Journal: Redox Biology

Article Title: A novel role of KEAP1/PGAM5 complex: ROS sensor for inducing mitophagy

doi: 10.1016/j.redox.2021.102186

Figure Lengend Snippet:

Article Snippet: FUNDC1 shRNA , Origene , Cat# TR702819.

Techniques: Recombinant, Lysis, Blocking Assay, Isolation, DC Protein Assay, Caspase-Glo Assay, shRNA, Generated, Plasmid Preparation, Synthesized, Variant Assay

Journal: Stroke and vascular neurology

Article Title: Src inhibition rescues FUNDC1-mediated neuronal mitophagy in ischaemic stroke.

doi: 10.1136/svn-2023-002606

Figure Lengend Snippet: Figure 1 Knockout of FUNDC1 has no effect on short-term outcomes of ischaemic stroke. (A) Cerebral blood flow (CBF) of WT and FUNDC1–/– mice. (B) Quantification for the variation of CBF at each time point of the perioperative period. n=5 mice for each genotype. (C) Representative TTC staining of brains from WT and FUNDC1–/– mice subjected to tMCAO at 24 hours after surgery. (D) Quantification for the infarcted volumes. n=10 mice for each genotype. (E) Degenerated neurons in the infarcted brains at 24 hours after reperfusion. (F) Quantification for the percentage of degenerated neurons. n=6 mice for each genotype. (G) Apoptosis in ipsilateral and contralateral brain tissues from WT and FUNDC1–/– mice subjected to tMCAO were detected by western blotting at 24 hours after reperfusion. (H) Semi-quantification for apoptosis-associated protein PARP, cleaved PARP, caspase 9, cleaved caspase 9, caspase 3 and cleaved caspase 3. n=6 mice for each group. (I) Comparison for neurological deficits including Bederson score (left) and Grip test (right). n=22 mice for each genotype. (J) Χ2 test fourfold tables for 24-hour death events in WT and FUNDC1–/– mice. DAPI: 4',6-diamidino-2-phenylindole; FJC, Fluore Jade C; FUNDC1, FUN14 domain- containing 1; KO: Knockout; PARP: poly ADP-ribose polymerase; tMCAO, transient middle cerebral artery occlusion; TTC, triphenyltetrazolium chloride; WT, wild-type.

Article Snippet: At the end of reperfusion, cells were fixed with 4% PFA and incubated with an antibody against FUNDC1 (1:100; CST, 49240).

Techniques: Knock-Out, Staining, Western Blot, Comparison

Journal: Stroke and vascular neurology

Article Title: Src inhibition rescues FUNDC1-mediated neuronal mitophagy in ischaemic stroke.

doi: 10.1136/svn-2023-002606

Figure Lengend Snippet: Figure 2 Absence of FUNDC1 does not influence long-term outcomes of ischaemic stroke. (A) 14-day survival rate after tMCAO. n=10 mice for each genotype. (B–C) Body weight recovery (B) and behavioural tests (C) including Garcia score, mNSS score, rotarod test, hanging test were assessed before the surgery (WT, n=10; FUNDC1−/−, n=10) and at 1 day (WT, n=10; FUNDC1−/−, n=8), 3-day (WT, n=7; FUNDC1−/−, n=7), 7-day (WT, n=6; FUNDC1−/−, n=6), 14-day (WT, n=5; FUNDC1−/−, n=5) after tMCAO. FUNDC1, FUN14 domain-containing 1; mNSS, Modified Neurological Severity Score; tMCAO, transient middle cerebral artery occlusion; WT, wild-type.

Article Snippet: At the end of reperfusion, cells were fixed with 4% PFA and incubated with an antibody against FUNDC1 (1:100; CST, 49240).

Techniques: Modification

Journal: Stroke and vascular neurology

Article Title: Src inhibition rescues FUNDC1-mediated neuronal mitophagy in ischaemic stroke.

doi: 10.1136/svn-2023-002606

Figure Lengend Snippet: Figure 3 Deletion of FUNDC1 does not affect neuronal mitophagy in vivo. (A) Time course of mitophagy in vivo was detected by western blotting. (B) Semi-quantification for western blotting detection in panel A. n=3 mice for each time point. (C) WT and FUNDC1−/− mice were subjected to sham surgery or tMCAO for 24 hours. Mitophagy was detected by western blotting. (D) Semi-quantification for western blotting detection in panel C. n=6 mice per group. (E) Brain sections from both genotypes of normal mice or mice with stroke were labelled with NeuN (red), LC3 (green), and Tomm20 (magenta). (F) Quantification of the overlap coefficient of LC3 and Tomm20. n=6 mice per group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. FUNDC1, FUN14 domain-containing 1; KO: Knockout; LC3, light chain 3; SQSTM1: Sequestosome 1; tMCAO, transient middle cerebral artery occlusion; WT, wild-type.

Article Snippet: At the end of reperfusion, cells were fixed with 4% PFA and incubated with an antibody against FUNDC1 (1:100; CST, 49240).

