Journal: Journal of Ginseng Research

Article Title: Rg1-R1 attenuates cardiac ischemia/reperfusion-induced endothelial cell injury through activating the ULK1/PGAM5-FUNDC1-mitophagy pathway

doi: 10.1016/j.jgr.2025.100973

Figure Lengend Snippet: Rg1-R1 alleviates H/R-induced endothelial cell mitochondrial dysfunction by culturing and isolating CMECs. (A) Changes in mitochondria and autophagic vesicles of endothelial cells in each group were observed using transmission electron microscopy. Red arrows indicate mitochondria and yellow arrows indicate mitochondria wrapped by autophagic lysosomes. (B) OCR measurement was used to observe cellular respiratory function and the production of ATP with or without Rg1-R1 treatment. (C) Mitochondria ROS were gauged by Mitosox™Red and cytoplasmic ROS were gauged by H2DCF-DA. (D) JC-1 was utilised to monitor the membrane potential of the mitochondria. (E) The openness of mPTP was determined based on the intensity of green fluorescence of calcein acetoxymethyl esterin mitochondria. (F) Expression of p-Drp1, Mff, Fis1, Mfn2, and Opa1 was examined by Western blot. Experiments were repeated at least three times and data are shown asmean ± SD (Three independent cellular alleles per group). ∗∗p < 0.01, ∗p < 0.05.

Article Snippet: After blockade treatment then incubated overnight with specific primary antibodies against ET-1(ab2786, Abcam, USA), e-NOS(#32027, CST, USA), Mitofusin-2 (#9482, CST, USA), OPA1 (#80471, CST, USA), Fis1 (#32525, CST, USA), p-DRP1(#4494, CST, USA), DRP1(#8570, CST, USA), FUNDC1(#49240, CST, USA), P-FUNDC1(PA5-114576, Invitrogen, China), P62 (ab109012, Abcam, USA), LC3B (#3868, CST, USA), ULK1(#8054, CST, USA), PGAM5(#63454, CST, USA), BNIP3(68091-1-Ig, Proteintech, China), BNIP3L(12986-1-AP, Proteintech, China), GAPDH (10494-1-AP, Proteintech, China).

Techniques: Transmission Assay, Electron Microscopy, Membrane, Fluorescence, Expressing, Western Blot

Journal: American journal of physiology. Heart and circulatory physiology

Article Title: High-fat, low-carbohydrate diet promotes arrhythmic death and increases myocardial ischemia-reperfusion injury in rats.

doi: 10.1152/ajpheart.00058.2014

Figure Lengend Snippet: Fig. 6. Effect of HFLCD on key mitochondrial fusion and fission regulatory proteins. A: real-time PCR measurement of mitofusin (Mfn)1, Mfn2, optic atrophy 1 (Opa1), fission1 (Fis1), and dynamin-related protein 1 (Drp1) transcript in hearts following I/R. B: Western blot analyses of Mfn1, Mfn2, Opa1, Fis1 and Drp1 from hearts following I/R. Values are means SE, n 4 to 5 each group, *P 0.05 vs. CONT.

Article Snippet: Western blots were conducted using commercial antibodies: carnitine palmitoyltransferase (CPT1B, CPT2), uncoupling proteins (UCP2, UCP3) (Alpha Diagnostic, San Antonio, TX); ATP synthase (MitoSciences, Eugene, OR); Mfn1, Opa1, GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA); Fis1, Drp1 (Thermo Fisher Scientific, Rockford, IL); Mfn2 (Abcam, Cambridge, MA); STIM1 (Cell Signaling Technology, Danvers, MA); and Orai1, Orai3 (ProSci, Poway, CA).

