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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 <t>siRNA</t> (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).

Fis1 Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "SENP3-FIS1 axis promotes mitophagy and cell survival under hypoxia."

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

Journal: Cell death & disease

doi: 10.1038/s41419-024-07271-8

Figure Legend 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).

Techniques Used: Expressing, Transfection, Inhibition

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).

Figure Legend 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).

Techniques Used: Expressing, Transfection

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).

Figure Legend 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).

Techniques Used: Knock-Out, Transfection

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).

Figure Legend 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).

Techniques Used: Expressing, Transfection

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).

Figure Legend 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).

Techniques Used: Transfection, Expressing

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

Techniques Used: Immunoprecipitation, Mutagenesis, Transfection, Knockdown, Expressing

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.

Figure Legend 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.

Techniques Used:

Transfection:

Article Title: SENP3-FIS1 axis promotes mitophagy and cell survival under hypoxia.
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). .. Hypoxia treatment of the cells was performed within a SCI-tive hypoxia workstation (Baker Ruskinn) set to humidified 1% (v/v) O2, and 5% (v/v) CO2 at 37 °C.

Article Title: Fragmentation level determines mitochondrial damage response and subsequently the fate of cancer cells exposed to carbon ions.
Article Snippet: a Institute of Modern Physics, Chinese Academy of Sciences; Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences; Key Laboratory of Basic Research on Heavy Ion Radiation Application in Medicine, Lanzhou; University of Chinese Academy of Sciences, Beijing, China; Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba, Japan

Cell Culture:

Control:

Image Search Results

a Example of hybrid sequence of miR-379 in pink and Fis1 in blue. b Alignment of miR-379 and its target site in the Fis1 3′-UTR (miRanda; microRNA.org). c Enrichment of AGO2 IP-seq RNA reads at miR-379 target site in Fis1 3′-UTR in WT mouse mesangial cells (MMC), with notable reduction in miR-379KO MMC. Two independent samples (A and B) from WT MMC (WT-A-IP and WT-B-IP) and miR-379KO MMC (379KO-A-IP and, 379KO-B-IP) were examined. d Enrichment of miR-379 RNA in AGO-IP in MMC isolated from kidney glomeruli of WT mice and significant reduction in miR-379KO MMC ( n = 3/group). e Significant increase of Fis1 gene expression in miR-379KO MMC compared to WT MMC, suggesting Fis1 is a target of miR-379 ( n = 6/group). f Significant decrease of WT Fis1 3′-UTR luciferase reporter activity by transfection with miR-379 mimic oligonucleotide, compared to mutant Fis1 3′-UTR reporter under similar conditions, further supporting Fis1 3′-UTR to a true target of miR-379. g Enrichment of AGO2 IP-seq RNA reads at the 3′-UTR of Txn1 gene in WT MMC and its significant reduction in miR-379KO MMC. Two independent samples (A and B) from WT MMC (WT-A-IP and WT-B-IP) and miR-379KO MMC (379KO-A-IP and 379KO-B-IP) were examined. h Significant decrease of WT Txn1 3′-UTR luciferase reporter activity induced by miR-379 mimics, compared with no change in mutant Txn1 3′-UTR reporter by miR-379 mimics. NC, negative control mimic; miR-379, miR-379 mimic. i RT-qPCR validation of the expression of enriched candidate genes identified by AGO2 IP-seq. RNA expression of all eight candidate miR-379 targets tested was decreased in AGO2-IP from miR-379KO MMC compared to WT MMC. Rab14 , Snrpe , Tcea1 , and Hmgb1 were used as negative controls because their enrichments in AGO2 IP-seq were not significantly changed between miR-379KO and WT MMC. Each dot indicates one biological repeat. Statistical analyses for two groups were performed by Student’s t-test, and for multiple comparisons one-way ANOVA with Tukey’s post hoc test was used. * P < 0.05, ** P < 0.01. All data are presented as mean ± SEM.

