Journal: Theranostics

Article Title: Bax inhibitor 1 preserves mitochondrial homeostasis in acute kidney injury through promoting mitochondrial retention of PHB2

doi: 10.7150/thno.40098

Figure Lengend Snippet: BI1 promotes import of PHB2 into mitochondria. (A-B) In vitro , after different times of IRI, proteins were isolated from tubule cells. Then, mitochondrial and cytosolic fractions were collected. PHB2 expression was determined using Western blots. VDAC was employed as the loading control for mitochondrial fraction whereas GAPDH was used as the marker of cytosolic fraction. (C-D) In vivo , proteins were isolated from reperfused kidney and mitochondrial and cytosolic fractions were collected. PHB2 expression was determined using Western blots. VDAC was utilized as the loading control for mitochondrial fraction whereas GAPDH was employed as the marker of cytosolic fraction. (E-F) siRNA against BI1 (BI1-si) and control siRNA (Ctrl-si) were transfected into primary tubule cells and then the expression of mitochondrial PHB2 (mito-PHB2) was determined. (G-H) In primary tubule cells from BI1 TG and WT mice, whole mitochondrial fraction (Whole) was firstly isolated and then mitochondrial outer-membrane (MOM) and mitoplast (inner-membrane plus matrix) fractions were collected. Western blotting was used to analyze the expression of PHB2 in whole, mitoplast and MOM fractions. ABCB10 was utilized as a loading control for mitoplast whereas VDAC was used as a MOM marker. (I-J) Under normal condition, BI1 siRNA (BI1-si), TIM23 siRNA (TIM23-si) and control siRNA (Ctrl-si) were transfected into primary tubule cells. Then, levels of PHB2 were determined. (K-L) Under mIRI condition, Myc-TIM23, HA-BI1 and vector were transfected into HK2 cells. Moreover, TIM23-si was employed to silence TIM23 in HK2 cells infected with HA-BI1 prior to determination of PHB2. Experiments were repeated for at least three times and data are shown as mean ± SEM (n = 6 mice or 3 independent cell isolations per group). * p <0.05.

Article Snippet: Similarly, BI1ΔN (30-237 aa), BI1ΔC (1-222 aa), BI1ΔTM (1-29 and 223-237 aa) and full length (1-237 aa) of BI1 were inserted into pcDNA3.1/HA (Invitrogen) to generate BI1 mutants. siRNAs against BI1 (BI1-si), TIM23 (TIM23-si), and PHB2 (PHB2-si) were designed and synthesized by GenePharma Co, Ltd. (Shanghai, China).

Techniques: In Vitro, Isolation, Expressing, Western Blot, Control, Marker, In Vivo, Transfection, Membrane, Plasmid Preparation, Infection

Journal: Theranostics

Article Title: Bax inhibitor 1 preserves mitochondrial homeostasis in acute kidney injury through promoting mitochondrial retention of PHB2

doi: 10.7150/thno.40098

Figure Lengend Snippet: BI1 interacts with PHB2 and promotes PHB2 localization into mitochondria under renal IRI. (A) Cell lysates from primary tubule cells were immunoprecipitated with the anti-BI1 or anti-PHB2 antibody, followed by immunoblotting with the anti-PHB2 or anti-BI1 antibody. IgG was employed as a control for the endogenous interaction assay between BI1 and PHB2. (B) Immunoblotting analysis of lysates after immunoprecipitation from HK2 cells transfected with exogenous HA-BI1 and Myc-PHB2. (C-D) Mapping of regions of PHB2. Different PHB2 mutants were transfected into HK2 cells. Then, immunoprecipitation, and immunoblot of cell lysates from HK2 cells. (E-F) Different BI1 mutants as indicated were transfected into HK2 cells, and then immunoprecipitation analyses were carried out. (G-H) Under mIRI, different BI1 mutants were transfected into HK2 cells. Besides, TIM23-siRNA was utilized to silence TIM23 in HK2 cells transfected with Myc-BI1 mutants. Experiments were repeated for at least three times and data are shown as mean ± SEM (n =3 independent cell isolations per group). * p <0.05.