Techniques: In Vivo, Western Blot, Knock-Out

Journal: Stroke and vascular neurology

Article Title: Src inhibition rescues FUNDC1-mediated neuronal mitophagy in ischaemic stroke.

doi: 10.1136/svn-2023-002606

Figure Lengend Snippet: Figure 4 Loss of FUNDC1 does not affect neuronal mitophagy and mitochondrial qualities in vitro. (A) Time course of neuronal mitophagy in vitro was detected by western blotting. (B) Semi-quantification for western blotting detection in panel A. n=3 for independent experiments. (C) Cortical neurons isolated from WT and FUNDC1–/– mice were subjected to control or OGD/R treatment for 9 hours. Mitophagy was detected by western blotting. (D) Semi-quantification for western blotting detection in panel C. n=3 per independent experiment. (E) Normal-cultured or OGD/R-treated neurons with two genotypes were labelled with MitoTracker (red) and GFP-LC3 (green), then visualised by confocal microscopy. (F) Quantification of the number of LC3 puncta per cell (left) and colocalisation coefficient of mitochondria and LC3 puncta (right). n=3 for independent experiment. **p<0.01, ***p<0.001, ****p<0.0001. FUNDC1, FUN14 domain-containing 1; GFP: Green Fluorescent Protein; KO: Knockout; LC3, light chain 3; OGD/R, oxygen glucose deprivation/reperfusion; SQSTM1: Sequestosome 1; WT, wild-type.

Article Snippet: At the end of reperfusion, cells were fixed with 4% PFA and incubated with an antibody against FUNDC1 (1:100; CST, 49240).

Techniques: In Vitro, Western Blot, Isolation, Control, Cell Culture, Confocal Microscopy, Knock-Out

Journal: Stroke and vascular neurology

Article Title: Src inhibition rescues FUNDC1-mediated neuronal mitophagy in ischaemic stroke.

doi: 10.1136/svn-2023-002606

Figure Lengend Snippet: Figure 5 FUNDC1 is inactivated in later stages of neuronal I/R injury. (A) Time course of FUNDC1 phosphorylation was detected by western blotting in vivo. (B) Semi-quantification for p-Tyr18 and total FUNDC1 in panel A. n=3 mice per time point. (C) Immunostaining of p-FUNDC1 (green) in neurons (red). (D) Quantification for proportion of p-FUNDC1 positive neurons. n=6 mice per time point. (E) Time course of FUNDC1 phosphorylation was detected by western blotting in vitro. n=3 for independent experiments. (F) Semi-quantification for LC3 II interacted with FUNDC1 as is shown in panel G. n=3 for independent experiments. (G) Isolated cortical neurons were subjected to 2 hours of OGD, followed by 0 hours, 6 hours, 9 hours or 12 hours of reperfusion. Interactions between FUNDC1 and LC3 were detected by co-immunoprecipitation. (H) Fluorescent staining of FUNDC1 (red) and GFP-LC3 (green) in isolated neurons subjected to control treatment, OGD/R 0 hours, or OGD/R 9 hours. (I) Quantification of the number of LC3 puncta per cell (left) and percentage of LC3 puncta colocalised with FUNDC1 (right). N=3 for independent experiment. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. FUNDC1, FUN14 domain-containing 1; Green Fluorescent Protein; I/R, ischaemia/reperfusion; LC3, light chain 3; OGD/R, oxygen glucose deprivation/reperfusion; tMCAO, transient middle cerebral artery occlusion; WT, wild-type.

Article Snippet: At the end of reperfusion, cells were fixed with 4% PFA and incubated with an antibody against FUNDC1 (1:100; CST, 49240).

Techniques: Phospho-proteomics, Western Blot, In Vivo, Immunostaining, In Vitro, Isolation, Immunoprecipitation, Staining, Control

Journal: Stroke and vascular neurology

Article Title: Src inhibition rescues FUNDC1-mediated neuronal mitophagy in ischaemic stroke.

doi: 10.1136/svn-2023-002606

Figure Lengend Snippet: Figure 6 Src is activated in later stages of neuronal ischaemia/reperfusion injury. (A) Src phosphorylation in vivo at different time points was detected by western blotting. (B) Semi-quantification for p-Tyr416 and total Src in panel A. n=3 mice per time point. (C) Immunostaining of p-Src (green) in neurons (red). (D) Quantification for proportion of p-Src positive neurons. n=6 mice per time point. (E) Src phosphorylation in vitro at different time points was detected by western blotting. n=3 for independent experiments. (F) Semi-quantification for Src interacted with FUNDC1 as is shown in panel G. n=3 for independent experiments. (G) Time course of FUNDC1-Src interaction in isolated neurons were detected by co-immunoprecipitation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. FUNDC1, FUN14 domain-containing 1; OGD, oxygen glucose deprivation; tMCAO, transient middle cerebral artery occlusion.

Article Snippet: At the end of reperfusion, cells were fixed with 4% PFA and incubated with an antibody against FUNDC1 (1:100; CST, 49240).