Journal: Communications Biology

Article Title: miR-379 deletion ameliorates features of diabetic kidney disease by enhancing adaptive mitophagy via FIS1

doi: 10.1038/s42003-020-01516-w

Figure Lengend Snippet: a Example of hybrid sequence of miR-379 in pink and Fis1 in blue. b Alignment of miR-379 and its target site in the Fis1 3′-UTR (miRanda; microRNA.org). c Enrichment of AGO2 IP-seq RNA reads at miR-379 target site in Fis1 3′-UTR in WT mouse mesangial cells (MMC), with notable reduction in miR-379KO MMC. Two independent samples (A and B) from WT MMC (WT-A-IP and WT-B-IP) and miR-379KO MMC (379KO-A-IP and, 379KO-B-IP) were examined. d Enrichment of miR-379 RNA in AGO-IP in MMC isolated from kidney glomeruli of WT mice and significant reduction in miR-379KO MMC ( n = 3/group). e Significant increase of Fis1 gene expression in miR-379KO MMC compared to WT MMC, suggesting Fis1 is a target of miR-379 ( n = 6/group). f Significant decrease of WT Fis1 3′-UTR luciferase reporter activity by transfection with miR-379 mimic oligonucleotide, compared to mutant Fis1 3′-UTR reporter under similar conditions, further supporting Fis1 3′-UTR to a true target of miR-379. g Enrichment of AGO2 IP-seq RNA reads at the 3′-UTR of Txn1 gene in WT MMC and its significant reduction in miR-379KO MMC. Two independent samples (A and B) from WT MMC (WT-A-IP and WT-B-IP) and miR-379KO MMC (379KO-A-IP and 379KO-B-IP) were examined. h Significant decrease of WT Txn1 3′-UTR luciferase reporter activity induced by miR-379 mimics, compared with no change in mutant Txn1 3′-UTR reporter by miR-379 mimics. NC, negative control mimic; miR-379, miR-379 mimic. i RT-qPCR validation of the expression of enriched candidate genes identified by AGO2 IP-seq. RNA expression of all eight candidate miR-379 targets tested was decreased in AGO2-IP from miR-379KO MMC compared to WT MMC. Rab14 , Snrpe , Tcea1 , and Hmgb1 were used as negative controls because their enrichments in AGO2 IP-seq were not significantly changed between miR-379KO and WT MMC. Each dot indicates one biological repeat. Statistical analyses for two groups were performed by Student’s t-test, and for multiple comparisons one-way ANOVA with Tukey’s post hoc test was used. * P < 0.05, ** P < 0.01. All data are presented as mean ± SEM.

Article Snippet: Mouse Fis1 siRNAs (double-stranded oligos were obtained from Thermo Fisher Scientific, Inc. Non-targeting siRNA controls were obtained from Thermo Fisher Scientific, Inc. MMC were trypsinized and resuspended in Basic Nucleofection Solution at 1 × 10 7 /ml.

Techniques: Sequencing, Isolation, Gene Expression, Luciferase, Activity Assay, Transfection, Mutagenesis, Negative Control, Quantitative RT-PCR, Biomarker Discovery, Expressing, RNA Expression

a Seahorse XF Cell Mito Stress test for mitochondrial function at basal conditions, ATP production, and maximal respiration levels using MMC from WT or miR-379KO mice cultured with normal glucose (NG, 5.5 mM) or high glucose (HG, 25 mM) for 72 h. b Oxygen consumption rates (OCRs) were calculated in basal and spare respiratory capacity (SRC) levels in NG and HG condition (20,000 cells per well of 96-well assay plate). c Representative images of IHC staining to detect FIS1 expression (brown color) in negative control (NC) and Fis1 siRNA (si-Fis1)-transfected MMC from WT or miR-379KO mice cultured with NG or HG. d Bar graph quantifications showing significant reduction in FIS1 levels in si-Fis1-transfected NG- or HG-treated WT and miR-379KO MMC compared to NC, and significant decrease in FIS1 levels in HG-treated WT MMC but not miR-379KO MMC ( n = 15 cells/group). The in vitro experiments were performed with at least three biological replicates. One-way ANOVA with Tukey’s post hoc test for multiple comparisons. ** P < 0.01, **** P < 0.0001. All data are presented as mean ± SEM. NC, negative control siRNA. si-Fis1, Fis1 siRNA.

