mKate2 coding sequence. Minus (–) indicates water-only control. (C) Frequency of indel formation in the Apoa1 3′ UTR by deep sequencing. (D) Frequency of Apoa1 alleles with NHEJ insertions of AAV genomes by ddPCR. (E) Frequency of correct HDR targeting of AAV-Donor by ddPCR. (F) Representative immunohistochemistry for mKate2-FLAG (brown cells) in Apoa1 -targeted mice. Scale bar is 100 μm. (G) Quantification of FLAG positive hepatocytes relative to total nuclei per field. (H) Western blot of mKate2-FLAG in liver lysates with β-tubulin (β-tub) as a loading control. (I and J) Western blot analysis of 2A-tagged (I) and total apoA1 (J) in plasma with alpha-1 antitrypsin (aat) as loading control. Four representative mice per group are shown in western blots. (K) Densitometry analysis of mKate2-FLAG in liver lysates relative to β-tub loading control. Densitometry analysis of apoA1-2A (L) and apoA1 (M) in plasma relative to aat loading control. Data are shown as mean ± standard deviation (n = 5; n = 4 in densitometry analyses), with significance determined by one-way ANOVA followed by Tukey test. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (A) Created with BioRender.

Article Snippet: Primary antibodies to the FLAG tag (1:5,000, rabbit, 600-401-383, Rockland), 2A peptide (1:5,000, rabbit, ABS31, Sigma-Aldrich), APOE (1:1,000, rabbit, ab52607, Abcam), FAH (1:1,000, rabbit, SAB2100745, Sigma-Aldrich), apoB (1:5,000, rabbit, K23300R, Meridian), apoA1 (1:5,000, rabbit, K23500R, Meridian), alpha-1 antitrypsin (Aat; 1:1,000, rabbit, 16382-1-AP, Proteintech), and beta tubulin (1:500, mouse, University of Iowa Developmental Studies Hybridoma Bank E7) were diluted in 1% BSA in PBS-T and membranes were incubated overnight at 4 degrees.

Techniques: Expressing, In Vivo, Injection, CRISPR, Control, Clinical Proteomics, Sequencing, Immunohistochemistry, Western Blot, Standard Deviation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Targeting the Apoa1 locus for liver-directed gene therapy

doi: 10.1016/j.omtm.2021.04.011

Figure Lengend Snippet: Improved targeting of the Apoa1 locus in neonatal mice injected at P4 (A) Experimental design and AAV-Donor scheme. C57BL/6J mice were subcutaneously injected with AAV-CRISPR (5 × 10 11 GC) and/or an AAV-Donor template (5 × 10 11 GC) encoding a far-red fluorescent protein (mKate2) at P4 and liver and plasma were evaluated for the expression of the mKate2 protein at 20 weeks post-injection. Control mice were injected with AAV-GFP (5 × 10 11 GC). (B) Integration PCR on liver showed two main products corresponding to correct HDR (1,139 bp) and NHEJ insertion (2,031 bp) of AAV-Donor at the Apoa1 locus. Minus (–) indicates water-only control. (C) Frequency of Apoa1 alleles with NHEJ insertions of AAV genomes by ddPCR. (D) Frequency of correct HDR targeting of AAV-Donor by ddPCR. (E) Indel formation in the 3′ UTR of Apoa1 by ICE analysis. (F) Representative immunohistochemistry for mKate2-FLAG (brown cells) in Apoa1 -targeted mice. Scale bar is 100 μm. (G) Quantification of FLAG-positive hepatocytes relative to total nuclei per field. (H) Western blot of mKate2-FLAG in liver lysates with β-tub as a loading control. (I and J) Western blot analysis of 2A-tagged (I) and total apoA1 (J) in plasma with aat as loading control. Aat shows a slight difference in protein expression based on sex: lanes 1, 4, 7, 8, 9, 10, 13, 14, and 15 are male mice; lanes 2, 3, 5, 6, 11, 12, and 16 are female mice. Four representative mice per group are shown in western blots. (K) Densitometry analysis of mKate2-FLAG in liver lysates relative to β-tub loading control. Densitometry analysis of apoA1-2A (L) and apoA1 (M) in plasma relative to aat loading control. Data are shown as mean ± standard deviation (n = 5; n = 4 in densitometry analyses), with significance determined by one-way ANOVA followed by Tukey test. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (A) Created with BioRender.