Techniques: Real-time Polymerase Chain Reaction, Western Blot

Journal: Cell death & disease

Article Title: SENP3-FIS1 axis promotes mitophagy and cell survival under hypoxia.

doi: 10.1038/s41419-024-07271-8

Figure Lengend Snippet: Fig. 3 Global deSUMOylation induces mitophagy and promotes HIM. A RNAi-mediated SUMO-2/3 depletion induces mitophagy and promotes HIM. HeLa cells expressing Mito-pHfluorin were transfected with Nsi or SUMO-2/3-specific siRNA (SUMO-2/3i). 48 h post-transfection the cells were exposed to normoxia or hypoxia (1% O2) for 24 h, and the cells were analysed 72 h post-transfection (Scale bar 10 µm). Histogram in the right panel shows average number of Mito-pHfluorin red puncta per cell for cells exposed to N or H for 24 h (n = 42–71, *p < 0.05; **p < 0.01; Ordinary One-way ANOVA followed by Sidak’s multiple comparisons test). B Global SUMOylation inhibition induces mitophagy and promotes HIM. HeLa cells were transfected with Mito-pHfluorin. 48 h post-transfection the cells were treated with DMSO or TAK-981 (100 nM) and exposed to normoxia or hypoxia (1% O2) for 24 h, and the cells were analysed 72 h post-transfection (Scale bar 10 µm). Histogram in the right panel shows relative mitophagy level per cell for cells treated with DMSO or TAK-981 under and exposed to N or H for 24 h (n = 62–81, *p < 0.05; ***p < 0.001; ****p < 0.0001; Ordinary One-way ANOVA by Sidak’s multiple comparisons test).

Article Snippet: DNA, siRNA or DNA & siRNA were transfected into HeLa cells, FIS1 KO HeLa cells or HEK293 using jetPRIME (Polyplus Transfection). siRNA duplexes Cell Death and Disease (2024) 15:881 used were as follows: non-specific siRNA (Eurofins Genomics), FIS1 siRNA used previously [23], FKBP8 siRNA (duplexes to target GAGUGGCUGGACAUUCUGG to silence FKBP8; synthesised by Eurofins Genomics), SENP1 siRNA (Santa Cruz sc-44449), SENP3 siRNA (Santa Cruz, sc-44451), SUMO1 siRNA (duplexes to target CCUUCAUAUUACCCUCUCC to silence SUMO1; synthesised by Eurofins Genomics), SUMO2/3 siRNA (duplexes to target GUCAAUGAGGCAGAUCAGA to silence SUMO2/3 [70]; synthesised by Eurofins Genomics), TBC1D15 siRNA (duplexes to target UCAACAAGAAGAACCAGG to silence TBC1D15; synthesised by Eurofins Genomics) and TBC1D17 siRNA (Santa Cruz sc-97889).

Techniques: Expressing, Transfection, Inhibition

Journal: Cell death & disease

Article Title: SENP3-FIS1 axis promotes mitophagy and cell survival under hypoxia.

doi: 10.1038/s41419-024-07271-8

Figure Lengend Snippet: Fig. 4 SENP3 plays an essential role in mitophagy induced by hypoxia. A RNAi-mediated SENP1 depletion does not appear to affect hypoxia-induced mitophagy in HeLa cells. HeLa cells expressing Mito-pHfluorin were transfected with Nsi or SENP1-specific siRNA (SENP1i). 48 h post-transfection the cells were exposed to normoxia or hypoxia (1% O2) for 24 h, and the cells were analysed 72 h post-transfection (Scale bar 10 µm). Histogram in the right panel shows relative mitophagy level per cell for cells exposed to N or H for 24 h (n = 32–55; N.S., non-significant; *p < 0.05; **p < 0.01; Ordinary One-way ANOVA by Sidak’s multiple comparisons test). B RNAi-mediated SENP3 depletion abolishes hypoxia-induced mitophagy in HeLa cells. HeLa cells expressing Mito-pHfluorin were transfected with Nsi or SENP3-specific siRNA (SENP3i). 48 h post-transfection the cells were exposed to normoxia or hypoxia (1% O2) for 24 h, and the cells were analysed 72 h post- transfection (Scale bar 10 µm). Histogram in the right panel shows average number of Mito-pHfluorin red puncta per cell for cells exposed to N or H for 24 h (n = 42–63, N.S., non-significant; *p < 0.05; ***p < 0.001; Ordinary One-way ANOVA by Sidak’s multiple comparisons test).