Article Snippet: Similarly, BI1ΔN (30-237 aa), BI1ΔC (1-222 aa), BI1ΔTM (1-29 and 223-237 aa) and full length (1-237 aa) of BI1 were inserted into pcDNA3.1/HA (Invitrogen) to generate BI1 mutants. siRNAs against BI1 (BI1-si), TIM23 (TIM23-si), and PHB2 (PHB2-si) were designed and synthesized by GenePharma Co, Ltd. (Shanghai, China).

Techniques: Immunoprecipitation, Western Blot, Control, Transfection

Journal: Theranostics

Article Title: Bax inhibitor 1 preserves mitochondrial homeostasis in acute kidney injury through promoting mitochondrial retention of PHB2

doi: 10.7150/thno.40098

Figure Lengend Snippet: PHB2 retention in mitochondria accounts for BI1-conferred renoprotection. (A) Prior to mIRI, HK2 cells were transfected with HA-BI1 or its mutants (HA-BI1ΔC and HA-BI1ΔN). Mitochondrial copy number was determined using qPCR. (B-C) Mitochondrial OCR was determined using an XFe96 extracellular flux analyzer in HK2 transfected with HA-BI1 and/or its mutants. (D) ELISA for caspase-9 activity. (E-F) Myc-labelled PHB2 mutants (Myc-PHB2ΔPHB, Myc-PHB2ΔC, Myc-PHB2ΔN) were infected into HK2 cells. Besides, HA-BI1 or vector were constructed into HK2 cells prior to mIRI. Mitochondrial membrane potential was recorded using JC-1 staining. (G-H) mt-Kemia assay for acidic mitochondria observation. The ratio of 534/458 nm was used to quantify acidic mitochondria index. (I-J) TUNEL assay for cell death. Number of TUNEL apoptotic cells were calculated. Experiments were repeated for at least three times and data are shown as mean ± SEM (n = 3 independent cell isolations per group). * p <0.05.

Article Snippet: Similarly, BI1ΔN (30-237 aa), BI1ΔC (1-222 aa), BI1ΔTM (1-29 and 223-237 aa) and full length (1-237 aa) of BI1 were inserted into pcDNA3.1/HA (Invitrogen) to generate BI1 mutants. siRNAs against BI1 (BI1-si), TIM23 (TIM23-si), and PHB2 (PHB2-si) were designed and synthesized by GenePharma Co, Ltd. (Shanghai, China).

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Activity Assay, Infection, Plasmid Preparation, Construct, Membrane, Staining, TUNEL Assay

Journal: Theranostics

Article Title: Bax inhibitor 1 preserves mitochondrial homeostasis in acute kidney injury through promoting mitochondrial retention of PHB2

doi: 10.7150/thno.40098

Figure Lengend Snippet: PHB2 knockdown abolishes BI1-induced renoprotection . (A-B) BI1 TG mice were subjected to intravenous injections of scramble control or PHB2-specific siRNA (Ctrl-si or PHB2-si) before IRI. Then, renal function was determined using levels of BUN and creatinine. (C-D) HE staining was used to observe the structural alterations of tubules following IRI or the whole kidney. Semiquantitative analysis of tubular injury (tubular atrophy or dilatation, loss of brush border, vacuolization, epithelial cell shedding, and denuded tubular basement membrane) scored as: 0, normal; 1, <10%; 2, 10%-25%; 3, 25%-50%; 4, 50%-75%; 5, 75%-100% of affected area from 20 random fields. (E-F) Tubule death was detected using TUNEL staining. AQP1 was employed to stain proximal tubules. (G-H) Immunofluorescence assay for F4/80 inflammatory cells. The immunosignal of F4/80 was used to evaluate kidney inflammation response. (I-J) RNA was isolated from reperfused kidneys and then transcriptional levels of Ccl2 and IL-6 were determined using qPCR. Experiments were repeated for at least three times and data are shown as mean ± SEM (n = 6 mice or 3 independent cell isolations per group). * p <0.05.

