Journal: International Journal of Medical Sciences

Article Title: CRISPR/Cas9-Driven Fndc5 Knockout Reveals Augmented Mitochondrial Structural and Dynamic Alterations in Diabetic Nephropathy

doi: 10.7150/ijms.131007

Figure Lengend Snippet: mRNA expression levels of Fndc5 (A) and mitochondrial function markers, including Dnm1l (B), Fis1 (C), and Mfn1 (D), in kidney samples from WT and Fndc5 KO mice, with or without DN induction, normalized to GAPDH. (E) Representative Western blot images showing protein levels of ATP5A1, ATP5B, and β-actin in kidney samples from WT and Fndc5 KO mice under control and DN conditions, with corresponding quantification normalized to β-actin. (F) Representative transmission electron microscopy images of mitochondrial morphology in proximal tubular cells of WT and Fndc5 KO mice, with or without DN induction. Red outlines demarcate representative mitochondria, while yellow lines highlight preserved mitochondrial cristae. Yellow arrows specifically indicate mitochondrial swelling characterized by an extensive loss of cristae, most notably in the Fndc5 KO DN group. Scale bars: 2 μm (low magnification) and 1 μm (high magnification). (G) Quantitative analysis of mitochondrial cristae number per mitochondrion in proximal tubular cells from WT and Fndc5 KO mice, with or without DN induction, based on transmission electron microscopy images. Data are shown as mean ± SEM (n = 4 for each group). *P < .05; **P < .01; ***P < .001.

Article Snippet: Membranes were blocked with 5% milk in TBST for 1 hour, then incubated with primary antibodies (nephrin, 1:1000 [Proteintech, 22912-1-AP]; podocin, 1: 1000 [ABclonal, A17337]; ATP5A1, 1:5,000 [ABclonal, A11217]; ATP5B, 1:10,000 [ABclonal, A11214]; β-actin, 1:10,000 [GeneTex, GTX109639]) at 4 °C overnight.

Techniques: Expressing, Western Blot, Control, Transmission Assay, Electron Microscopy

Journal: International Journal of Medical Sciences

Article Title: CRISPR/Cas9-Driven Fndc5 Knockout Reveals Augmented Mitochondrial Structural and Dynamic Alterations in Diabetic Nephropathy

doi: 10.7150/ijms.131007

Figure Lengend Snippet: Integrated schematic model linking FNDC5 to mitochondrial integrity in diabetic nephropathy (DN). FNDC5 may sustain the expression of PGC-1α, which serves as its own upstream regulator, either directly or through an AMPK-mediated positive feedback loop. PGC-1α is a master regulator of mitochondrial biogenesis; additionally, FNDC5 may modulate mitochondrial function via the p38 MAPK axis. In FNDC5-deficient DN mice, the loss of FNDC5 and the subsequent decrease in PGC-1α levels impair mitochondrial homeostasis. This condition is characterized by structural alterations such as disrupted cristae, increased mitochondrial fission signaling ( Dnm1l and Fis1 ), and reduced fusion signaling ( Mfn1 ). Furthermore, expression of ATP synthase subunits ATP5A1 and ATP5B is reduced. These changes within the 'vicious cycle' of mitochondrial failure lead to fragmentation of mitochondrial networks, increased oxidative damage as shown by 4-HNE IHC, and DNA damage detected by TUNEL assay. Collectively, these cellular stressors accelerate the progression of DN, resulting in kidney structural injury characterized by mesangial expansion, glomerular basement membrane thickening, and podocyte effacement.

Article Snippet: Membranes were blocked with 5% milk in TBST for 1 hour, then incubated with primary antibodies (nephrin, 1:1000 [Proteintech, 22912-1-AP]; podocin, 1: 1000 [ABclonal, A17337]; ATP5A1, 1:5,000 [ABclonal, A11217]; ATP5B, 1:10,000 [ABclonal, A11214]; β-actin, 1:10,000 [GeneTex, GTX109639]) at 4 °C overnight.

Techniques: Expressing, TUNEL Assay, Membrane

Journal: Aging (Albany NY)

Article Title: PGC-1α alleviates mitochondrial dysfunction via TFEB-mediated autophagy in cisplatin-induced acute kidney injury

doi: 10.18632/aging.202653

Figure Lengend Snippet: PGC-1α positively regulate the TFEB in HK2 cells. ( A ) HK2 Cell lysates were immunoprecipitated (IP) with an anti-PGC-1α or an anti-TFEB antibody, then immunoblotted (IB) with TFEB and PGC-1α antibodies. Anti- IgG antibody as a negative control. ( B , C ) The expression of TFEB and PGC-1α was measured by western blotting. ( D ) Luciferase activity in HK-2 cells transfected with the TFEB promoter-reporter construct linked to luciferase along with PGC-1α plasmids or control empty vectors. ( E ) Representative immunofluorescence images of TFEB in HK2 cells. Scar bar: 20 μm. Data are provided as the mean ± SEM, n=3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. over con ; # P < 0.05, ## P < 0.01, ### P < 0.001 vs si con (Con, control; si con , sicontrol; over con , overcontrol).