Journal: Communications Biology

Article Title: miR-379 deletion ameliorates features of diabetic kidney disease by enhancing adaptive mitophagy via FIS1

doi: 10.1038/s42003-020-01516-w

Figure Lengend Snippet: a Seahorse XF Cell Mito Stress test for mitochondrial function at basal conditions, ATP production, and maximal respiration levels using MMC from WT or miR-379KO mice cultured with normal glucose (NG, 5.5 mM) or high glucose (HG, 25 mM) for 72 h. b Oxygen consumption rates (OCRs) were calculated in basal and spare respiratory capacity (SRC) levels in NG and HG condition (20,000 cells per well of 96-well assay plate). c Representative images of IHC staining to detect FIS1 expression (brown color) in negative control (NC) and Fis1 siRNA (si-Fis1)-transfected MMC from WT or miR-379KO mice cultured with NG or HG. d Bar graph quantifications showing significant reduction in FIS1 levels in si-Fis1-transfected NG- or HG-treated WT and miR-379KO MMC compared to NC, and significant decrease in FIS1 levels in HG-treated WT MMC but not miR-379KO MMC ( n = 15 cells/group). The in vitro experiments were performed with at least three biological replicates. One-way ANOVA with Tukey’s post hoc test for multiple comparisons. ** P < 0.01, **** P < 0.0001. All data are presented as mean ± SEM. NC, negative control siRNA. si-Fis1, Fis1 siRNA.

Techniques: Cell Culture, Immunohistochemistry, Expressing, Negative Control, Transfection, In Vitro

a Representative images of WT and miR-379KO MMC transfected with the DsRed2-Mito-7 plasmid, which fluorescently labels mitochondria with red emission spectra (quantification in c ). WT and miR-379KO MMC treated with NC siRNA or Fis1 siRNA were transfected with DsRed2-Mito-7 reporter. Upper panel (with NC siRNA): in HG conditions (25 mM glucose), mitochondrial signal intensity (red fluorescence) was significantly reduced compared to NG (5.5 mM glucose) conditions in both WT and miR-379KO MMC, but to a lesser extent in miR-379KO MMC. Lower panel: with si-Fis1: WT-MMC cells treated with si-Fis1 show decreased intensity in mitochondrial fluorescent signals in NG and HG conditions. miR-379KO MMC with si-Fis1 under HG conditions depicted more significant changes than under NG conditions. The degree of reduction of fluorescence (mitochondrial quality) under HG conditions was much lower in miR-379KO MMC compared to WT MMC. b Representative images showing adaptive mitophagy in MMC examined by expressing the pCLBW-cox8-EGFP-mCherry reporter (quantification in d ). WT and miR-379KO MMC MMC treated with NC siRNA or Fis1 siRNA were transfected with pCLBW-cox8-EGFP-mCherry and then treated with HG (25 mM) or NG (5.5 mM) for 5 days at 37 °C and 5% CO 2 . Upper panels (with NC siRNA): adaptive mitophagy shows marked decrease in WT-MMC after 5 days of HG treatment (decrease in red mCherry fluorescence) but not in NG; lower panels (with si-Fis1): Fis1 siRNA significantly reduced mitophagy in WT MMC in NG and HG conditions, but no significant changes were detected in miR-379KO MMC even under HG conditions. Adaptive mitophagy was significantly reduced only in miR-379KO MMC treated with Fis1 siRNA in HG conditions relative to NC under NG conditions. c Bar graph quantification of DsRed2-Mito-7 staining data (shown in a ) based on analysis of integrated density ( n = 6 cells/group). d Bar graph of quantitative analysis of the number of red-only puncta per cell from data in b ( n = 3–8 cells/group). These in vitro experiments in MMC were performed with at least three biological replicates. One-way ANOVA with Tukey’s post hoc tests for multiple comparisons in panels c and d . * P < 0.05, ** P < 0.01, **** P < 0.0001. All data are presented as mean ± SEM. NC, negative control siRNA. si-Fis1, Fis1 siRNA. Scale bar, 50 µm.

Journal: Communications Biology

Article Title: miR-379 deletion ameliorates features of diabetic kidney disease by enhancing adaptive mitophagy via FIS1