Techniques: Phospho-proteomics, In Vivo, Western Blot, Immunostaining, In Vitro, Isolation, Immunoprecipitation

Journal: Stroke and vascular neurology

Article Title: Src inhibition rescues FUNDC1-mediated neuronal mitophagy in ischaemic stroke.

doi: 10.1136/svn-2023-002606

Figure Lengend Snippet: Figure 7 Pharmacological inhibition of Src rescues FUNDC1-mediated mitophagy in neurons subjected to ischaemia/ reperfusion injury. (A) Neurons subjected to OGD/R were treated with vehicle or PP1, and were labelled using antibodies against FUNDC1 (red) and GFP-LC3 (green). (B) Quantification of the number of LC3 puncta per cell (left) and percentage of LC3 puncta colocalised with FUNDC1 (right). n=3 for independent experiment. (C) Mitophagy in mice with both genotypes treated by PP1 in brain samples was detected by western blotting. (D) Semi-quantification for western blotting detection in panel C. n=6 mice per group. (E) Representative TTC staining of brains from vehicle-treated WT mice (n=5), FUNDC1–/– mice (n=5), PP1-treated WT mice (n=6), and PP1-treated FUNDC1–/– mice (n=6). (F) Quantification of infarcted volume. (G) Comparison of neurological deficits among vehicle-treated WT mice (n=11), FUNDC1–/– mice (n=11) PP1-treated WT mice (n=12), and PP1- treated FUNDC1–/– mice (n=12). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. FUNDC1, FUN14 domain-containing 1; GFP: Green Fluorescent Protein; OGD/R, oxygen glucose deprivation/reperfusion; KO: Knockout; LC3, light chain 3; SQSTM1: Sequestosome 1; tMCAO, transient middle cerebral artery occlusion; WT, wild-type.

Article Snippet: At the end of reperfusion, cells were fixed with 4% PFA and incubated with an antibody against FUNDC1 (1:100; CST, 49240).

Techniques: Inhibition, Western Blot, Staining, Comparison, Knock-Out

Journal: Stroke and vascular neurology

Article Title: Src inhibition rescues FUNDC1-mediated neuronal mitophagy in ischaemic stroke.

doi: 10.1136/svn-2023-002606

Figure Lengend Snippet: Figure 8 Working model of FUNDC1 inactivation by Src during neuronal I/R injury (created with BioRender.com). Although neuronal I/R induces mitophagy, hyperactivated Src inactivates FUNDC1, leading to FUNDC1 exclusion from mitophagy. Even in the absence of FUNDC1, neurons can initiate mitophagy through the BNIP3L/Nix and PINK1/Parkin pathways. FUNDC1, FUN14 domain-containing 1; I/R, ischaemia/reperfusion.

Article Snippet: At the end of reperfusion, cells were fixed with 4% PFA and incubated with an antibody against FUNDC1 (1:100; CST, 49240).

Techniques:

Journal: Open Medicine

Article Title: Methylation regulation for FUNDC1 stability in childhood leukemia was up-regulated and facilitates metastasis and reduces ferroptosis of leukemia through mitochondrial damage by FBXL2

doi: 10.1515/med-2023-0810

Figure Lengend Snippet: Expression levels of FUNDC1 in CL. FUNDC1 mRNA expression (a) and (b), ROC (c) in patients with CL; FUNDC1 mRNA and protein expression (d) and (e) in leukemia cells. Normal, normal serum sample; Cancer, patients with CL; ** p < 0.01 compared with normal serum sample or HS-5 cells.

Article Snippet: FUNDC1 plasmids (sc-428195, Santa Cruz Biotechnology, Inc.) or si-FUNDC1 plasmids (sc-145273, Santa Cruz Biotechnology, Inc.) were transfected into GC cell lines using Lipofectamine 2000.

Techniques: Expressing

Journal: Open Medicine

Article Title: Methylation regulation for FUNDC1 stability in childhood leukemia was up-regulated and facilitates metastasis and reduces ferroptosis of leukemia through mitochondrial damage by FBXL2

doi: 10.1515/med-2023-0810

Figure Lengend Snippet: FUNDC1 up-regulation promotes leukemia metastasis. FUNDC1 mRNA expression (a), cell growth (b), metastasis (c), and EDU assay (d) in vitro model of leukemia by FUNDC1 up-regulation; FUNDC1 mRNA expression (e), cell growth (f), metastasis (g), and EDU assay (h) in vitro model of leukemia by FUNDC1 down-regulation. (i). Fluorescence images showing reduced proliferation in leukemia cells with FUNDC1 knockdown, as evidenced by fewer EDU-positive cells. (j). Micrographs illustrating decreased migration of leukemia cells after FUNDC1 knockdown. Vector, negative control group; FUNDC1, over-expression of FUNDC1 group; Si-nc, si-negative control group; Si-FUNDC1, down-regulation of FUNDC1 group; ** p < 0.01 compared with negative control group or si-negative control group.

Article Snippet: FUNDC1 plasmids (sc-428195, Santa Cruz Biotechnology, Inc.) or si-FUNDC1 plasmids (sc-145273, Santa Cruz Biotechnology, Inc.) were transfected into GC cell lines using Lipofectamine 2000.