Article Snippet: The membranes were blocked with 5% skim milk at room temperature for 2 h. This was followed by overnight incubation at 4° C with monoclonal antibodies against PGC-1α (1:500 dilution, A12348; ABclonal), TFEB (1:1000 dilution, 4240S; CST), TFEB (1:500 dilution, A7311; ABclonal), P62 (1:1000 dilution, 109012; Abcam), LC3B (1:1000 dilution, 2775S; CST), Bax (1:500 dilution, A0207; ABclonal), Bcl-2 (1:1000 dilution, A11025; ABclonal), Ndufs4 (1:500 dilution, A6390; ABclonal), and ATP5b (1:500 dilution, A5769; ABclonal).

Techniques: Immunoprecipitation, Negative Control, Expressing, Western Blot, Luciferase, Activity Assay, Transfection, Construct, Control, Immunofluorescence

Journal: Aging (Albany NY)

Article Title: PGC-1α alleviates mitochondrial dysfunction via TFEB-mediated autophagy in cisplatin-induced acute kidney injury

doi: 10.18632/aging.202653

Figure Lengend Snippet: PGC-1α activates autophagy by modulating TFEB. ( A ) The expression of the proteins P62 and LC3 was measured by western blotting in HK2 cells exposed to cisplatin (5 μM) in the presence or absence of HCQ (30 μM) for 48 h. ( B ) HK2 cells were exposed to cisplatin in the presence or absence of ZLN005 (10 μM) for 48 h, and the expression of the proteins TFEB, P62 and LC3was measured by western blotting. ( C ) HK2 cells treated with ZLN005 (10 μM) and p62 mRNA was measured by real-time PCR. ( D ) Representative images of the colocalization between LC3 and mitochondria. Data are provided as the mean ± SEM, n=3 independent experiments. *P < 0.05, **P < 0.01 vs. Con; &P < 0.05, &&P < 0.01 vs. Cisp. (Con, control; Zln, ZLN005; Cisp, cisplatin; C+Z, cisplatin + ZLN005; HCQ, hydroxychloroquine; C+H, cisplatin + hydroxychloroquine).

Article Snippet: The membranes were blocked with 5% skim milk at room temperature for 2 h. This was followed by overnight incubation at 4° C with monoclonal antibodies against PGC-1α (1:500 dilution, A12348; ABclonal), TFEB (1:1000 dilution, 4240S; CST), TFEB (1:500 dilution, A7311; ABclonal), P62 (1:1000 dilution, 109012; Abcam), LC3B (1:1000 dilution, 2775S; CST), Bax (1:500 dilution, A0207; ABclonal), Bcl-2 (1:1000 dilution, A11025; ABclonal), Ndufs4 (1:500 dilution, A6390; ABclonal), and ATP5b (1:500 dilution, A5769; ABclonal).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Aging (Albany NY)

Article Title: PGC-1α alleviates mitochondrial dysfunction via TFEB-mediated autophagy in cisplatin-induced acute kidney injury

doi: 10.18632/aging.202653

Figure Lengend Snippet: Silencing of TFEB partially abolishes the protective effects of ZLN005 in cisplatin-treated HK2 cells. HK2 cells were transfected with control siRNA (si con , black column) or TFEB siRNA (si tfeb , white column) for 6 h and treated with cisplatin in the presence or absence of ZLN005 for 48 h. ( A ) The expression of autophagy-related protein (TFEB, P62 and LC3) was measured by western blotting. ( B ) The expression of mitochondria-related proteins (ATP5b and Ndufs4) was measured by western blotting. ( C ) Mitochondrial ROS (mtROS) were measured by incubation with Mito-SOX Red. ( D ) The expression of apoptosis-related proteins was measured by western blotting. ( E ) The effects of ZLN005 on cisplatin-induced apoptosis were determined by flow cytometry. ( F ) ATP levels were measured by using an ATP Assay kit. Data are provided as the mean ± SEM, n=3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Con; & P < 0.05, && P < 0.01 vs. Cisp; # P < 0.05, ## P < 0.01 vs. si tfeb. (Con, control; Zln, ZLN005; Cisp, cisplatin; C+Z, cisplatin + ZLN005).

Article Snippet: The membranes were blocked with 5% skim milk at room temperature for 2 h. This was followed by overnight incubation at 4° C with monoclonal antibodies against PGC-1α (1:500 dilution, A12348; ABclonal), TFEB (1:1000 dilution, 4240S; CST), TFEB (1:500 dilution, A7311; ABclonal), P62 (1:1000 dilution, 109012; Abcam), LC3B (1:1000 dilution, 2775S; CST), Bax (1:500 dilution, A0207; ABclonal), Bcl-2 (1:1000 dilution, A11025; ABclonal), Ndufs4 (1:500 dilution, A6390; ABclonal), and ATP5b (1:500 dilution, A5769; ABclonal).