Article Snippet: Similarly, BI1ΔN (30-237 aa), BI1ΔC (1-222 aa), BI1ΔTM (1-29 and 223-237 aa) and full length (1-237 aa) of BI1 were inserted into pcDNA3.1/HA (Invitrogen) to generate BI1 mutants. siRNAs against BI1 (BI1-si), TIM23 (TIM23-si), and PHB2 (PHB2-si) were designed and synthesized by GenePharma Co, Ltd. (Shanghai, China).

Techniques: Knockdown, Control, Staining, Membrane, TUNEL Assay, Immunofluorescence, Isolation

Journal: Theranostics

Article Title: Bax inhibitor 1 preserves mitochondrial homeostasis in acute kidney injury through promoting mitochondrial retention of PHB2

doi: 10.7150/thno.40098

Figure Lengend Snippet: Schematic diagram depicting proposed BI1-PHB2 signaling modality in AKI. In physiological settings (as shown in the right panel), BI1 interacts with and therefore promotes PHB2 retention into mitochondria with the assistance of the mitochondrial transport protein TIM23, preserving mitochondrial homeostasis and tubular viability. Pathological stress such as IRI (as shown on the left panel) suffers from loss of BI1, leading to poor MOM localization and translocation of PHB2 into MIM. As a result, PHB2 is lost into cytoplasm (loss of mitochondrial retention) to trigger mitochondrial damage.

Article Snippet: Similarly, BI1ΔN (30-237 aa), BI1ΔC (1-222 aa), BI1ΔTM (1-29 and 223-237 aa) and full length (1-237 aa) of BI1 were inserted into pcDNA3.1/HA (Invitrogen) to generate BI1 mutants. siRNAs against BI1 (BI1-si), TIM23 (TIM23-si), and PHB2 (PHB2-si) were designed and synthesized by GenePharma Co, Ltd. (Shanghai, China).

Techniques: Preserving, Translocation Assay

Journal: Molecular Therapy

Article Title: Targeted Gene Delivery to the Enteric Nervous System Using AAV: A Comparison Across Serotypes and Capsid Mutants

doi: 10.1038/mt.2015.7

Figure Lengend Snippet: Transduction efficiency of recombinant adeno-associated virus (AAV) in the myenteric plexus (MP) of the enteric nervous system: a comparison across serotypes 1, 2, 5, 6, 8, and 9 . Adult male rats received direct injections of AAV (6 × 5 µl injections at 1.3 × 10 12 vg/ml) into the wall of the descending colon and were sacrificed one month later, at which time the MP was dissected and native fluorescence was viewed under a fluorescent microscope. ( a ) Transduction efficiency of AAV1, 2, 5, 6, 8, and 9, was measured by the quantification of green fluorescent protein (GFP) expressing cells in the MP. Columns represent mean number of GFP-positive cells, expressed as cells/mm 2 , + 1 SEM ( n = 4–6/group). AAV6 and 9 exhibited significantly greater numbers of GFP positive cells than AAV1 and AAV2. ( b ) The percent of HUc-positive cells transduced for each respective serotype. Table represents percent of HUc cells colocalized with GFP ± SEM ( n = 4–6/group). ( c–h ) Shows representative images of GFP-positive cells in the MP following transduction by AAV1, 2, 5, 8, and 9. Scale bar in ( h ) represents 100 µm and applies to panels ( c–g ). * Indicates significantly different than AAV1 and AAV2 ( P < 0.05).

Article Snippet: Recombinant adeno-associated virus (AAV) is one of the most widely used gene therapy systems in research and clinical trials., , AAV is favored over other viral vectors due to its lack of pathogenicity and the ability to establish expression within both dividing and mature nondividing cells of multiple lineages.