Techniques: Expressing, EdU Assay, In Vitro, Fluorescence, Knockdown, Migration, Plasmid Preparation, Negative Control, Over Expression

Journal: Open Medicine

Article Title: Methylation regulation for FUNDC1 stability in childhood leukemia was up-regulated and facilitates metastasis and reduces ferroptosis of leukemia through mitochondrial damage by FBXL2

doi: 10.1515/med-2023-0810

Figure Lengend Snippet: FUNDC1 up-regulation reduces ferroptosis by inducing mitochondrial damage. JC-1 levels (a), MPT (b), mitochondrial damage (c), PI-positive cells (d), iron content (e), caspase-3/9 activity (f) and (g), LDH activity levels (h), GSH levels (i), and GPX4 protein expression (j) in vitro model of leukemia by FUNDC1 up-regulation; JC-1 levels (k), MPT (i), mitochondrial damage (l), mitochondrial damage (m), PI-positive cells (n), iron content (o), caspase-3/9 activity (p) and (q), LDH activity levels (r), GSH levels (s), and GPX4 protein expression (t) in vitro model of leukemia by FUNDC1 down-regulation. Vector, negative control group; FUNDC1, over-expression of FUNDC1 group; Si-nc, si-negative control group; Si-FUNDC1, down-regulation of FUNDC1 group; ** p < 0.01 compared with negative control group or si-negative control group.

Article Snippet: FUNDC1 plasmids (sc-428195, Santa Cruz Biotechnology, Inc.) or si-FUNDC1 plasmids (sc-145273, Santa Cruz Biotechnology, Inc.) were transfected into GC cell lines using Lipofectamine 2000.

Techniques: Activity Assay, Expressing, In Vitro, Plasmid Preparation, Negative Control, Over Expression

Journal: Open Medicine

Article Title: Methylation regulation for FUNDC1 stability in childhood leukemia was up-regulated and facilitates metastasis and reduces ferroptosis of leukemia through mitochondrial damage by FBXL2

doi: 10.1515/med-2023-0810

Figure Lengend Snippet: FUNDC1 up-regulation induces FBXL2 expression in a leukemia model. FBXL2 mRNA expression (a), FBXL2/FUNDC1 protein expression (b) and (c), FBXL2/FUNDC1 expression confocal microscope (d), FBXL2 protein interlinked with FUNDC1 protein (e), FBXL2 ubiquitination (f). Vector, negative control group; FUNDC1, over-expression of FUNDC1 group; Si-nc, si-negative control group; Si-FUNDC1, down-regulation of FUNDC1 group; ** p < 0.01 compared with negative control group or si-negative control group.

Article Snippet: FUNDC1 plasmids (sc-428195, Santa Cruz Biotechnology, Inc.) or si-FUNDC1 plasmids (sc-145273, Santa Cruz Biotechnology, Inc.) were transfected into GC cell lines using Lipofectamine 2000.

Techniques: Expressing, Microscopy, Ubiquitin Proteomics, Plasmid Preparation, Negative Control, Over Expression

Journal: Open Medicine

Article Title: Methylation regulation for FUNDC1 stability in childhood leukemia was up-regulated and facilitates metastasis and reduces ferroptosis of leukemia through mitochondrial damage by FBXL2

doi: 10.1515/med-2023-0810

Figure Lengend Snippet: Mitochondrial damage regulates the effects of FUNDC1 up-regulation on ferroptosis in leukemia. JC-1 levels (a), MPT (b), mitochondrial damage (c), PI-positive cells (d), LDH activity levels (e), iron content (f), GSH levels (g), and GPX4 protein expression (h) in vitro model of leukemia by FUNDC1 up-regulation and activator; JC-1 levels (i), MPT (j), mitochondrial damage (k), PI-positive cells (l), LDH activity levels (m), iron content (n), GSH levels (o), and GPX4 protein expression (p) in vitro model of leukemia by FUNDC1 down-regulation and inhibitor. Vector, negative control group; FUNDC1, over-expression of FUNDC1 group; Si-nc, si-negative control group; si-FUNDC1, down-regulation of FUNDC1 group; ** p < 0.01 compared with negative control group or si-negative control group; ## p < 0.01 compared with FUNDC1 or si-FUNDC1 group.

Article Snippet: FUNDC1 plasmids (sc-428195, Santa Cruz Biotechnology, Inc.) or si-FUNDC1 plasmids (sc-145273, Santa Cruz Biotechnology, Inc.) were transfected into GC cell lines using Lipofectamine 2000.

Techniques: Activity Assay, Expressing, In Vitro, Plasmid Preparation, Negative Control, Over Expression

Journal: Open Medicine

Article Title: Methylation regulation for FUNDC1 stability in childhood leukemia was up-regulated and facilitates metastasis and reduces ferroptosis of leukemia through mitochondrial damage by FBXL2

doi: 10.1515/med-2023-0810

Figure Lengend Snippet: Mitochondrial damage regulates the effects of FUNDC1 up-regulation on metastasis in leukemia. Cell growth (a), metastasis (b), and EDU assay (c) in vitro model of leukemia by FUNDC1 up-regulation; cell growth (d), metastasis (e), and EDU assay (f) in vitro model of leukemia by FUNDC1 down-regulation. Vector, negative control group; FUNDC1, over-expression of FUNDC1 group; Si-nc, si-negative control group; Si-FUNDC1, down-regulation of FUNDC1 group; ** p < 0.01 compared with negative control group or si-negative control group.