doi: 10.1038/s42003-020-01516-w

Figure Lengend Snippet: a Representative images of WT and miR-379KO MMC transfected with the DsRed2-Mito-7 plasmid, which fluorescently labels mitochondria with red emission spectra (quantification in c ). WT and miR-379KO MMC treated with NC siRNA or Fis1 siRNA were transfected with DsRed2-Mito-7 reporter. Upper panel (with NC siRNA): in HG conditions (25 mM glucose), mitochondrial signal intensity (red fluorescence) was significantly reduced compared to NG (5.5 mM glucose) conditions in both WT and miR-379KO MMC, but to a lesser extent in miR-379KO MMC. Lower panel: with si-Fis1: WT-MMC cells treated with si-Fis1 show decreased intensity in mitochondrial fluorescent signals in NG and HG conditions. miR-379KO MMC with si-Fis1 under HG conditions depicted more significant changes than under NG conditions. The degree of reduction of fluorescence (mitochondrial quality) under HG conditions was much lower in miR-379KO MMC compared to WT MMC. b Representative images showing adaptive mitophagy in MMC examined by expressing the pCLBW-cox8-EGFP-mCherry reporter (quantification in d ). WT and miR-379KO MMC MMC treated with NC siRNA or Fis1 siRNA were transfected with pCLBW-cox8-EGFP-mCherry and then treated with HG (25 mM) or NG (5.5 mM) for 5 days at 37 °C and 5% CO 2 . Upper panels (with NC siRNA): adaptive mitophagy shows marked decrease in WT-MMC after 5 days of HG treatment (decrease in red mCherry fluorescence) but not in NG; lower panels (with si-Fis1): Fis1 siRNA significantly reduced mitophagy in WT MMC in NG and HG conditions, but no significant changes were detected in miR-379KO MMC even under HG conditions. Adaptive mitophagy was significantly reduced only in miR-379KO MMC treated with Fis1 siRNA in HG conditions relative to NC under NG conditions. c Bar graph quantification of DsRed2-Mito-7 staining data (shown in a ) based on analysis of integrated density ( n = 6 cells/group). d Bar graph of quantitative analysis of the number of red-only puncta per cell from data in b ( n = 3–8 cells/group). These in vitro experiments in MMC were performed with at least three biological replicates. One-way ANOVA with Tukey’s post hoc tests for multiple comparisons in panels c and d . * P < 0.05, ** P < 0.01, **** P < 0.0001. All data are presented as mean ± SEM. NC, negative control siRNA. si-Fis1, Fis1 siRNA. Scale bar, 50 µm.

Techniques: Transfection, Plasmid Preparation, Fluorescence, Expressing, Staining, In Vitro, Negative Control

a , b Expression of indicated miRNAs (miRs) in control and STZ-treated WT and miR-379KO mice at a , 6 and b , 24 weeks after diabetes onset ( n = 4–6/group). c Expression of glomerular Chop , an ER stress-responsive transcription factor ( n = 5–8/group, n = 6–8/group, and n = 5–6/group for 1, 6, and 24 weeks of diabetes, respectively). Results are expressed as fold over WT-Con, after normalization with internal control U6. d – f Glomerular expression of profibrotic genes, Tgf-β1 , Col1a2 , Col4a1 , Ctgf , and Fn1 at 1, 6, and 24 weeks ( d – f ) after diabetes onset ( n = 5–8/group, n = 5–8/group, and n = 5–6/group for 1, 6, and 24 weeks of diabetes, respectively). g – i Glomerular expression of miR-379 target genes. g Edem3 , h Fis1 , and i Txn1 were measured in WT and miR-379KO mice ( n = 5–8/group, n = 6–8/group, and n = 5–6/group at 1, 6, and 24 weeks of diabetes, respectively). Results are expressed as fold over WT-Con, after normalization with internal control Cypa . Each dot indicates the value from each mouse. Statistical analyses were performed by one-way ANOVA with post hoc Tukey’s test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All data are presented as mean ± SEM.

Journal: Communications Biology

Article Title: miR-379 deletion ameliorates features of diabetic kidney disease by enhancing adaptive mitophagy via FIS1

doi: 10.1038/s42003-020-01516-w

Figure Lengend Snippet: a , b Expression of indicated miRNAs (miRs) in control and STZ-treated WT and miR-379KO mice at a , 6 and b , 24 weeks after diabetes onset ( n = 4–6/group). c Expression of glomerular Chop , an ER stress-responsive transcription factor ( n = 5–8/group, n = 6–8/group, and n = 5–6/group for 1, 6, and 24 weeks of diabetes, respectively). Results are expressed as fold over WT-Con, after normalization with internal control U6. d – f Glomerular expression of profibrotic genes, Tgf-β1 , Col1a2 , Col4a1 , Ctgf , and Fn1 at 1, 6, and 24 weeks ( d – f ) after diabetes onset ( n = 5–8/group, n = 5–8/group, and n = 5–6/group for 1, 6, and 24 weeks of diabetes, respectively). g – i Glomerular expression of miR-379 target genes. g Edem3 , h Fis1 , and i Txn1 were measured in WT and miR-379KO mice ( n = 5–8/group, n = 6–8/group, and n = 5–6/group at 1, 6, and 24 weeks of diabetes, respectively). Results are expressed as fold over WT-Con, after normalization with internal control Cypa . Each dot indicates the value from each mouse. Statistical analyses were performed by one-way ANOVA with post hoc Tukey’s test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All data are presented as mean ± SEM.