Techniques: Transduction, Recombinant, Virus, Comparison, Fluorescence, Microscopy, Expressing

Journal: Molecular Therapy

Article Title: Targeted Gene Delivery to the Enteric Nervous System Using AAV: A Comparison Across Serotypes and Capsid Mutants

doi: 10.1038/mt.2015.7

Figure Lengend Snippet: Transduction efficiency of recombinant adeno-associated virus (AAV) in the submucosal plexus (SMP) of the enteric nervous system: a comparison across serotypes 1, 2, 5, 6, 8, and 9 . Adult male rats received direct injections of AAV (6 × 5 µl injections at 1.3 × 10 12 vg/ml) into the wall of the descending colon and were sacrificed one month later, at which time the SMP was dissected and viewed under a fluorescent microscope. ( a ) Transduction efficiency of AAV1, 2, 5, 6, 8, and 9 was measured by the quantification of green fluorescent protein (GFP) expressing cells in the SMP. Columns represent mean number of GFP-positive cells, expressed as cells/mm 2 , + 1 SEM ( n = 4–6/group). AAV6 and 9 exhibited significantly greater numbers of GFP-positive cells than AAV1 and AAV2. ( b ) The percent of HUc-positive cells transduced for each respective serotype. Table represents the percent of HUc cells colocalized with GFP ± SEM ( n = 4–6/group). ( c–h ) Representative images of GFP-positive cells in the SMP following transduction by AAV1, 2, 5, 8, and 9. Scale bar in ( h ) represents 100 µm and applies to panels ( c–g ). * Indicates significantly different than AAV1 and AAV2 ( P < 0.05).

Article Snippet: Recombinant adeno-associated virus (AAV) is one of the most widely used gene therapy systems in research and clinical trials., , AAV is favored over other viral vectors due to its lack of pathogenicity and the ability to establish expression within both dividing and mature nondividing cells of multiple lineages.

Techniques: Transduction, Recombinant, Virus, Comparison, Microscopy, Expressing

Journal: Molecular Therapy

Article Title: Targeted Gene Delivery to the Enteric Nervous System Using AAV: A Comparison Across Serotypes and Capsid Mutants

doi: 10.1038/mt.2015.7

Figure Lengend Snippet: Neuronal tropism of recombinant adeno-associated virus (AAV) transduction in the myenteric plexus (MP): serotypes 1, 2, 5, 6, 8, and 9 . Adult male rats received direct injections of AAV (6 × 5 µl injections at 1.3 × 10 12 vg/ml) into the wall of the descending colon and were sacrificed one month later, at which time the MP was dissected. MP tissue was stained for the neuronal marker HUc (red) and the enteric glia marker, glial fibrillary acidic protein (GFAP; blue). Following the MP was viewed under a fluorescent microscope and the transduction of neurons in the MP was determined by examining the colocalization of green fluorescent protein (GFP; green) expression in HUc-positive cells (colocalization appears yellow). Neuronal transduction was observed for all serotypes examined. Representative images of neuronal transduction are shown for AAV1 ( a–d ), AAV2 ( e–h ), AAV5 ( i–l ), AAV6 ( m–p ), AAV8 ( q–t ), and AAV9 ( u–x ). Scale bar in ( x ) represents 100 µm and applies to panels ( a–w ).

Article Snippet: Recombinant adeno-associated virus (AAV) is one of the most widely used gene therapy systems in research and clinical trials., , AAV is favored over other viral vectors due to its lack of pathogenicity and the ability to establish expression within both dividing and mature nondividing cells of multiple lineages.

Techniques: Recombinant, Virus, Transduction, Staining, Marker, Microscopy, Expressing

Journal: Molecular Therapy

Article Title: Targeted Gene Delivery to the Enteric Nervous System Using AAV: A Comparison Across Serotypes and Capsid Mutants

doi: 10.1038/mt.2015.7

Figure Lengend Snippet: Neuronal tropism of recombinant adeno-associated virus (AAV) transduction in the submucosal plexus (SMP): serotypes 1, 2, 5, 6, 8, and 9 . Adult male rats received direct injections of AAV (6 × 5 µl injections at 1.3 × 10 12 vg/ml) into the wall of the descending colon and were sacrificed one month later, at which time the SMP was dissected. SMP tissue was stained for the neuronal marker HUc (red) and the enteric glia marker, glial fibrillary acidic protein (GFAP; blue). Following, the SMP was viewed under a fluorescent microscope and the transduction of neurons in the SMP was determined by examining the colocalization of green fluorescent protein (GFP; green) expression in HUc-positive cells (colocalization appears in yellow). Neuronal transduction was observed for all serotypes examined. Representative images of neuronal transduction are shown for AAV1 ( a–d ), AAV2 ( e–h ), AAV5 ( i–l ), AAV6 ( m–p ), AAV8 ( q–t ), and AAV9 ( u–x ). Scale bar in ( x ) represents 100 µm and applies to panels ( a–w ).