Article Snippet: FUNDC1 plasmids (sc-428195, Santa Cruz Biotechnology, Inc.) or si-FUNDC1 plasmids (sc-145273, Santa Cruz Biotechnology, Inc.) were transfected into GC cell lines using Lipofectamine 2000.

Techniques: EdU Assay, In Vitro, Plasmid Preparation, Negative Control, Over Expression

Journal: Open Medicine

Article Title: Methylation regulation for FUNDC1 stability in childhood leukemia was up-regulated and facilitates metastasis and reduces ferroptosis of leukemia through mitochondrial damage by FBXL2

doi: 10.1515/med-2023-0810

Figure Lengend Snippet: Methylation controls FUNDC1 stability. m6A modification site of FUNDC1 (a), METTL3-mediated FUNDC1 m6A modifications (b) and (c), the position of m6A motifs within FUNDC1 transcript sequence (d), luciferase reporter activity level (e), m6A levels of FUNDC1 (f) and (g), ** p < 0.01 compared with vector or negative or IgG group.

Article Snippet: FUNDC1 plasmids (sc-428195, Santa Cruz Biotechnology, Inc.) or si-FUNDC1 plasmids (sc-145273, Santa Cruz Biotechnology, Inc.) were transfected into GC cell lines using Lipofectamine 2000.

Techniques: Methylation, Modification, Sequencing, Luciferase, Activity Assay, Plasmid Preparation

Journal: Science signaling

Article Title: The mitophagy effector FUNDC1 controls mitochondrial reprogramming and cellular plasticity in cancer cells

doi: 10.1126/scisignal.aaz8240

Figure Lengend Snippet: (A) PC3 cells transfected with control non-targeting siRNA (siCtrl) or FUNDC1-directed pooled siRNA (siFND1) were labeled with Talin-RFP and analyzed for focal adhesion (FA) dynamics by time-lapse videomicroscopy. Representative images at 0 h and 2 h from 3 independent experiments are shown. (B) The rate (events/h) of FA assembly (top) or disassembly (bottom) was quantified from the cells in (A). Each point corresponds to FA events in an individual cell (11–14 cells per condition). Mean±SD (N=3 independent experiments per group). *, p=0.02–0.04. (C and D) PC3 cells transfected with siCtrl or siFND1 were analyzed for cell motility in 2D contour plots by time-lapse video microscopy (C) with quantification of speed of cell movements (D, top) and distance traveled by individual cells (D, bottom). Each tracing in (C) corresponds to the movements of an individual cell (49 cells per condition) in a representative experiment. The cutoff velocities for slow (blue, <0.6 μm/min)- or fast (orange, >0.6 μm/min)-moving cells are indicated. Mean±SD (N=3 independent experiments per group). ***, p<0.0001. (E) PC3 cells transfected with siCtrl or siFND1 were analyzed for 2D chemotaxis in a rose plot (top) with quantification of the forward migration index (bottom). Arrows indicate the direction of the chemotactic gradient. Each point corresponds to an individual cell (89 cells per condition; N=3 independent experiments per group). (F) PC3 cells transfected with siCtrl or siFND1 were analyzed for invasion across Matrigel-coated Transwell inserts. Each point corresponds to an individual determination. Mean±SD (N=3 independent experiments per group). ***, p=0.0001. (G) The indicated prostate cancer cell lines were transfected with vector or FND1 cDNA and analyzed for Matrigel invasion. Mean±SD (N=3 independent experiments per group). **, p=0.001; ***, p <0.0001 – 0.0005. (H and I) PC3 cells transfected with vector or FND1 cDNA were analyzed by Western blotting (H, representative blot of three independent experiments) and the intensity of phosphorylated (p) FAK protein band was quantified by densitometry (I). Mean±SD (N=3 independent experiments per group). ***, p=0.0008. (J) PC3 cells transfected with siCtrl or siFND1 were analyzed for Matrigel invasion in the presence or absence of FAK-directed siRNA (siFAK). Mean±SD (N=3 independent experiments per group). ***, p<0.0001.

Article Snippet: Antibodies to FUNDC1 (Aviva System Biology, diluted 1:500), Drp1 (clone D6C7, Cell Signaling cat. #8570 diluted 1:1000), Ser 616 -phosphorylated Drp1 (Cell Signaling cat. #3455 diluted 1:1000), Drp1 (Cell Signaling cat. #5391 diluted 1:1000), Tyr 925 -phosphorylated FAK (Cell Signaling cat. #3284 diluted 1:1000), FAK (Cell Signaling cat. n. #3285), FLAG (clone M-2, Sigma cat. #F1804 diluted 1:5000) and β-actin (clone AC-15, Sigma cat. #A5441 diluted 1:100000) were used for Western blotting.