Techniques: Expressing, Control

a , b EDEM3 ( n = 32 and 50 glomeruli/group for 6 and 24 weeks, respectively), c , d FIS1 ( n = 50 glomeruli/group), e , f TXN1 ( n = 30 and 50 glomeruli/group for 6 and 24 weeks, respectively), and g , h PGC-1a ( n = 50 glomeruli/group) protein in kidney cortex sections from WT and miR-379KO mice at 6 and 24 weeks after diabetes onset. Scale bar, 50 µm. Bar graph results are expressed as fold over WT-Con. Statistical analyses were performed by one-way ANOVA with post hoc Tukey’s test for multiple comparisons. * P < 0.05, ** P < 0.01, **** P < 0.0001. All data are presented as mean ± SEM.

Journal: Communications Biology

Article Title: miR-379 deletion ameliorates features of diabetic kidney disease by enhancing adaptive mitophagy via FIS1

doi: 10.1038/s42003-020-01516-w

Figure Lengend Snippet: a , b EDEM3 ( n = 32 and 50 glomeruli/group for 6 and 24 weeks, respectively), c , d FIS1 ( n = 50 glomeruli/group), e , f TXN1 ( n = 30 and 50 glomeruli/group for 6 and 24 weeks, respectively), and g , h PGC-1a ( n = 50 glomeruli/group) protein in kidney cortex sections from WT and miR-379KO mice at 6 and 24 weeks after diabetes onset. Scale bar, 50 µm. Bar graph results are expressed as fold over WT-Con. Statistical analyses were performed by one-way ANOVA with post hoc Tukey’s test for multiple comparisons. * P < 0.05, ** P < 0.01, **** P < 0.0001. All data are presented as mean ± SEM.

Techniques:

Diabetic conditions induce expression of the miR-379 cluster via TGF-β signaling. This increase includes upregulation of miR-379 and subsequent downregulation of its targets ( Edem3 , Fis1 , Txn1 ) related to ER stress, adaptive mitophagy, mitochondrial dysfunction, and oxidant stress, which are directly involved in the development of early features of DKD, such as ECM accumulation, glomerular hypertrophy, and fibrosis. Activated ER stress can also increase CHOP transcription factor and the miR-379 cluster miRNAs, creating a positive feedback. Genetic deletion of miR-379 can diminish these alterations induced by diabetes, interrupt the auto-feedback, and protect against DKD progression.

Journal: Communications Biology

Article Title: miR-379 deletion ameliorates features of diabetic kidney disease by enhancing adaptive mitophagy via FIS1

doi: 10.1038/s42003-020-01516-w

Figure Lengend Snippet: Diabetic conditions induce expression of the miR-379 cluster via TGF-β signaling. This increase includes upregulation of miR-379 and subsequent downregulation of its targets ( Edem3 , Fis1 , Txn1 ) related to ER stress, adaptive mitophagy, mitochondrial dysfunction, and oxidant stress, which are directly involved in the development of early features of DKD, such as ECM accumulation, glomerular hypertrophy, and fibrosis. Activated ER stress can also increase CHOP transcription factor and the miR-379 cluster miRNAs, creating a positive feedback. Genetic deletion of miR-379 can diminish these alterations induced by diabetes, interrupt the auto-feedback, and protect against DKD progression.

Techniques: 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. 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).

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).

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).

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).

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).

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.

Techniques:

(A) 72-hour knockdown of FATP3 and FATP4 with 25 nM siRNA reduced FA uptake 25–33% as compared to scrambled control siRNA (siC).

Journal: Cell metabolism

Article Title: Local Mitochondrial ATP Production Regulates Endothelial Fatty Acid Uptake and Transport.

doi: 10.1016/j.cmet.2020.05.018

Figure Lengend Snippet: (A) 72-hour knockdown of FATP3 and FATP4 with 25 nM siRNA reduced FA uptake 25–33% as compared to scrambled control siRNA (siC).