Article Snippet: DNA, siRNA or DNA & siRNA were transfected into HeLa cells, FIS1 KO HeLa cells or HEK293 using jetPRIME (Polyplus Transfection). siRNA duplexes Cell Death and Disease (2024) 15:881 used were as follows: non-specific siRNA (Eurofins Genomics), FIS1 siRNA used previously [23], FKBP8 siRNA (duplexes to target GAGUGGCUGGACAUUCUGG to silence FKBP8; synthesised by Eurofins Genomics), SENP1 siRNA (Santa Cruz sc-44449), SENP3 siRNA (Santa Cruz, sc-44451), SUMO1 siRNA (duplexes to target CCUUCAUAUUACCCUCUCC to silence SUMO1; synthesised by Eurofins Genomics), SUMO2/3 siRNA (duplexes to target GUCAAUGAGGCAGAUCAGA to silence SUMO2/3 [70]; synthesised by Eurofins Genomics), TBC1D15 siRNA (duplexes to target UCAACAAGAAGAACCAGG to silence TBC1D15; synthesised by Eurofins Genomics) and TBC1D17 siRNA (Santa Cruz sc-97889).

Techniques: Expressing, Transfection

Journal: Cell death & disease

Article Title: SENP3-FIS1 axis promotes mitophagy and cell survival under hypoxia.

doi: 10.1038/s41419-024-07271-8

Figure Lengend Snippet: Fig. 5 FIS1 is essential for mitophagy induced by hypoxia in HeLa cells. A Genetic depletion of FIS1 abolishes hypoxia-induced mitophagy. Wild-type (WT; FIS1+/+) or FIS1 knockout (KO; FIS1−/−) HeLa cells were transfected with Mito-pHfluorin. 48 h post-transfection the cells were exposed to normoxia or hypoxia (1% O2) for 24 h, and the cells were analysed 72 h post-transfection (Scale bar 10 µm). Histogram in the right panel shows relative mitophagy level per cell for cells exposed to N or H for 24 h (n = 33–64, N.S., non-significant; **p < 0.01; ****p < 0.0001; Ordinary one-way ANOVA followed by Sidak’s multiple comparisons test). B RNAi-mediated FIS1 depletion prevents hypoxia-induced mitophagy. HeLa cells were transfected with Mito-pHfluorin and Nsi or FIS1-specific siRNA, and the cells were analysed 72 h post-transfection (Scale bar 10 µm). Histogram in the right panel shows relative mitophagy level per cell for cells exposed to N or H for 24 h (n = 54–61, N.S., non-significant; ****p < 0.0001; Ordinary one-way ANOVA followed by Sidak’s multiple comparisons test).

Article Snippet: DNA, siRNA or DNA & siRNA were transfected into HeLa cells, FIS1 KO HeLa cells or HEK293 using jetPRIME (Polyplus Transfection). siRNA duplexes Cell Death and Disease (2024) 15:881 used were as follows: non-specific siRNA (Eurofins Genomics), FIS1 siRNA used previously [23], FKBP8 siRNA (duplexes to target GAGUGGCUGGACAUUCUGG to silence FKBP8; synthesised by Eurofins Genomics), SENP1 siRNA (Santa Cruz sc-44449), SENP3 siRNA (Santa Cruz, sc-44451), SUMO1 siRNA (duplexes to target CCUUCAUAUUACCCUCUCC to silence SUMO1; synthesised by Eurofins Genomics), SUMO2/3 siRNA (duplexes to target GUCAAUGAGGCAGAUCAGA to silence SUMO2/3 [70]; synthesised by Eurofins Genomics), TBC1D15 siRNA (duplexes to target UCAACAAGAAGAACCAGG to silence TBC1D15; synthesised by Eurofins Genomics) and TBC1D17 siRNA (Santa Cruz sc-97889).