Techniques: Transfection, Labeling, Microscopy, Chemotaxis Assay, Migration, Plasmid Preparation, Western Blot

Journal: Science signaling

Article Title: The mitophagy effector FUNDC1 controls mitochondrial reprogramming and cellular plasticity in cancer cells

doi: 10.1126/scisignal.aaz8240

Figure Lengend Snippet: (A) PC3 cells were fractionated in submitochondrial compartments and analyzed by Western blotting. MTE, mitochondrial extracts; OM, outer membrane; IM, inner membrane; IMS, intermembrane space; Mat, matrix. Data are representative of 2 independent experiments. (B) Ingenuity pathway analysis of a mitochondrial FUNDC1 interactome identified by LC-MS/MS proteomics (N=1 independent experiment). The fold-induction for FUNDC1-associated proteins compared to Flag-vector are indicated with red color intensity. (C) PC3 cells transfected with Flag-vector or Flag-FND1 cDNA were immunoprecipitated (IP) with an antibody to Flag and immune complexes were analyzed by Western blotting. Data are representative of 2 independent experiments. WCE, whole cell extracts. (D) PC3 cells were immunoprecipitated with non-binding IgG or an antibody to endogenous ATP5C1 and immune complexes were analyzed by Western blotting. Data are representative of 2 independent experiments. WCE, whole cell extracts. (E) PC3 cells transfected with siCtrl or siFND1 were reconstituted with FND1 cDNA and analyzed for complex V (C.V) activity. Tracings from two independent experiments (Exp) are shown. Mean±SD of 3 technical replicates per experiment. (F) Citrate synthase-normalized complex V (C.V) activity as in (E) was quantified. Two independent experiments (Exp) are shown. Mean±SD of 3 technical replicates per experiment. (G) PC3 cells stably transduced with pLKO or shFND1 were reconstituted with Flag-LonP1 cDNA, and Flag-eluted LonP1 immune complexes (Elu) (top, representative blot of 3 independent experiments) were analyzed for LonP1 proteolytic activity (bottom). TCE, total cell extracts. Mean±SD (N=3 independent experiments per group). (H and I) PC3 cells transfected with siCtrl or siFND1 were reconstituted with LonP1 cDNA, extracted at increasing concentrations of NP-40 and detergent-insoluble protein bands corresponding to complex V subunits, ATP5C1, ATP5O and ATP5B were analyzed by Western blotting (H, representative blot of 2 independent experiments) and quantified by densitometry (I). VDAC was a control. Two independent experiments (Exp) are shown (I).

Article Snippet: Antibodies to FUNDC1 (Aviva System Biology, diluted 1:500), Drp1 (clone D6C7, Cell Signaling cat. #8570 diluted 1:1000), Ser 616 -phosphorylated Drp1 (Cell Signaling cat. #3455 diluted 1:1000), Drp1 (Cell Signaling cat. #5391 diluted 1:1000), Tyr 925 -phosphorylated FAK (Cell Signaling cat. #3284 diluted 1:1000), FAK (Cell Signaling cat. n. #3285), FLAG (clone M-2, Sigma cat. #F1804 diluted 1:5000) and β-actin (clone AC-15, Sigma cat. #A5441 diluted 1:100000) were used for Western blotting.

Techniques: Western Blot, Liquid Chromatography with Mass Spectroscopy, Plasmid Preparation, Transfection, Immunoprecipitation, Binding Assay, Activity Assay, Stable Transfection, Transduction

Journal: Science signaling

Article Title: The mitophagy effector FUNDC1 controls mitochondrial reprogramming and cellular plasticity in cancer cells

doi: 10.1126/scisignal.aaz8240

Figure Lengend Snippet: (A and B) PC3 (top), C42B (middle) or DU145 (bottom) cells were transfected with vector or FND1 cDNA and analyzed for Matrigel invasion (A) or cell proliferation by direct cell counting (B). Mean±SD (N=3 or 5 independent experiments per condition). **, p=0.001; ***, p <0.0001–0.0005. (C and D) PC3 cells stably transduced with pLKO or FUNDC1-directed shRNA (shFND1) were analyzed for colony formation (C) and quantified after 14 d (D). Mean±SD (N=3 independent experiments per group). ***, p<0.0001. (E) PC3 cells transduced with pLKO or shFND1 were engrafted subcutaneously onto the flanks of immunocompromised mice (N=10 animals/group) and tumor growth was quantified at the indicated time intervals. Each line corresponds to an individual tumor. The mean±SD tumor size (mm3) for each animal group harvested at day 27 is indicated, p<0.0001. (F) Representative tumor samples from each animal group in (E) were excised at the end of the experiment and analyzed for Ki67 staining by immunohistochemistry. Representative images are shown. Scale bar, 100 μm. (G and H) Representative lung samples isolated from each animal group in (E) were stained with an antibody to human mitochondria by immunohistochemistry (G, representative images) and the number of lung metastatic foci was quantified (H). Scale bar, 100 μm. Mean±SD (N=25 determinations per group). ***, p<0.0001. (I) PC3 cells stably transduced with pLKO or shFND1 were injected into the spleen of immunocompromised mice (N=5 animals per group) and metastatic foci to the liver were quantified after 11 days by histology. Mean±SD (N=8–10 determinations per group). ***, p=0.0009. (J and K) RNA-seq expression data from 33 cancer types (TCGA) for genes (Spearman r>0.2; p<0.05) positively (J, GO:0046034, ATP metabolism) or negatively (K, GO:0000302, response to ROS; GO:0048870, cell motility; GO:0008283, cell proliferation) correlated with FUNDC1 (FND1) expression.