Article Snippet: SiRNA targeting human FIS1 , MilliporeSigma , SASI_Hs01_00171952.

Techniques: Knockdown, Control

Journal: Cell metabolism

Article Title: Local Mitochondrial ATP Production Regulates Endothelial Fatty Acid Uptake and Transport.

doi: 10.1016/j.cmet.2020.05.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: SiRNA targeting human FIS1 , MilliporeSigma , SASI_Hs01_00171952.

Techniques: Purification, Virus, Recombinant, Transfection, Infection, Plasmid Preparation, Drug discovery, Gel Extraction, Cloning, ATP Bioluminescent Assay, Mutagenesis, CyQUANT Assay, Proliferation Assay, Isolation, Negative Control, Sequencing, Synthesized, Software, Membrane

Effect of Fis1/Drp1 siRNA on high glucose (HG)-induced overexpression of Fis1 and Drp1 expression in rat retinal endothelial cells (RRECs). ( A ) Representative Western blot (WB) image shows Fis1 and Drp1 expression is significantly upregulated in cells grown in HG medium. In parallel, cells grown in HG and transfected with Fis1 siRNA, Drp1 siRNA, or both siRNAs showed corresponding decreases in Fis1 or Drp1 expression. Graphical illustration of cumulative data shows Drp1 ( B ) and Fis1 ( C ) are significantly upregulated in cells grown in HG condition and reduced in cells transfected with Drp1 and Fis1 siRNAs. Data are expressed as mean ± SD. * p < 0.05. n = 6.

Journal: Cells

Article Title: Downregulation of Drp1 and Fis1 Inhibits Mitochondrial Fission and Prevents High Glucose-Induced Apoptosis in Retinal Endothelial Cells

doi: 10.3390/cells9071662

Figure Lengend Snippet: Effect of Fis1/Drp1 siRNA on high glucose (HG)-induced overexpression of Fis1 and Drp1 expression in rat retinal endothelial cells (RRECs). ( A ) Representative Western blot (WB) image shows Fis1 and Drp1 expression is significantly upregulated in cells grown in HG medium. In parallel, cells grown in HG and transfected with Fis1 siRNA, Drp1 siRNA, or both siRNAs showed corresponding decreases in Fis1 or Drp1 expression. Graphical illustration of cumulative data shows Drp1 ( B ) and Fis1 ( C ) are significantly upregulated in cells grown in HG condition and reduced in cells transfected with Drp1 and Fis1 siRNAs. Data are expressed as mean ± SD. * p < 0.05. n = 6.

Article Snippet: To investigate the effect of downregulating HG-induced overexpression of mitochondrial fission genes on apoptosis, cells were grown in high glucose medium and transfected with Fis1 siRNA or Drp1 siRNA (Ambion) individually, with both siRNAs in combination, or with scrambled siRNA (Qiagen), using Lipofectin reagent.

Techniques: Over Expression, Expressing, Western Blot, Transfection

Effect of reducing HG-induced overexpression of fission genes on apoptosis. ( A ) Representative WB image shows cleaved caspase-3 and Bax expression are significantly upregulated in cells grown in HG medium. In contrast, cells grown in HG medium and transfected with Fis1 siRNA, Drp1 siRNA, or both siRNAs exhibit reduced levels of these pro-apoptotic genes. Graphical illustration of cumulative data shows ( B ) cleaved caspase-3 and ( C ) Bax levels are downregulated in cells transfected with Fis1 siRNA, Drp1 siRNA, or both siRNAs compared with those of cells grown in HG medium alone. Data are expressed as mean ± SD. * p < 0.05. n = 6.

Journal: Cells

Article Title: Downregulation of Drp1 and Fis1 Inhibits Mitochondrial Fission and Prevents High Glucose-Induced Apoptosis in Retinal Endothelial Cells

doi: 10.3390/cells9071662

Figure Lengend Snippet: Effect of reducing HG-induced overexpression of fission genes on apoptosis. ( A ) Representative WB image shows cleaved caspase-3 and Bax expression are significantly upregulated in cells grown in HG medium. In contrast, cells grown in HG medium and transfected with Fis1 siRNA, Drp1 siRNA, or both siRNAs exhibit reduced levels of these pro-apoptotic genes. Graphical illustration of cumulative data shows ( B ) cleaved caspase-3 and ( C ) Bax levels are downregulated in cells transfected with Fis1 siRNA, Drp1 siRNA, or both siRNAs compared with those of cells grown in HG medium alone. Data are expressed as mean ± SD. * p < 0.05. n = 6.