Article Snippet: Recombinant adeno-associated virus (AAV) is one of the most widely used gene therapy systems in research and clinical trials., , AAV is favored over other viral vectors due to its lack of pathogenicity and the ability to establish expression within both dividing and mature nondividing cells of multiple lineages.

Techniques: Recombinant, Virus, Transduction, Staining, Marker, Microscopy, Expressing

Journal: Molecular Therapy

Article Title: Targeted Gene Delivery to the Enteric Nervous System Using AAV: A Comparison Across Serotypes and Capsid Mutants

doi: 10.1038/mt.2015.7

Figure Lengend Snippet: Glial tropism of recombinant adeno-associated virus (AAV) transduction . Adult male rats received direct injections of AAV (6 × 5 µl injections at 1.3 × 10 12 vg/ml) into the wall of the descending colon and were sacrificed one month later, at which time the submucosal plexus (SMP) and myenteric plexus (MP) were dissected. MP and SMP tissue was stained for the enteric glia marker, glial fibrillary acidic protein (GFAP; blue). Following, tissue was viewed under a fluorescent microscope and the transduction of glia was determined by examining the colocalization of green fluorescent protein (GFP; green) expression in GFAP-positive enteric glia (colocalization appears turquoise). Micrographs also show the neuronal marker HUc (red). Glia transduction was rare and was only observed in AAV serotypes 1, 5, 6, 8 and AAV8-doubleY-F+T-V. AAV6 showed far more glial transduction than any other serotype. Morphologically, most glial transduction appeared to occur within reactive, protoplasmic or mucosal, enteric glia ( d,h,p, and t) . In contrast, AAV6 was able to transduce intraganglionic enteric glia ( l ). Representative images of glial transduction are shown for AAV1 in the MP ( a–d ), AAV5 in the MP ( e–h ), AAV6 in the SMP ( i–l) , AAV8 in the SMP ( m–p ), and AAV8-doubleY-F+T-V in the SMP ( q–t ). Scale bar in ( d,t ) represents 50 µm and applies to ( a–c ) and ( q–s ). Scale bars in ( h,l,p ) represent 100 µm and apply to ( e–g , i–k,m–o ).

Article Snippet: Recombinant adeno-associated virus (AAV) is one of the most widely used gene therapy systems in research and clinical trials., , AAV is favored over other viral vectors due to its lack of pathogenicity and the ability to establish expression within both dividing and mature nondividing cells of multiple lineages.

Techniques: Recombinant, Virus, Transduction, Staining, Marker, Microscopy, Expressing

Journal: Molecular Therapy

Article Title: Targeted Gene Delivery to the Enteric Nervous System Using AAV: A Comparison Across Serotypes and Capsid Mutants