Article Snippet: Primary antibodies to the FLAG tag (1:5,000, rabbit, 600-401-383, Rockland), 2A peptide (1:5,000, rabbit, ABS31, Sigma-Aldrich), APOE (1:1,000, rabbit, ab52607, Abcam), FAH (1:1,000, rabbit, SAB2100745, Sigma-Aldrich), apoB (1:5,000, rabbit, K23300R, Meridian), apoA1 (1:5,000, rabbit, K23500R, Meridian), alpha-1 antitrypsin (Aat; 1:1,000, rabbit, 16382-1-AP, Proteintech), and beta tubulin (1:500, mouse, University of Iowa Developmental Studies Hybridoma Bank E7) were diluted in 1% BSA in PBS-T and membranes were incubated overnight at 4 degrees.

Techniques: Injection, CRISPR, Clinical Proteomics, Expressing, Control, Immunohistochemistry, Western Blot, Standard Deviation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Targeting the Apoa1 locus for liver-directed gene therapy

doi: 10.1016/j.omtm.2021.04.011

Figure Lengend Snippet: Sustained expression of factor IX over time in Apoa1 -targeted mice (A) AAV-Donor scheme. (B) Adult C57BL/6J mice were intraperitoneally injected with AAV-CRISPR (5 × 10 11 GC) and/or an AAV-Donor (5 × 10 11 GC) encoding human FIX . Control mice were injected with AAV-GFP (5 × 10 11 GC). Plasma was collected every 2 to 4 weeks up to 24 weeks post-injection for analysis of human FIX protein levels. (C and D) Western blot analysis of 2A-tagged (C) and total apoA1 (D) in plasma isolated at 24 weeks post-injection, with aat as loading control. Five representative mice per group are shown in western blots. (E and F) Densitometry analysis of apoA1-2A (E) and apoA1 (F) in plasma relative to aat loading control. (G) Quantitative measurement of plasma FIX over time by ELISA (green line, control; blue line, Donor; red line, CRISPR + Donor mice). Data are shown as mean ± standard deviation (n = 5 in densitometry analyses; n = 6 in FIX ELISA). A one-way and two-way ANOVA followed by Tukey test were used respectively for densitometry analyses (E and F) and FIX ELISA (G). ∗p < 0.05 CRISPR + Donor versus Donor; ∗∗p < 0.01 CRISPR + Donor versus Donor; ∗∗∗p < 0.001 CRISPR + Donor versus Donor, # p < 0.0001 Control versus Donor and CRISPR + Donor, ∗∗∗∗p < 0.0001. (A and B) Created with BioRender.

Article Snippet: Primary antibodies to the FLAG tag (1:5,000, rabbit, 600-401-383, Rockland), 2A peptide (1:5,000, rabbit, ABS31, Sigma-Aldrich), APOE (1:1,000, rabbit, ab52607, Abcam), FAH (1:1,000, rabbit, SAB2100745, Sigma-Aldrich), apoB (1:5,000, rabbit, K23300R, Meridian), apoA1 (1:5,000, rabbit, K23500R, Meridian), alpha-1 antitrypsin (Aat; 1:1,000, rabbit, 16382-1-AP, Proteintech), and beta tubulin (1:500, mouse, University of Iowa Developmental Studies Hybridoma Bank E7) were diluted in 1% BSA in PBS-T and membranes were incubated overnight at 4 degrees.

Techniques: Expressing, Injection, CRISPR, Control, Clinical Proteomics, Western Blot, Isolation, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Targeting the Apoa1 locus for liver-directed gene therapy