Article Snippet: Antibodies to FUNDC1 (Aviva System Biology, diluted 1:500), Drp1 (clone D6C7, Cell Signaling cat. #8570 diluted 1:1000), Ser 616 -phosphorylated Drp1 (Cell Signaling cat. #3455 diluted 1:1000), Drp1 (Cell Signaling cat. #5391 diluted 1:1000), Tyr 925 -phosphorylated FAK (Cell Signaling cat. #3284 diluted 1:1000), FAK (Cell Signaling cat. n. #3285), FLAG (clone M-2, Sigma cat. #F1804 diluted 1:5000) and β-actin (clone AC-15, Sigma cat. #A5441 diluted 1:100000) were used for Western blotting.

Techniques: Transfection, Plasmid Preparation, Cell Counting, Stable Transfection, Transduction, shRNA, Staining, Immunohistochemistry, Isolation, Injection, RNA Sequencing Assay, Expressing

Journal: Science signaling

Article Title: The mitophagy effector FUNDC1 controls mitochondrial reprogramming and cellular plasticity in cancer cells

doi: 10.1126/scisignal.aaz8240

Figure Lengend Snippet: (A) Heatmap of changes in metabolite levels in PC3 cells transfected with siCtrl or siFND1. Fold changes, p values and false discovery rate (FDR) are indicated. Data are from a representative experiment (siCtrl) or two independent experiments in triplicate (siFND1, 1A-C; 2A-C). (B) Schematic diagram of mitochondrial bioenergetics and ROS pathways affected by FUNDC1 silencing as in (A). (C) PC3 cells transfected with siCtrl or siFND1were analyzed for oxygen consumption rates (OCR) on a Seahorse XFe96 Bioenergetics Flux Analyzer. Tracings from two independent experiments (Exp) are shown. Mean±SD of 3 technical replicates per experiment. (D) PC3 cells transfected with siCtrl or siFND1were analyzed for MitoSox reactivity and mitochondrial membrane potential (TMRE) by flow cytometry. Mean±SD (N=3–4 independent experiments per group). **, p=0.001. (E) PC3 cells transfected with siCtrl or siFND1were analyzed for MitoSox reactivity in the presence or absence of the superoxide scavenger, MnTBAP, by flow cytometry. Mean±SD (N=3 independent experiments per group). **, p=0.005; *, p=0.03. (F) PC3 cells transfected with siCtrl or siFND1were analyzed for speed of mitochondrial movements (top) or distance traveled by individual mitochondria (bottom) with or without MnTBAP. Each symbol corresponds to the tracked movement of an individual mitochondrion. Data are representative of 3 independent experiments. The mean±SD of mitochondrial speed (siCtrl compared to siFND1, p<0.0001; siFND1 compared to siFND1+MnTBAP, p=0.002) and distance traveled (siCtrl compared to siFND1, p=0.001; siFND1 compared to siFND1+MnTBAP, p=0.0005) are indicated. (G) siCtrl- or siFND1-transfected PC3 cells were analyzed for cellular motility in 2D contour plots in the presence or absence of MnTBAP. Each tracing corresponds to the movements of an individual cell. The cut-off velocities for slow (<0.25 μm/min)- or fast (>0.25 μm/min)-moving cells are indicated. Data are representative of 3 independent experiments per group. The speed (μm/min) of cell motility (siCtrl compared to siFND1, p<0.0001; siFND1 compared to siFND1+MnTBAP, p=0.006) and distance (μm) traveled (siCtrl compared to siFND1, p<0.0001; siFND1 compared to siFND1+MnTBAP, p=0.006) are indicated. (H) PC3 cells transfected with siCtrl or siFND1 were analyzed for Matrigel invasion in the presence or absence of MnTBAP. Mean±SD (N=3 independent experiments per group). ***, p<0.0001. (I) PC3 cells transfected with siCtrl or siFND1 were analyzed for cell proliferation in the presence or absence of MnTBAP by direct cell counting. Mean±SD (N=4 independent experiments per group). **, p=0.03.

Article Snippet: Antibodies to FUNDC1 (Aviva System Biology, diluted 1:500), Drp1 (clone D6C7, Cell Signaling cat. #8570 diluted 1:1000), Ser 616 -phosphorylated Drp1 (Cell Signaling cat. #3455 diluted 1:1000), Drp1 (Cell Signaling cat. #5391 diluted 1:1000), Tyr 925 -phosphorylated FAK (Cell Signaling cat. #3284 diluted 1:1000), FAK (Cell Signaling cat. n. #3285), FLAG (clone M-2, Sigma cat. #F1804 diluted 1:5000) and β-actin (clone AC-15, Sigma cat. #A5441 diluted 1:100000) were used for Western blotting.