Techniques: Over Expression, Expressing, Transfection

Reducing fission protects against HG-induced mitochondrial fragmentation. ( A ) Representative images of mitochondrial networks of RRECs grown in normal medium, HG, HG transfected with DRP1 siRNA, HG transfected with FIS1 siRNA, HG transfected with DRP1 siRNA and FIS 1 siRNA (Combo siRNA), or HG transfected with scrambled siRNA. Cells grown in HG medium or HG medium transfected with scrambled siRNA show fragmented mitochondrial networks, while cells grown in N medium or cells transfected with siRNAs exhibit tubular, elongated mitochondrial morphology. The large inset represents an enlarged view of the corresponding image (indicated by dotted lines) in the small inset in each panel. Scale bar = 20 μm. ( B ) Graphical illustration of cumulative data shows that reducing fission genes, Drp1 and Fis1, significantly prevents HG-induced mitochondrial fragmentation. Data are expressed as mean ± SD. * p < 0.05. n = 6. FF, form factor; AR, aspect ratio.

Journal: Cells

Article Title: Downregulation of Drp1 and Fis1 Inhibits Mitochondrial Fission and Prevents High Glucose-Induced Apoptosis in Retinal Endothelial Cells

doi: 10.3390/cells9071662

Figure Lengend Snippet: Reducing fission protects against HG-induced mitochondrial fragmentation. ( A ) Representative images of mitochondrial networks of RRECs grown in normal medium, HG, HG transfected with DRP1 siRNA, HG transfected with FIS1 siRNA, HG transfected with DRP1 siRNA and FIS 1 siRNA (Combo siRNA), or HG transfected with scrambled siRNA. Cells grown in HG medium or HG medium transfected with scrambled siRNA show fragmented mitochondrial networks, while cells grown in N medium or cells transfected with siRNAs exhibit tubular, elongated mitochondrial morphology. The large inset represents an enlarged view of the corresponding image (indicated by dotted lines) in the small inset in each panel. Scale bar = 20 μm. ( B ) Graphical illustration of cumulative data shows that reducing fission genes, Drp1 and Fis1, significantly prevents HG-induced mitochondrial fragmentation. Data are expressed as mean ± SD. * p < 0.05. n = 6. FF, form factor; AR, aspect ratio.

Techniques: Transfection

Reducing fission protects against HG-induced apoptosis. ( A ) Representative images of TUNEL-positive RRECs (white arrows) grown in normal medium, HG, HG transfected with DRP1 siRNA, HG transfected with FIS1 siRNA, HG transfected with DRP1 siRNA and FIS 1 siRNA, or HG transfected with scrambled siRNA. Scale bar = 50 μm. ( B ) Graphical illustration of cumulative data indicates that cells grown in HG medium or HG medium transfected with scrambled siRNA exhibit an increased number of TUNEL-positive cells. In parallel, downregulation of DRP1 and FIS1 fission genes significantly protects against HG-induced apoptotic cell death. Data are expressed as mean ± SD. * p < 0.05. n = 6.

Journal: Cells

Article Title: Downregulation of Drp1 and Fis1 Inhibits Mitochondrial Fission and Prevents High Glucose-Induced Apoptosis in Retinal Endothelial Cells

doi: 10.3390/cells9071662

Figure Lengend Snippet: Reducing fission protects against HG-induced apoptosis. ( A ) Representative images of TUNEL-positive RRECs (white arrows) grown in normal medium, HG, HG transfected with DRP1 siRNA, HG transfected with FIS1 siRNA, HG transfected with DRP1 siRNA and FIS 1 siRNA, or HG transfected with scrambled siRNA. Scale bar = 50 μm. ( B ) Graphical illustration of cumulative data indicates that cells grown in HG medium or HG medium transfected with scrambled siRNA exhibit an increased number of TUNEL-positive cells. In parallel, downregulation of DRP1 and FIS1 fission genes significantly protects against HG-induced apoptotic cell death. Data are expressed as mean ± SD. * p < 0.05. n = 6.