doi: 10.1038/mt.2015.7

Figure Lengend Snippet: Time course and titer response of recombinant adeno-associated virus (AAV) serotype 9 in the myenteric plexus (MP) and submucosal plexus (SMP) of the enteric nervous system . Time course ( a–h ): Adult male rats received direct injections of high titer AAV9 (6 × 5 µl injections at 1.2 × 10 13 vg/ml) into the wall of the descending colon and were sacrificed 2, 4, or 6 weeks postsurgery, at which time the MP and SMP were dissected and viewed under a fluorescent microscope. Transduction efficiency of the 2-week ( a , dark blue columns), 4-week ( a , light blue columns), and 6-week groups ( a , turquoise columns) was measured by the quantification of green fluorescent protein (GFP) expressing cells in the MP ( a , left column grouping) and SMP ( a , right column grouping). Columns represent mean number of GFP-positive cells, expressed as cells/mm 2 , + 1 SEM ( n = 4–6/group). The 4- and 6-week groups had significantly more GFP-positive cells in both the MP and SMP than the 2-week group. No difference between the 4- and 6-week groups was observed. ( b ) The percent of HUc-positive cells expressing GFP ± SEM ( n = 4–6/group). Representative images of GFP (green) and HUc (red) positive cells are shown for the 2-week ( c ), 4-week ( d ), and 6-week ( e ) group in the MP and the 2-week ( f ), 4-week ( g ), and 6-week ( h ) groups in the SMP. * Indicates significantly different than AAV9 at 2 weeks ( P < 0.05). Titer response ( i–p ): Adult male rats received direct injections of AAV9 at a low titer (6 × 5 µl injections at 1.3 × 10 12 vg/ml), an intermediate titer (6 × 5 μl injection at 6.25 × 10 12 vg/ml) or a high titer (6 × 5 µl injections at 1.2 × 10 13 vg/ml) into the wall of the descending colon and were sacrificed 1 month later, at which time the MP and SMP were dissected and viewed under a fluorescent microscope. Transduction efficiency of low-titer AAV9 ( i , dark blue columns), intermediate titer AAV9 ( i , light blue columns) and high-titer AAV9 ( i , turquoise columns), were measured by the quantification of green fluorescent protein (GFP) expressing cells in the MP ( i , left column grouping) and SMP ( i , right column grouping). Columns represent mean number of GFP-positive cells, expressed as cells/mm 2 , + 1 SEM ( n = 4–6/group). The intermediate (6.25 × 10 12 ) and high titer (1.2 × 10 13 ) AAV9 groups had significantly more GFP-positive cells in both the MP and SMP than the low titer (1.3 × 10 12 ) AAV9 groups. ( j ) The percent of HUc-positive cells expressing GFP ± SEM ( n = 4–6/group). Representative images of GFP (green) and HUc (red) positive cells are shown for the low titer ( k ), intermediate titer ( l ), and high titer ( m ) groups in the MP and the low titer ( n ), intermediate titer ( o ), and high titer ( p ) groups in the SMP. * Indicates significantly different than AAV9 at 1.2 × 10 12 vg/ml ( P < 0.05). Scale bar in ( h and g ) represents 100 µm and applies to all panels.

Article Snippet: Recombinant adeno-associated virus (AAV) is one of the most widely used gene therapy systems in research and clinical trials., , AAV is favored over other viral vectors due to its lack of pathogenicity and the ability to establish expression within both dividing and mature nondividing cells of multiple lineages.

Techniques: Recombinant, Virus, Microscopy, Transduction, Expressing, Injection

Journal: Molecular Therapy

Article Title: Targeted Gene Delivery to the Enteric Nervous System Using AAV: A Comparison Across Serotypes and Capsid Mutants

doi: 10.1038/mt.2015.7

Figure Lengend Snippet: The spread of a single injection of recombinant adeno-associated virus (AAV) serotype 9 in the myenteric plexus (MP) and submucosal plexus (SMP) of the enteric nervous system . Adult male rats received direct injections of AAV9 (1.2 × 10 13 vg/ml) into the wall of the descending colon and were sacrificed one month later, at which time the MP and SMP were dissected and viewed under a fluorescent microscope. The spread of transduction was determined by quantifying the area of the MP and SMP containing green fluorescent protein (GFP)-positive cells. Panel ( a ) and ( b ) show representative images of vector spread and transduction following a single injection of AAV9 within the MP and SMP, respectively. Representative images are shown with axis to indicate longitudinal (x-axis) and perpendicular (y-axis) spread. Tick marks on the axis represent 1 mm. The circle in the center of the axis represents the approximate location of the injection site. Insets represent high magnification images of the area within the white box in a and b . Scale bars in ( a ) and ( b ) represent 1 mm. Scale bars in the insets in ( a ) and ( b ) represent 50 μm.

Article Snippet: Recombinant adeno-associated virus (AAV) is one of the most widely used gene therapy systems in research and clinical trials., , AAV is favored over other viral vectors due to its lack of pathogenicity and the ability to establish expression within both dividing and mature nondividing cells of multiple lineages.

Techniques: Injection, Recombinant, Virus, Microscopy, Transduction, Plasmid Preparation