Techniques: Transfection, Control, Expressing, Western Blot, Incubation, Flow Cytometry, ATP Assay

Journal: Aging (Albany NY)

Article Title: PGC-1α alleviates mitochondrial dysfunction via TFEB-mediated autophagy in cisplatin-induced acute kidney injury

doi: 10.18632/aging.202653

Figure Lengend Snippet: ZLN005 treatment alleviates renal injury in cisplatin-induced AKI mice via PGC-1a/TFEB pathway. The male C57BL/6 mice were injected once with cisplatin (16mg/kg, i.p.) to induce AKI, followed by ZLN005 treatment (15mg/kg/d, i.g.) for 4 days. ( A ) Western blots of TFEB, P62 and LC3 levels in kidney. ( B ) Representative TEM micrographs of mouse renal tubular epithelial cell mitochondria from each group. Scale bar, 2 μm (wireframe indicates the magnified image). ( C ) The expression of mitochondria-related proteins (ATP5b and Ndufs4) was measured by western blotting. ( D ) For the measurement of mitochondrial ROS (mtROS), frozen sections of freshly renal tissues were stained with Mito-SOX Red (2.5 μM) 15 min at 37° C and determined by confocal microscope. Scar bar: 20 μm. Data are provided as the mean ± SEM, n=3 independent experiments. *P < 0.05, **P < 0.01 vs. Con; & P < 0.05, && P < 0.01 vs. Cisp. (NC, normal control; Cisp, cisplatin; C+Z, cisplatin + ZLN005).

Article Snippet: The membranes were blocked with 5% skim milk at room temperature for 2 h. This was followed by overnight incubation at 4° C with monoclonal antibodies against PGC-1α (1:500 dilution, A12348; ABclonal), TFEB (1:1000 dilution, 4240S; CST), TFEB (1:500 dilution, A7311; ABclonal), P62 (1:1000 dilution, 109012; Abcam), LC3B (1:1000 dilution, 2775S; CST), Bax (1:500 dilution, A0207; ABclonal), Bcl-2 (1:1000 dilution, A11025; ABclonal), Ndufs4 (1:500 dilution, A6390; ABclonal), and ATP5b (1:500 dilution, A5769; ABclonal).

Techniques: Injection, Western Blot, Expressing, Staining, Microscopy, Control

Journal: Mitochondrial communications

Article Title: Structural determinants of mitochondrial STAT3 targeting and function

doi: 10.1016/j.mitoco.2024.01.001

Figure Lengend Snippet: (A) Graphic representation of recombinant STAT3 constructs. Mitochondrial extracts from MDA-MB-231 cells expressing MTS.STAT3.Bio (B) or MTS.TruncSTAT3.Bio (C) were immunoprecipitated with anti-Flag antibodies and immunoblotted with either STAT3 or Flag antibodies as indicated. Asterix (*) marks endogenous STAT3. (D) Cytosolic (Cytosol) or mitochondrial (Mitochondria) extracts generated by 2 distinct purification protocols from HEK293T cells expressing either full length STAT3 (WTSTAT3) or STAT3 missing the first 132 amino acids (ΔNST3) were analyzed by SDS-PAGE followed by immunoblotting using antibodies against STAT3, SDHA, UBQCR2 and Tubulin. SE: short exposure. LE: long exposure.

Article Snippet: Unless otherwise specified, all antibodies and detection reagents were used at 1:2000 dilution prepared in 5 % BSA in TBST: STAT3-alpha (D1A5) (CST, Danvers, MA, USA, catalog no. 8768S), STAT3 (124H6) (CST, catalog no. 9139S), pS727-STAT3 (CST, catalog no. 9134) NDUFV2 (ABclonal, Woburn, MA, USA, catalog no. A7442), NDUFS2 (ABclonal, catalog no. A12858), SDHA (ABclonal, catalog no. A2594), Tubulin (Sigma, catalog no. 26628228), ERK1 (ABclonal catalog no. A0228), ATP5A1 (ABclonal catalog no. A5884), COXVa (ABclonal catalog no. A6437), ATP5B (Abclonal catalog no. A5769), PDE (Abclonal catalog no. A20888), Cytc1 (ABclonal catalog no. A10449), TFAm (ABclonal catalog no. A1926), Tom20 (ABclonal catalog no. A18047), Flag (Sigma-Aldrich catalog no. F1804), UBQCR2 (ABclonal catalog no. A4181), COX4I1 (ABclonal catalog no. A6564), CalR (ABclonal catalog no. 1066).