Techniques: Knock-Out, Transfection

Journal: Cell death & disease

Article Title: SENP3-FIS1 axis promotes mitophagy and cell survival under hypoxia.

doi: 10.1038/s41419-024-07271-8

Figure Lengend Snippet: Fig. 6 SUMOylatable FIS1 is required for SENP3 regulation of mitophagy induced by hypoxia. A Expressing SUMOylation-deficient CFP- FIS1 K149R rescues hypoxia-induced mitophagy in SENP3-KD HeLa cells. HeLa cells expressing Mito-pHfluorin were transfected with Nsi or SENP3i (50 nM), together with CFP, CFP-FIS1 or CFP-FIS1 K149R. 48 h post-transfection the cells were exposed to hypoxia (1% O2) for 24 h, and the cells were analysed 72 h post-transfection (Scale bar 10 µm). Histogram in the right panel shows relative mitophagy level per cell for cells exposed to N or H for 24 h (n = 48-74; N.S., non-significant; ***p < 0.001 ****p < 0.0001; Ordinary one-way ANOVA followed by Sidak’s multiple comparisons test). B Expressing or CFP-FIS1-SUMO2ΔGG abolishes hypoxia-induced mitophagy. HeLa cells expressing Mito-pHfluorin were transfected CFP, CFP-FIS1 or CFP-FIS1-SUMO2ΔGG. 48 h post-transfection the cells were exposed to normoxia or hypoxia (1% O2) for 24 h. and the cells were analysed 72 h post-transfection (Scale bar 10 µm). Histogram in the right panel shows relative mitophagy level per cell for cells exposed to N or H for 24 h (n = 46–58; N.S., non-significant; *p < 0.05 **p < 0.01 ****p < 0.0001; Ordinary one-way ANOVA followed by Sidak’s multiple comparisons test).

Article Snippet: DNA, siRNA or DNA & siRNA were transfected into HeLa cells, FIS1 KO HeLa cells or HEK293 using jetPRIME (Polyplus Transfection). siRNA duplexes Cell Death and Disease (2024) 15:881 used were as follows: non-specific siRNA (Eurofins Genomics), FIS1 siRNA used previously [23], FKBP8 siRNA (duplexes to target GAGUGGCUGGACAUUCUGG to silence FKBP8; synthesised by Eurofins Genomics), SENP1 siRNA (Santa Cruz sc-44449), SENP3 siRNA (Santa Cruz, sc-44451), SUMO1 siRNA (duplexes to target CCUUCAUAUUACCCUCUCC to silence SUMO1; synthesised by Eurofins Genomics), SUMO2/3 siRNA (duplexes to target GUCAAUGAGGCAGAUCAGA to silence SUMO2/3 [70]; synthesised by Eurofins Genomics), TBC1D15 siRNA (duplexes to target UCAACAAGAAGAACCAGG to silence TBC1D15; synthesised by Eurofins Genomics) and TBC1D17 siRNA (Santa Cruz sc-97889).

Techniques: Expressing, Transfection

Journal: Cell death & disease

Article Title: SENP3-FIS1 axis promotes mitophagy and cell survival under hypoxia.