doi: 10.1016/j.omtm.2021.04.011

Figure Lengend Snippet: Reduction of plasma lipids in a mouse model of hypercholesterolemia through Apoa1 -targeting (A) AAV-Donor scheme. (B) P4 Apoe −/− male pups were subcutaneously injected with AAV-CRISPR (5 × 10 11 GC) and an AAV-Donor (5 × 10 11 GC) encoding human APOE or saline (control). Mice were fed a western diet starting at weaning for 20 weeks. Plasma was collected every 2 to 4 weeks up to 23 weeks of age. (C) Integration PCR on liver DNA showed two main products corresponding to HDR (1,289 bp) and NHEJ (2,213 bp) insertion of AAV-Donor in the Apoa1 cut site. Minus (–) indicates a water-only PCR control. (D–G) Western blot analysis of 2A-tagged apoA1 (D), APOE (E), total apoA1 (F), and apoB-48 and apoB-100 (G) in plasma isolated at endpoint, with aat as loading control. Eight representative mice per group are shown in western blots. (H–K) Densitometry analysis of apoA1-2A (H), APOE (I), apoA1 (J), and apoB-48 (K) in plasma relative to aat loading control. (L and M) Plasma total cholesterol (L) and triglycerides (M) measurement over time (green line, control; red line, CRISPR + Donor mice). Data are shown as mean ± standard deviation (n = 8 for densitometry analyses; n = 11 control and 9 CRISPR + Donor mice for lipid analyses). Significance was determined by a two-tailed Student’s t test in densitometry analyses (H–K). A two-way ANOVA followed by Bonferroni test was used for plasma lipid analyses in (L) and (M). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001. (A and B) Created with BioRender.

Article Snippet: Primary antibodies to the FLAG tag (1:5,000, rabbit, 600-401-383, Rockland), 2A peptide (1:5,000, rabbit, ABS31, Sigma-Aldrich), APOE (1:1,000, rabbit, ab52607, Abcam), FAH (1:1,000, rabbit, SAB2100745, Sigma-Aldrich), apoB (1:5,000, rabbit, K23300R, Meridian), apoA1 (1:5,000, rabbit, K23500R, Meridian), alpha-1 antitrypsin (Aat; 1:1,000, rabbit, 16382-1-AP, Proteintech), and beta tubulin (1:500, mouse, University of Iowa Developmental Studies Hybridoma Bank E7) were diluted in 1% BSA in PBS-T and membranes were incubated overnight at 4 degrees.

Techniques: Clinical Proteomics, Injection, CRISPR, Saline, Control, Western Blot, Isolation, Standard Deviation, Two Tailed Test

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Targeting the Apoa1 locus for liver-directed gene therapy

doi: 10.1016/j.omtm.2021.04.011

Figure Lengend Snippet: Correction of HT-I through targeted integration at the Apoa1 locus (A) Adult Fah −/− mice were intraperitoneally injected with either saline (control) or AAV-CRISPR plus an AAV-Donor encoding a human FAH transgene (5 × 10 11 GC each; CRISPR + Donor). NTBC was withdrawn at 7 days post-injection (time 0). Body weight was monitored over time up to 40 days and liver and plasma samples were collected for analyses. (B) Body weight ratios normalized to time zero (red line, CRISPR + Donor; green line, control mice). (C) Kaplan-Meier curve showing disease-free survival. Mice were euthanized upon loss of >20% body weight (red line, CRISPR + Donor; green line, control mice). One control mouse was found dead at day 30. (D) Western blot analysis of FAH in liver lysates with β-tub as a loading control. Plus (+) indicates a wild-type mouse liver as a positive control for endogenous Fah levels. (E) Representative FAH and hematoxylin and eosin staining in liver from control mice. (F) Representative FAH and hematoxylin and eosin staining in liver from CRISPR + Donor mice. Red squares are amplified in the images on the right. Scale bar is 100 μm. (A) Created with BioRender.

Article Snippet: Primary antibodies to the FLAG tag (1:5,000, rabbit, 600-401-383, Rockland), 2A peptide (1:5,000, rabbit, ABS31, Sigma-Aldrich), APOE (1:1,000, rabbit, ab52607, Abcam), FAH (1:1,000, rabbit, SAB2100745, Sigma-Aldrich), apoB (1:5,000, rabbit, K23300R, Meridian), apoA1 (1:5,000, rabbit, K23500R, Meridian), alpha-1 antitrypsin (Aat; 1:1,000, rabbit, 16382-1-AP, Proteintech), and beta tubulin (1:500, mouse, University of Iowa Developmental Studies Hybridoma Bank E7) were diluted in 1% BSA in PBS-T and membranes were incubated overnight at 4 degrees.