Techniques: Recombinant, Construct, Expressing, Immunoprecipitation, Generated, Purification, SDS Page, Western Blot

Journal: Mitochondrial communications

Article Title: Structural determinants of mitochondrial STAT3 targeting and function

doi: 10.1016/j.mitoco.2024.01.001

Figure Lengend Snippet: (A) Graphic representation of recombinant STAT3 constructs. Mitochondrial extracts from MDA-MB-231 cells expressing MTS.STAT3.Bio (B) or MTS.TruncSTAT3.Bio (C) were immunoprecipitated with anti-Flag antibodies and immunoblotted with either STAT3 or Flag antibodies as indicated. Asterix (*) marks endogenous STAT3. (D) Cytosolic (Cytosol) or mitochondrial (Mitochondria) extracts generated by 2 distinct purification protocols from HEK293T cells expressing either full length STAT3 (WTSTAT3) or STAT3 missing the first 132 amino acids (ΔNST3) were analyzed by SDS-PAGE followed by immunoblotting using antibodies against STAT3, SDHA, UBQCR2 and Tubulin. SE: short exposure. LE: long exposure.

Article Snippet: Unless otherwise specified, all antibodies and detection reagents were used at 1:2000 dilution prepared in 5 % BSA in TBST: STAT3-alpha (D1A5) (CST, Danvers, MA, USA, catalog no. 8768S), STAT3 (124H6) (CST, catalog no. 9139S), pS727-STAT3 (CST, catalog no. 9134) NDUFV2 (ABclonal, Woburn, MA, USA, catalog no. A7442), NDUFS2 (ABclonal, catalog no. A12858), SDHA (ABclonal, catalog no. A2594), Tubulin (Sigma, catalog no. 26628228), ERK1 (ABclonal catalog no. A0228), ATP5A1 (ABclonal catalog no. A5884), COXVa (ABclonal catalog no. A6437), ATP5B (Abclonal catalog no. A5769), PDE (Abclonal catalog no. A20888), Cytc1 (ABclonal catalog no. A10449), TFAm (ABclonal catalog no. A1926), Tom20 (ABclonal catalog no. A18047), Flag (Sigma-Aldrich catalog no. F1804), UBQCR2 (ABclonal catalog no. A4181), COX4I1 (ABclonal catalog no. A6564), CalR (ABclonal catalog no. 1066).

Techniques: Recombinant, Construct, Expressing, Immunoprecipitation, Generated, Purification, SDS Page, Western Blot

Journal: Mitochondrial communications

Article Title: Structural determinants of mitochondrial STAT3 targeting and function

doi: 10.1016/j.mitoco.2024.01.001

Figure Lengend Snippet: Volcano plot of protein abundance found respectively in NP-40 extracted fraction (NP-40) and SDS-extracted fraction (SDS). Arrow indicates STAT3. Reactome 2022 pathway analysis of proteins enriched in the NP-40 fractions (B) or SDS fractions (C).

Article Snippet: Unless otherwise specified, all antibodies and detection reagents were used at 1:2000 dilution prepared in 5 % BSA in TBST: STAT3-alpha (D1A5) (CST, Danvers, MA, USA, catalog no. 8768S), STAT3 (124H6) (CST, catalog no. 9139S), pS727-STAT3 (CST, catalog no. 9134) NDUFV2 (ABclonal, Woburn, MA, USA, catalog no. A7442), NDUFS2 (ABclonal, catalog no. A12858), SDHA (ABclonal, catalog no. A2594), Tubulin (Sigma, catalog no. 26628228), ERK1 (ABclonal catalog no. A0228), ATP5A1 (ABclonal catalog no. A5884), COXVa (ABclonal catalog no. A6437), ATP5B (Abclonal catalog no. A5769), PDE (Abclonal catalog no. A20888), Cytc1 (ABclonal catalog no. A10449), TFAm (ABclonal catalog no. A1926), Tom20 (ABclonal catalog no. A18047), Flag (Sigma-Aldrich catalog no. F1804), UBQCR2 (ABclonal catalog no. A4181), COX4I1 (ABclonal catalog no. A6564), CalR (ABclonal catalog no. 1066).

Techniques: Quantitative Proteomics

Journal: Mitochondrial communications

Article Title: Structural determinants of mitochondrial STAT3 targeting and function

doi: 10.1016/j.mitoco.2024.01.001

Figure Lengend Snippet: (A) Graphic representation of recombinant STAT3 constructs. Mitochondrial extracts from MDA-MB-231 cells expressing MTS.STAT3.Bio (B) or MTS.TruncSTAT3.Bio (C) were immunoprecipitated with anti-Flag antibodies and immunoblotted with either STAT3 or Flag antibodies as indicated. Asterix (*) marks endogenous STAT3. (D) Cytosolic (Cytosol) or mitochondrial (Mitochondria) extracts generated by 2 distinct purification protocols from HEK293T cells expressing either full length STAT3 (WTSTAT3) or STAT3 missing the first 132 amino acids (ΔNST3) were analyzed by SDS-PAGE followed by immunoblotting using antibodies against STAT3, SDHA, UBQCR2 and Tubulin. SE: short exposure. LE: long exposure.