doi: 10.1038/s41419-024-07271-8

Figure Lengend Snippet: Fig. 7 FIS1 is required for TBC1D17 regulation of HIM. A RNAi-mediated TBC1D15 depletion does not affect hypoxia-induced mitophagy in HeLa cells. HeLa cells were transfected with Mito-pHfluorin and Nsi or TBC1D15-specific siRNA (TBC1D15i; 20 nM). 48 h post-transfection the cells were exposed to normoxia or hypoxia (1% O2) for 24 h, and the cells were analysed 72 h post-transfection (Scale bar 10 µm). Histogram in the right panel shows relative mitophagy level per cell for cells exposed to N or H for 24 h (n = 39–59, N.S., non-significant; ***p < 0.001; ****p < 0.0001; Ordinary one-way ANOVA followed by Sidak’s multiple comparisons test). B RNAi-mediated TBC1D17 depletion promotes hypoxia-induced mitophagy in HeLa cells. HeLa cells were transfected with Mito-pHfluorin and Nsi or TBC1D17-specific siRNA (TBC1D17i; 20 nM). 48 h post-transfection the cells were exposed to normoxia or hypoxia (1% O2) for 24 h, and the cells were analysed 72 h post- transfection (Scale bar 10 µm). Histogram in the right panel shows relative mitophagy level per cell for cells exposed to N or H for 24 h (n = 42–56, N.S. non-significant; *p < 0.05; ****p < 0.0001; Ordinary one-way ANOVA followed by Sidak’s multiple comparisons test). C HeLa cells expressing Mito-pHfluorin were transfected with Nsi, TBC1D17i (20 nM), and/or FIS1-specific siRNA (50 nM). 48 h post-transfection the cells were exposed to normoxia or hypoxia (1% O2) for 24 h, and the cells were analysed 72 h post-transfection (Scale bar 10 µm). Histogram in the right panel shows relative mitophagy level per cell for cells exposed to N or H for 24 h (n = 52–62; N.S., non-significant; *p < 0.05; **p < 0.01; Ordinary one-way ANOVA followed by Sidak’s multiple comparisons test). D Hypoxia does not appear to affect the colocalisation of TBC1D17 with FIS1 in the Nsi/Nsi cells shown in (C). Relative fluorescence intensity of each channel as points along the white lines shown in the lower graphs for normoxia and hypoxia, respectively (Scale bar 10 µm).

Article Snippet: DNA, siRNA or DNA & siRNA were transfected into HeLa cells, FIS1 KO HeLa cells or HEK293 using jetPRIME (Polyplus Transfection). siRNA duplexes Cell Death and Disease (2024) 15:881 used were as follows: non-specific siRNA (Eurofins Genomics), FIS1 siRNA used previously [23], FKBP8 siRNA (duplexes to target GAGUGGCUGGACAUUCUGG to silence FKBP8; synthesised by Eurofins Genomics), SENP1 siRNA (Santa Cruz sc-44449), SENP3 siRNA (Santa Cruz, sc-44451), SUMO1 siRNA (duplexes to target CCUUCAUAUUACCCUCUCC to silence SUMO1; synthesised by Eurofins Genomics), SUMO2/3 siRNA (duplexes to target GUCAAUGAGGCAGAUCAGA to silence SUMO2/3 [70]; synthesised by Eurofins Genomics), TBC1D15 siRNA (duplexes to target UCAACAAGAAGAACCAGG to silence TBC1D15; synthesised by Eurofins Genomics) and TBC1D17 siRNA (Santa Cruz sc-97889).

Techniques: Transfection, Expressing

Journal: Cell death & disease

Article Title: SENP3-FIS1 axis promotes mitophagy and cell survival under hypoxia.

doi: 10.1038/s41419-024-07271-8

Figure Lengend Snippet: Fig. 8 SUMOylatable FIS1 is required for FIS1-TBC1D17 interaction. A, B Hypoxia induces FIS1-TBC1D17 interaction. HeLa cells (A) or GSCs (B) were exposed to normoxia or hypoxia (1% O2) for 24 h. FIS1 was enriched through immunoprecipitation (IP). Lysate (input) and IP samples were immunoblotted as indicated. C TBC1D17 interacts with GST-FIS1 but not GST-FIS1 K149R mutant in FIS1 KO HeLa cells under hypoxia. FIS1 KO HeLa cells were transfected with GST, GST-FIS1 or GST-FIS1 K149R mutant. 48 h post-transfection the cells were exposed to normoxia or hypoxia (1% O2) for 24 h. GST-tagged proteins were enriched through GST-Pulldown (PD), Lysate (input) and GST-PD samples were immunoblotted as indicated. D SENP3 knockdown increases TBC1D17 interaction with GST-FIS1 in FIS1 KO HeLa cells exposed to hypoxia (1% O2) for 24 h. FIS1 KO HeLa cells expressing GST-FIS1 were transfected with Nsi or SENP3i. 48 h post-transfection the cells were lysed and GST- tagged proteins were enriched through GST-PD, Lysate (input) and GST-PD samples were immunoblotted as indicated. E Hypoxia induces FIS1 SUMO2/3-ylation in HeLa cells. HeLa cells were exposed to normoxia or hypoxia (1% O2) in the absence or presence of TAK981 (100 nM) for 24 h. FIS1 was enriched through IP. Lysate (input) and IP samples were immunoblotted as indicated. F Hypoxia reduces the levels of cytoplasmic SENP3 in HeLa cells. HeLa cells were exposed to normoxia or hypoxia (1% O2) for 24 h. Samples of whole cell lysate (WCL), cytoplasmic or nuclear fraction was prepared and blotted as indicated, SENP3 levels were normalised to β-Actin in WCL, α-Tubulin in the cytoplasmic fraction, and H2AX in the nuclear fraction (n = 5 biological replicates; N.S., not statistically significant; ***p < 0.001; Paired t-test).