Techniques: TUNEL Assay, Transfection

( A ) Reducing mitochondrial fission protects against HG-induced decrease in oxygen consumption rate (OCR) in RRECs. At the fifth time point, steady-state oxygen consumption was measured. Subsequently, oligomycin was injected (vertical line O) to inhibit ATP synthase, and carbonyl cyanide-4-[trifluoromethoxy] phenylhydrazone (FCCP) was injected (vertical line F) to uncouple mitochondria and obtain maximal oxygen consumption rate at the eleventh time point. Antimycin A was injected (vertical line A) to confirm that the respiration changes were primarily owing to mitochondrial respiration. The cumulative OCR data are represented by the bar graph that shows RRECs grown in N media, HG media, HG media transfected with DRP1 siRNA, HG media transfected with FIS1 siRNA, HG media transfected with both DRP1 siRNA and FIS1 siRNA (Combo siRNA), or HG transfected with scrambled siRNA. Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01. n = 6. ( B ) Reducing mitochondrial fission protects against HG-induced increase in extracellular acidification (ECAR) in RRECs. Steady-state extracellular acidification rate was measured at the fifth time point. This was followed by the injection of glucose (vertical line G) to trigger glycolysis under basal conditions, and oligomycin was injected (vertical line O) to inhibit ATP synthase to assess glycolytic capacity at the eleventh time point. 2-deoxyglucose, a glucose analogue, was then injected (vertical line 2-DG) to inhibit glycolysis and to confirm that the respiration changes were primarily owing to glycolysis. The cumulative ECAR data are represented by the bar graph that shows RRECs grown in normal medium, HG, HG transfected with DRP1 siRNA, HG transfected with FIS1 siRNA, HG media transfected with both DRP1 siRNA and FIS1 siRNA (Combo siRNA), or HG transfected with scrambled siRNA. Lines G, O, and 2-DG indicate injections of glucose, oligomycin, and 2-deoxyglucose, respectively. Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01. n = 6.

Journal: Cells

Article Title: Downregulation of Drp1 and Fis1 Inhibits Mitochondrial Fission and Prevents High Glucose-Induced Apoptosis in Retinal Endothelial Cells

doi: 10.3390/cells9071662

Figure Lengend Snippet: ( A ) Reducing mitochondrial fission protects against HG-induced decrease in oxygen consumption rate (OCR) in RRECs. At the fifth time point, steady-state oxygen consumption was measured. Subsequently, oligomycin was injected (vertical line O) to inhibit ATP synthase, and carbonyl cyanide-4-[trifluoromethoxy] phenylhydrazone (FCCP) was injected (vertical line F) to uncouple mitochondria and obtain maximal oxygen consumption rate at the eleventh time point. Antimycin A was injected (vertical line A) to confirm that the respiration changes were primarily owing to mitochondrial respiration. The cumulative OCR data are represented by the bar graph that shows RRECs grown in N media, HG media, HG media transfected with DRP1 siRNA, HG media transfected with FIS1 siRNA, HG media transfected with both DRP1 siRNA and FIS1 siRNA (Combo siRNA), or HG transfected with scrambled siRNA. Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01. n = 6. ( B ) Reducing mitochondrial fission protects against HG-induced increase in extracellular acidification (ECAR) in RRECs. Steady-state extracellular acidification rate was measured at the fifth time point. This was followed by the injection of glucose (vertical line G) to trigger glycolysis under basal conditions, and oligomycin was injected (vertical line O) to inhibit ATP synthase to assess glycolytic capacity at the eleventh time point. 2-deoxyglucose, a glucose analogue, was then injected (vertical line 2-DG) to inhibit glycolysis and to confirm that the respiration changes were primarily owing to glycolysis. The cumulative ECAR data are represented by the bar graph that shows RRECs grown in normal medium, HG, HG transfected with DRP1 siRNA, HG transfected with FIS1 siRNA, HG media transfected with both DRP1 siRNA and FIS1 siRNA (Combo siRNA), or HG transfected with scrambled siRNA. Lines G, O, and 2-DG indicate injections of glucose, oligomycin, and 2-deoxyglucose, respectively. Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01. n = 6.

Techniques: Injection, Transfection

Review

fis1 sirna (Santa Cruz Biotechnology)

Structured Review

Images

1) Product Images from "SENP3-FIS1 axis promotes mitophagy and cell survival under hypoxia."

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