Article Snippet: Unless otherwise specified, all antibodies and detection reagents were used at 1:2000 dilution prepared in 5 % BSA in TBST: STAT3-alpha (D1A5) (CST, Danvers, MA, USA, catalog no. 8768S), STAT3 (124H6) (CST, catalog no. 9139S), pS727-STAT3 (CST, catalog no. 9134) NDUFV2 (ABclonal, Woburn, MA, USA, catalog no. A7442), NDUFS2 (ABclonal, catalog no. A12858), SDHA (ABclonal, catalog no. A2594), Tubulin (Sigma, catalog no. 26628228), ERK1 (ABclonal catalog no. A0228), ATP5A1 (ABclonal catalog no. A5884), COXVa (ABclonal catalog no. A6437), ATP5B (Abclonal catalog no. A5769), PDE (Abclonal catalog no. A20888), Cytc1 (ABclonal catalog no. A10449), TFAm (ABclonal catalog no. A1926), Tom20 (ABclonal catalog no. A18047), Flag (Sigma-Aldrich catalog no. F1804), UBQCR2 (ABclonal catalog no. A4181), COX4I1 (ABclonal catalog no. A6564), CalR (ABclonal catalog no. 1066).

Techniques: Recombinant, Construct, Expressing, Immunoprecipitation, Generated, Purification, SDS Page, Western Blot

Journal: Mitochondrial communications

Article Title: Structural determinants of mitochondrial STAT3 targeting and function

doi: 10.1016/j.mitoco.2024.01.001

Figure Lengend Snippet: In vitro translated 35 S-labeled GFP, STAT3, Slc and Slc-GFP were incubated with freshly prepared mitochondria. Input fractions (I) were resolved on SDS-page alongside the mitochondria imported fraction (M) to probe mitochondrial import efficiency. (B) Graphic map of the different functional domains of STAT3 (upper panel). Description of STAT3 linker domain mutants M1 to M5. Residues highlighted in red were mutated to alanine (lower panel). (C) In vitro mitochondrial import of STAT3 domains F1 to F6 fused to GFP (as described in A). (D) In vitro mitochondrial import of mutant M1 to M5 (as described in B) of linker domain F4 fused to GFP (as described in A). Lanes 1–2 showing fragment F4 (delineated by a dotted line) is the same image as lanes 11–12 of panel C, reshown to allow direct comparison. (E) As in (D), in vitro mitochondrial export of F4-GFP compared to its N-terminal fragment (F4-GFP N-term) and C-terminal fragment (F4-GFP C-term).

Article Snippet: Unless otherwise specified, all antibodies and detection reagents were used at 1:2000 dilution prepared in 5 % BSA in TBST: STAT3-alpha (D1A5) (CST, Danvers, MA, USA, catalog no. 8768S), STAT3 (124H6) (CST, catalog no. 9139S), pS727-STAT3 (CST, catalog no. 9134) NDUFV2 (ABclonal, Woburn, MA, USA, catalog no. A7442), NDUFS2 (ABclonal, catalog no. A12858), SDHA (ABclonal, catalog no. A2594), Tubulin (Sigma, catalog no. 26628228), ERK1 (ABclonal catalog no. A0228), ATP5A1 (ABclonal catalog no. A5884), COXVa (ABclonal catalog no. A6437), ATP5B (Abclonal catalog no. A5769), PDE (Abclonal catalog no. A20888), Cytc1 (ABclonal catalog no. A10449), TFAm (ABclonal catalog no. A1926), Tom20 (ABclonal catalog no. A18047), Flag (Sigma-Aldrich catalog no. F1804), UBQCR2 (ABclonal catalog no. A4181), COX4I1 (ABclonal catalog no. A6564), CalR (ABclonal catalog no. 1066).

Techniques: In Vitro, Labeling, Incubation, SDS Page, Functional Assay, Mutagenesis, Comparison

Journal: Mitochondrial communications

Article Title: Structural determinants of mitochondrial STAT3 targeting and function

doi: 10.1016/j.mitoco.2024.01.001

Figure Lengend Snippet: (A) Cytoplasmic (CYTO) and mitochondrial extracts (MITO) as well as remaining residual pellet (PEL) of MDA-MB-231 cells deficient for endogenous STAT3 but ectopically expressing WT STAT3 (WT) or STAT3 mutated on C687 (C687S) or STAT3 doubly mutated on C687 and C712 (C687/C712S) were analyzed by SDS-PAGE and western blotting and probed with antibodies against STAT3, Cox4l1 and calreticulin (CalR). (B) Quantification of the STAT3 target gene SOCS3 mRNA levels by RT-qPCR normalized to B2M mRNA levels in STAT3 WT cells (WT), STAT3 KO cells (KO), STAT3 KO cells ectopically expressing either WT STAT3 (KO + WT) or STAT3 carrying the double mutation C6787S/C712S (KO+2CS). (C) Complex I activity of the set of cell lines described in B.