Article Snippet: DNA, siRNA or DNA & siRNA were transfected into HeLa cells, FIS1 KO HeLa cells or HEK293 using jetPRIME (Polyplus Transfection). siRNA duplexes Cell Death and Disease (2024) 15:881 used were as follows: non-specific siRNA (Eurofins Genomics), FIS1 siRNA used previously [23], FKBP8 siRNA (duplexes to target GAGUGGCUGGACAUUCUGG to silence FKBP8; synthesised by Eurofins Genomics), SENP1 siRNA (Santa Cruz sc-44449), SENP3 siRNA (Santa Cruz, sc-44451), SUMO1 siRNA (duplexes to target CCUUCAUAUUACCCUCUCC to silence SUMO1; synthesised by Eurofins Genomics), SUMO2/3 siRNA (duplexes to target GUCAAUGAGGCAGAUCAGA to silence SUMO2/3 [70]; synthesised by Eurofins Genomics), TBC1D15 siRNA (duplexes to target UCAACAAGAAGAACCAGG to silence TBC1D15; synthesised by Eurofins Genomics) and TBC1D17 siRNA (Santa Cruz sc-97889).

Techniques: Immunoprecipitation, Mutagenesis, Transfection, Knockdown, Expressing

Journal: Cell death & disease

Article Title: SENP3-FIS1 axis promotes mitophagy and cell survival under hypoxia.

doi: 10.1038/s41419-024-07271-8

Figure Lengend Snippet: Fig. 10 Schematic representation of a proposed SUMO2/3-dependent mitophagy/cell survival pathway under hypoxia. In response to hypoxia, FIS1 SUMO2/3-ylation increases due to reduced cytoplasmic SENP3. SUMO2/3-ylated FIS1 interacts with TBC1D17. With the associated FIS1, TBC1D17 inhibits the levels of hypoxia-induced mitophagy. Moreover, FIS1 deSUMO2/3-ylation mediated by residual cytoplasmic SENP3 is essential for maintaining hypoxia-induced mitophagy for cell survival.

Article Snippet: DNA, siRNA or DNA & siRNA were transfected into HeLa cells, FIS1 KO HeLa cells or HEK293 using jetPRIME (Polyplus Transfection). siRNA duplexes Cell Death and Disease (2024) 15:881 used were as follows: non-specific siRNA (Eurofins Genomics), FIS1 siRNA used previously [23], FKBP8 siRNA (duplexes to target GAGUGGCUGGACAUUCUGG to silence FKBP8; synthesised by Eurofins Genomics), SENP1 siRNA (Santa Cruz sc-44449), SENP3 siRNA (Santa Cruz, sc-44451), SUMO1 siRNA (duplexes to target CCUUCAUAUUACCCUCUCC to silence SUMO1; synthesised by Eurofins Genomics), SUMO2/3 siRNA (duplexes to target GUCAAUGAGGCAGAUCAGA to silence SUMO2/3 [70]; synthesised by Eurofins Genomics), TBC1D15 siRNA (duplexes to target UCAACAAGAAGAACCAGG to silence TBC1D15; synthesised by Eurofins Genomics) and TBC1D17 siRNA (Santa Cruz sc-97889).

Techniques: