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