Article Snippet: Unless otherwise specified, all antibodies and detection reagents were used at 1:2000 dilution prepared in 5 % BSA in TBST: STAT3-alpha (D1A5) (CST, Danvers, MA, USA, catalog no. 8768S), STAT3 (124H6) (CST, catalog no. 9139S), pS727-STAT3 (CST, catalog no. 9134) NDUFV2 (ABclonal, Woburn, MA, USA, catalog no. A7442), NDUFS2 (ABclonal, catalog no. A12858), SDHA (ABclonal, catalog no. A2594), Tubulin (Sigma, catalog no. 26628228), ERK1 (ABclonal catalog no. A0228), ATP5A1 (ABclonal catalog no. A5884), COXVa (ABclonal catalog no. A6437), ATP5B (Abclonal catalog no. A5769), PDE (Abclonal catalog no. A20888), Cytc1 (ABclonal catalog no. A10449), TFAm (ABclonal catalog no. A1926), Tom20 (ABclonal catalog no. A18047), Flag (Sigma-Aldrich catalog no. F1804), UBQCR2 (ABclonal catalog no. A4181), COX4I1 (ABclonal catalog no. A6564), CalR (ABclonal catalog no. 1066).

Techniques: Expressing, SDS Page, Western Blot, Quantitative RT-PCR, Mutagenesis, Activity Assay

Journal: Mitochondrial communications

Article Title: Structural determinants of mitochondrial STAT3 targeting and function

doi: 10.1016/j.mitoco.2024.01.001

Figure Lengend Snippet: (A) Cytoplasmic (CytoSTAT3) and mitochondrial (MitoSTAT3) protein extracts from MDA-MB-231 cells were separated by isoelectric focusing followed by SDS-PAGE and then analyzed by immunoblotting for STAT3. (B) MDA-MB-231 cells either carrying WT STAT3 (WT) or deleted for STAT3 (KO) were treated with Calyculin A (CalA) or left untreated. Cytoplasmic (cyto) and mitochondrial (mito) extracts were separated on SDS-PAGE and probed for pS727-STAT3, total STAT3 (STAT3) or NDUFV2 by western blot. (C) Cytoplasmic and mitochondrial extracts from livers of WT mice (WT) and mice homozygous for a knock-in mutant of STAT3 where serine727 is replaced by alanine (SA) were analyzed by SDS-PAGE and immunoblotting using antibodies against STAT3, F1 ATPase and ERK1.

Article Snippet: Unless otherwise specified, all antibodies and detection reagents were used at 1:2000 dilution prepared in 5 % BSA in TBST: STAT3-alpha (D1A5) (CST, Danvers, MA, USA, catalog no. 8768S), STAT3 (124H6) (CST, catalog no. 9139S), pS727-STAT3 (CST, catalog no. 9134) NDUFV2 (ABclonal, Woburn, MA, USA, catalog no. A7442), NDUFS2 (ABclonal, catalog no. A12858), SDHA (ABclonal, catalog no. A2594), Tubulin (Sigma, catalog no. 26628228), ERK1 (ABclonal catalog no. A0228), ATP5A1 (ABclonal catalog no. A5884), COXVa (ABclonal catalog no. A6437), ATP5B (Abclonal catalog no. A5769), PDE (Abclonal catalog no. A20888), Cytc1 (ABclonal catalog no. A10449), TFAm (ABclonal catalog no. A1926), Tom20 (ABclonal catalog no. A18047), Flag (Sigma-Aldrich catalog no. F1804), UBQCR2 (ABclonal catalog no. A4181), COX4I1 (ABclonal catalog no. A6564), CalR (ABclonal catalog no. 1066).

Techniques: SDS Page, Western Blot, Knock-In, Mutagenesis

Journal: Mitochondrial communications

Article Title: Structural determinants of mitochondrial STAT3 targeting and function

doi: 10.1016/j.mitoco.2024.01.001

Figure Lengend Snippet: Volcano plot of protein abundance found respectively in NP-40 extracted fraction (NP-40) and SDS-extracted fraction (SDS). Arrow indicates STAT3. Reactome 2022 pathway analysis of proteins enriched in the NP-40 fractions (B) or SDS fractions (C).

Article Snippet: Unless otherwise specified, all antibodies and detection reagents were used at 1:2000 dilution prepared in 5 % BSA in TBST: STAT3-alpha (D1A5) (CST, Danvers, MA, USA, catalog no. 8768S), STAT3 (124H6) (CST, catalog no. 9139S), pS727-STAT3 (CST, catalog no. 9134) NDUFV2 (ABclonal, Woburn, MA, USA, catalog no. A7442), NDUFS2 (ABclonal, catalog no. A12858), SDHA (ABclonal, catalog no. A2594), Tubulin (Sigma, catalog no. 26628228), ERK1 (ABclonal catalog no. A0228), ATP5A1 (ABclonal catalog no. A5884), COXVa (ABclonal catalog no. A6437), ATP5B (Abclonal catalog no. A5769), PDE (Abclonal catalog no. A20888), Cytc1 (ABclonal catalog no. A10449), TFAm (ABclonal catalog no. A1926), Tom20 (ABclonal catalog no. A18047), Flag (Sigma-Aldrich catalog no. F1804), UBQCR2 (ABclonal catalog no. A4181), COX4I1 (ABclonal catalog no. A6564), CalR (ABclonal catalog no. 1066).