Techniques: Injection, Saline, Control, CRISPR, Clinical Proteomics, Western Blot, Positive Control, Staining, Amplification

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Differential Proteomic Analysis of Gender-dependent Hepatic Tumorigenesis in Hras12V Transgenic Mice *

doi: 10.1074/mcp.M116.065474

Figure Lengend Snippet: Validation of the differentially expressed proteins that were identified by MALDI-TOF/TOF-MS. Seven proteins (HSP90B1, beta-actin, albumin, APOA1, CALR, FABP5, and PRDX6) were randomly selected from the differently expressed proteins that were identified by MALDI-TOF/TOF-MS and were validated in transgenic and nontransgenic males (A) and females (B) by Western blot assay. Wt: the liver tissue of wild-type mice; P: the peritumor tissue of transgenic mice; T: the tumor tissue of transgenic mice; Non-Tg, C57BL/6J wild-type nontransgenic mice; Tg: transgenic mice. The numbers indicate different individuals. Bradford reagent was used as the loading control.

Article Snippet: Primary and secondary antibodies: anti-phoshpo-MEK-1/2 polyclonal antibody (diluted 1:1000, Elabscience, Wuhan, China), anti-phoshpo-ERK1/2 monoclonal antibody (diluted 1:2000, Cell Signaling Technology, MA), anti-phoshpo-PI3 Kinase polyclonal antibody (diluted 1:1000, Cell Signaling Technology), anti-phoshpo-Akt monoclonal antibody (diluted 1:2000, Cell Signaling Technology), anti-phoshpo-mTOR monoclonal antibody (diluted 1:1000, Cell Signaling Technology), anti-APOA1 monoclonal antibody (diluted 1:1000, Proteintech, Wuhan, China), anti-β-actin polyclonal antibody (diluted 1:5000, Bioworld Technology Co., Ltd., MN), anti-HSP90B1 polyclonal antibody (diluted 1:1000, Elabscience), anti-albumin polyclonal antibody (diluted 1:1000, Abclonal, Boston, MA), anti-calreticulin polyclonal antibody (diluted 1:1000, Abways, Shanghai, China), anti-fatty acid binding protein 5 polyclonal antibody (diluted 1:500, Proteintech), anti-peroxiredoxin VI monoclonal antibody (diluted 1:2000, Abfrontier, Santiago, CA), anti-mouse-IgG antibody (diluted 1:5000, Bioworld) and anti-rabbit-IgG antibody (diluted 1:5000, Bioworld).

Techniques: Transgenic Assay, Western Blot

Journal: PLoS ONE

Article Title: Novel Apo E-Derived ABCA1 Agonist Peptide (CS-6253) Promotes Reverse Cholesterol Transport and Induces Formation of preβ-1 HDL In Vitro

doi: 10.1371/journal.pone.0131997

Figure Lengend Snippet: Non denaturing gradient gel electrophoresis (4–20%) is used to separate CS-6253 complexes, apo A-I and apo E. CS-6253 was immunoblotted using antibody against human apo A-I, human apo E and against CS-6253. Normolipidemic human plasma was used as positive control. Molecular weight markers were revealed by Ponceau S. Left panel shows dose response relationship with an antibody directed against CS-6253. Middle panel shows anti-apo A-I antibody does not cross-react with CS-6253; similarly, right panel shows antibody against apo E does not react against CS-6253.

Article Snippet: Purified human plasma apo A-I (Biodesign) and apo E (Biodesign) were resolubilized in 4 M guanidine-HCL and dialyzed extensively against Tris buffer, (10 mm Tris, 150 mm NaCl; pH 8.2) [ , ].

Techniques: Denaturing Gradient Gel Electrophoresis, Positive Control, Molecular Weight

Journal: PLoS ONE

Article Title: Novel Apo E-Derived ABCA1 Agonist Peptide (CS-6253) Promotes Reverse Cholesterol Transport and Induces Formation of preβ-1 HDL In Vitro