Techniques:

Journal: Mitochondrial communications

Article Title: Structural determinants of mitochondrial STAT3 targeting and function

doi: 10.1016/j.mitoco.2024.01.001

Figure Lengend Snippet: (A) Graphic representation of recombinant STAT3 constructs. Mitochondrial extracts from MDA-MB-231 cells expressing MTS.STAT3.Bio (B) or MTS.TruncSTAT3.Bio (C) were immunoprecipitated with anti-Flag antibodies and immunoblotted with either STAT3 or Flag antibodies as indicated. Asterix (*) marks endogenous STAT3. (D) Cytosolic (Cytosol) or mitochondrial (Mitochondria) extracts generated by 2 distinct purification protocols from HEK293T cells expressing either full length STAT3 (WTSTAT3) or STAT3 missing the first 132 amino acids (ΔNST3) were analyzed by SDS-PAGE followed by immunoblotting using antibodies against STAT3, SDHA, UBQCR2 and Tubulin. SE: short exposure. LE: long exposure.

Article Snippet: Unless otherwise specified, all antibodies and detection reagents were used at 1:2000 dilution prepared in 5 % BSA in TBST: STAT3-alpha (D1A5) (CST, Danvers, MA, USA, catalog no. 8768S), STAT3 (124H6) (CST, catalog no. 9139S), pS727-STAT3 (CST, catalog no. 9134) NDUFV2 (ABclonal, Woburn, MA, USA, catalog no. A7442), NDUFS2 (ABclonal, catalog no. A12858), SDHA (ABclonal, catalog no. A2594), Tubulin (Sigma, catalog no. 26628228), ERK1 (ABclonal catalog no. A0228), ATP5A1 (ABclonal catalog no. A5884), COXVa (ABclonal catalog no. A6437), ATP5B (Abclonal catalog no. A5769), PDE (Abclonal catalog no. A20888), Cytc1 (ABclonal catalog no. A10449), TFAm (ABclonal catalog no. A1926), Tom20 (ABclonal catalog no. A18047), Flag (Sigma-Aldrich catalog no. F1804), UBQCR2 (ABclonal catalog no. A4181), COX4I1 (ABclonal catalog no. A6564), CalR (ABclonal catalog no. 1066).

Techniques: Recombinant, Construct, Expressing, Immunoprecipitation, Generated, Purification, SDS Page, Western Blot

Journal: Mitochondrial communications

Article Title: Structural determinants of mitochondrial STAT3 targeting and function

doi: 10.1016/j.mitoco.2024.01.001

Figure Lengend Snippet: In vitro translated 35 S-labeled GFP, STAT3, Slc and Slc-GFP were incubated with freshly prepared mitochondria. Input fractions (I) were resolved on SDS-page alongside the mitochondria imported fraction (M) to probe mitochondrial import efficiency. (B) Graphic map of the different functional domains of STAT3 (upper panel). Description of STAT3 linker domain mutants M1 to M5. Residues highlighted in red were mutated to alanine (lower panel). (C) In vitro mitochondrial import of STAT3 domains F1 to F6 fused to GFP (as described in A). (D) In vitro mitochondrial import of mutant M1 to M5 (as described in B) of linker domain F4 fused to GFP (as described in A). Lanes 1–2 showing fragment F4 (delineated by a dotted line) is the same image as lanes 11–12 of panel C, reshown to allow direct comparison. (E) As in (D), in vitro mitochondrial export of F4-GFP compared to its N-terminal fragment (F4-GFP N-term) and C-terminal fragment (F4-GFP C-term).

Article Snippet: Unless otherwise specified, all antibodies and detection reagents were used at 1:2000 dilution prepared in 5 % BSA in TBST: STAT3-alpha (D1A5) (CST, Danvers, MA, USA, catalog no. 8768S), STAT3 (124H6) (CST, catalog no. 9139S), pS727-STAT3 (CST, catalog no. 9134) NDUFV2 (ABclonal, Woburn, MA, USA, catalog no. A7442), NDUFS2 (ABclonal, catalog no. A12858), SDHA (ABclonal, catalog no. A2594), Tubulin (Sigma, catalog no. 26628228), ERK1 (ABclonal catalog no. A0228), ATP5A1 (ABclonal catalog no. A5884), COXVa (ABclonal catalog no. A6437), ATP5B (Abclonal catalog no. A5769), PDE (Abclonal catalog no. A20888), Cytc1 (ABclonal catalog no. A10449), TFAm (ABclonal catalog no. A1926), Tom20 (ABclonal catalog no. A18047), Flag (Sigma-Aldrich catalog no. F1804), UBQCR2 (ABclonal catalog no. A4181), COX4I1 (ABclonal catalog no. A6564), CalR (ABclonal catalog no. 1066).

Techniques: In Vitro, Labeling, Incubation, SDS Page, Functional Assay, Mutagenesis, Comparison

Journal: Mitochondrial communications

Article Title: Structural determinants of mitochondrial STAT3 targeting and function

doi: 10.1016/j.mitoco.2024.01.001

Figure Lengend Snippet: (A) Cytoplasmic (CYTO) and mitochondrial extracts (MITO) as well as remaining residual pellet (PEL) of MDA-MB-231 cells deficient for endogenous STAT3 but ectopically expressing WT STAT3 (WT) or STAT3 mutated on C687 (C687S) or STAT3 doubly mutated on C687 and C712 (C687/C712S) were analyzed by SDS-PAGE and western blotting and probed with antibodies against STAT3, Cox4l1 and calreticulin (CalR). (B) Quantification of the STAT3 target gene SOCS3 mRNA levels by RT-qPCR normalized to B2M mRNA levels in STAT3 WT cells (WT), STAT3 KO cells (KO), STAT3 KO cells ectopically expressing either WT STAT3 (KO + WT) or STAT3 carrying the double mutation C6787S/C712S (KO+2CS). (C) Complex I activity of the set of cell lines described in B.

Article Snippet: Unless otherwise specified, all antibodies and detection reagents were used at 1:2000 dilution prepared in 5 % BSA in TBST: STAT3-alpha (D1A5) (CST, Danvers, MA, USA, catalog no. 8768S), STAT3 (124H6) (CST, catalog no. 9139S), pS727-STAT3 (CST, catalog no. 9134) NDUFV2 (ABclonal, Woburn, MA, USA, catalog no. A7442), NDUFS2 (ABclonal, catalog no. A12858), SDHA (ABclonal, catalog no. A2594), Tubulin (Sigma, catalog no. 26628228), ERK1 (ABclonal catalog no. A0228), ATP5A1 (ABclonal catalog no. A5884), COXVa (ABclonal catalog no. A6437), ATP5B (Abclonal catalog no. A5769), PDE (Abclonal catalog no. A20888), Cytc1 (ABclonal catalog no. A10449), TFAm (ABclonal catalog no. A1926), Tom20 (ABclonal catalog no. A18047), Flag (Sigma-Aldrich catalog no. F1804), UBQCR2 (ABclonal catalog no. A4181), COX4I1 (ABclonal catalog no. A6564), CalR (ABclonal catalog no. 1066).

Techniques: Expressing, SDS Page, Western Blot, Quantitative RT-PCR, Mutagenesis, Activity Assay

Journal: Mitochondrial communications

Article Title: Structural determinants of mitochondrial STAT3 targeting and function

doi: 10.1016/j.mitoco.2024.01.001

Figure Lengend Snippet: (A) Cytoplasmic (CytoSTAT3) and mitochondrial (MitoSTAT3) protein extracts from MDA-MB-231 cells were separated by isoelectric focusing followed by SDS-PAGE and then analyzed by immunoblotting for STAT3. (B) MDA-MB-231 cells either carrying WT STAT3 (WT) or deleted for STAT3 (KO) were treated with Calyculin A (CalA) or left untreated. Cytoplasmic (cyto) and mitochondrial (mito) extracts were separated on SDS-PAGE and probed for pS727-STAT3, total STAT3 (STAT3) or NDUFV2 by western blot. (C) Cytoplasmic and mitochondrial extracts from livers of WT mice (WT) and mice homozygous for a knock-in mutant of STAT3 where serine727 is replaced by alanine (SA) were analyzed by SDS-PAGE and immunoblotting using antibodies against STAT3, F1 ATPase and ERK1.

Article Snippet: Unless otherwise specified, all antibodies and detection reagents were used at 1:2000 dilution prepared in 5 % BSA in TBST: STAT3-alpha (D1A5) (CST, Danvers, MA, USA, catalog no. 8768S), STAT3 (124H6) (CST, catalog no. 9139S), pS727-STAT3 (CST, catalog no. 9134) NDUFV2 (ABclonal, Woburn, MA, USA, catalog no. A7442), NDUFS2 (ABclonal, catalog no. A12858), SDHA (ABclonal, catalog no. A2594), Tubulin (Sigma, catalog no. 26628228), ERK1 (ABclonal catalog no. A0228), ATP5A1 (ABclonal catalog no. A5884), COXVa (ABclonal catalog no. A6437), ATP5B (Abclonal catalog no. A5769), PDE (Abclonal catalog no. A20888), Cytc1 (ABclonal catalog no. A10449), TFAm (ABclonal catalog no. A1926), Tom20 (ABclonal catalog no. A18047), Flag (Sigma-Aldrich catalog no. F1804), UBQCR2 (ABclonal catalog no. A4181), COX4I1 (ABclonal catalog no. A6564), CalR (ABclonal catalog no. 1066).

Techniques: SDS Page, Western Blot, Knock-In, Mutagenesis