doi: 10.1371/journal.pone.0131997

Figure Lengend Snippet: A. Time-course activity of ABCA1-mediated cholesterol efflux to lipid-free CS-6253 (30 μg/ml) and apo A-I (10 μg/ml), using BHK cells expressing ABCA1, and control cells (unstimulated BHK-ABCA1 and BHK-mock cells). Cholesterol efflux from either ABCA1 cells or control cells to apo A-I (closed circles), and CS-6253 (closed triangles) was determined at the indicated time points. B. Dose dependent ABCA1 mediated cholesterol efflux in BHK-ABCA1 induced with mifepristone. Kinetic parameters for ABCA1-mediated cholesterol efflux from BHK-ABCA1 cells to apo A-I: K m = 4.53±0.67 μg/ml (0.15±0.02 μM), V max = 14.85±0.02% efflux/4h, and relative catalytic efficiency: V max /K m = 3.27. CS-6253: K m = 2.27±0.16 μg/ml (0.73±0.05 μM), V max = 15.25±0.05% efflux/4h, and relative catalytic efficiency: V max /K m = 6.71. ATI-5261: K m = 1.04 ± 0.16 μg/ml (0.37 ± 0.04 μM), V max = 14.48 ± 0.29% efflux/ h, and relative catalytic efficiency: V max /K m = 13.92. Apo E: K m = 10.30±2.84 μg/ml (0.21±0.05 μM), V max = 11.45±1.16% efflux/4h, and relative catalytic efficiency: V max /K m = 1.11. C. Ability of CS-6253 to stimulate cholesterol efflux from human macrophages THP-1. Foam cells were incubated with CS-6253 or apoA-I at equimolar ratio (0.96 μM) for 4h. Cholesterol efflux induced by CS-6253 and apoA-I were compared to control cells incubated alone. P<0.001 by Student’s t-test. D. Membrane ABCA1 levels assessment by Western blotting assays. ABCA1 was detected from THP-1 foam cells lysis at 4°C with lysis buffer containing 0.5% n-dodecylmaltoside in the presence of a protease inhibitor mixture followed by low speed centrifugation to remove cell debris. Protein concentration was determined by standard assay (Bio-Rad). The supernatants were then separated by SDS-PAGE (4–22.5%) in duplicate. After electrophoresis, ABCA1 was detected by an anti-ABCA1 antibody. GAPDH was used as loading control. E. Quantification of FC and CE in foam cells after interaction with CS-6253 and apoA-I. After a period of 4h cholesterol efflux, THP-1 foam cell lipids were extracted with hexane: isopropanol (3v/2v) as under ‘‘Material and Methods’. 3[H]-FC and 3[H]-CE were separated by TLC and located by exposure to iodine vapor, and were scraped off into liquid scintillating vials and assayed for radioactivity. Results are from a single experiment using triplicate wells and the mean (±SD) is presented. P<0.0001 versus control sample untreated THP-1 cells for cholesterol efflux. P<0.05 versus FC or CE in untreated cells, by Student’s t-test.

Article Snippet: Purified human plasma apo A-I (Biodesign) and apo E (Biodesign) were resolubilized in 4 M guanidine-HCL and dialyzed extensively against Tris buffer, (10 mm Tris, 150 mm NaCl; pH 8.2) [ , ].

Techniques: Activity Assay, Expressing, Incubation, Western Blot, Lysis, Protease Inhibitor, Centrifugation, Protein Concentration, SDS Page, Electrophoresis, Radioactivity

Journal: PLoS ONE

Article Title: Novel Apo E-Derived ABCA1 Agonist Peptide (CS-6253) Promotes Reverse Cholesterol Transport and Induces Formation of preβ-1 HDL In Vitro

doi: 10.1371/journal.pone.0131997

Figure Lengend Snippet: BHK-ABCA1 cells were plated in 24-well plates and stimulated for 20 h. Cells were then incubated with 2 μg/ml of 125 I-apo A-I for 2 h at 37°C with increasing molar concentrations relative to apo A-I of either CS-6253 peptide, ATI-5261, human apo E, and unlabelled apo A-I (0, 0.0035, 0.017, 0.035, 0.071, 0.17, 0.35, 1.79 μM). Cells were then washed rapidly three times with ice-cold PBS/BSA and then PBS alone. 125 I-apo A-I cell associated was determined as described under “Experimental Procedures.” The values shown represent the mean ± S.D from triplicate wells. The 100% of control value measured in the absence of competitors was 0.21 ng of apo A-I /μg cell protein. Values of IC 50 shown were determined using the Graph Pad Prism 6 software.

Article Snippet: Purified human plasma apo A-I (Biodesign) and apo E (Biodesign) were resolubilized in 4 M guanidine-HCL and dialyzed extensively against Tris buffer, (10 mm Tris, 150 mm NaCl; pH 8.2) [ , ].

Techniques: Incubation, Software