Techniques: Transfection, Flow Cytometry, Cell Counting

Journal: Nature Communications

Article Title: FUNDC1-dependent mitochondria-associated endoplasmic reticulum membranes are involved in angiogenesis and neoangiogenesis

doi: 10.1038/s41467-021-22771-3

Figure Lengend Snippet: a HUVECs were transfected with scrambled (scr) siRNA or FUN14 domain-containing 1 (FUNDC1) siRNA for 24 h. The protein levels of FUNDC1 were determined by western blot assays ( n = 5 independent experiments). b HUVECs were transfected with Scr siRNA or FUNDC1 siRNA for 24 h. Representative confocal images are shown. Quantification of endoplasmic reticulum (ER)–mitochondria contacts using Pearson’s coefficient. ( n = 5 independent experiments). Scale bars, 10 µm. c HUVECs transfected with Scr siRNA or FUNDC1 siRNA were plated onto matrigel. Representative images of tube formation are shown (left). Tube length/field in 10 random microscopic fields per group was measured by NIH ImageJ and statistically analyzed ( n = 5 independent experiments) (right). Scale bar, 100 µm. d HUVECs were transfected with Scr siRNA or FUNDC1 siRNA for 24 h. Three-dimensional spheroids were generated (left), and representative images of spheroid-sprouting were analyzed ( n = 5 independent experiments) (right). Scale bar, 100 µm. e Representative confocal images of a P5 retina lobe stained with IsoB4 showing reduced number of sprouts (yellow stars) at the vascular front of FUNDC1 f/Y Cdh5 + retinas compared with FUNDC1 f/Y Cdh5 − retinas. ( n = 5 mice/group). Scale bars, 500 mm. f Matrigel plugs implanted into FUNDC1 f/Y Cdh5 − and FUNDC1 f/Y Cdh5 + mice for 10 days. Quantification of hemoglobin (Hb) extracted from matrigel plugs from FUNDC1 f/Y Cdh5 − and FUNDC1 f/Y Cdh5 + mice ( n = 5 mice/group). g CD31 immunostaining of matrigel plugs implanted into FUNDC1 f/Y Cdh5 − and FUNDC1 f/Y Cdh5 + mice for 10 days (left). Quantification of the relative CD31-positive area (fold-over control) (right). ( n = 5 mice/group). Scale bar, 100 µm. h Representative images showing blood flow reperfusion assessed by Doppler laser ultrasound on day 10 after ischemic injury. ( n = 5 mice/group). The ratio of ischemic/non-ischemic perfusion was quantitatively analyzed. i Representative images showing anti-CD31 immunostaining in gastrocnemius muscle on day 10 after femoral artery ligation. ( n = 5 mice/group). Scale bar, 100 µm. j , k Tumors following subcutaneous injection of Lewis lung carcinoma (LLC) tumor cells into the abdomen of FUNDC1 f/Y Cdh5 − and FUNDC1 f/Y Cdh5 + mice. After 28 days, tumors were harvested and quantified. Scale bar, 100 µm. j Representative images of tumors ( n = 5 mice/group). k Immunostaining of LLC tumor sections and quantification of relative CD31-positive areas. ( n = 5 mice/group). Scale bar, 100 µm. Statistical significance was assessed using two-tailed t-tests to compare two groups. * p < 0.05 vs Scr siRNA or Fundc1 f/Y Cdh5 − . All values are mean ± S.D.

Article Snippet: Antibodies against the following proteins were used as the primary antibodies: CD31 (BD Pharmingen, San Jose, CA; 550274; Cell Signaling Technology, Danvers, MA;77699); β-actin and GAPDH (Santa Cruz Biotechnology, Dallas, Texas; sc-47778 and sc-137179); PCNA, Calreticulin, Cyt C, VDAC1, mitofusin-2 (MFN2), SRF, phosphorylated (p) SRF, VEGFR1, VEGFR2, pVEGFR2, and VEGFR3 (Cell Signaling Technology, Danvers, MA; 13110, 12238, 4280,4661, 9482, 5147,4261, 2893, 2478, 2479, 3408); IP3R1 (Abcam, Cambridge, MA; ab5804; ThermoFisher, Waltham, MA, PA1-901); calnexin (Abcam, Cambridge, MA; ab22595); FUNDC1 (Aviva Systems Biology, San Diego, CA; ARP53281), FUNDC1 (Novus Biologicals, LLC, Littleton CO, NBP1-81063; EMD Millipore Corporation, Temecula, CA, ABC506).

Techniques: Transfection, Western Blot, Generated, Staining, Immunostaining, Control, Ligation, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: FUNDC1-dependent mitochondria-associated endoplasmic reticulum membranes are involved in angiogenesis and neoangiogenesis

doi: 10.1038/s41467-021-22771-3

Figure Lengend Snippet: a Angiogenesis PCR array for FUNDC1 f/Y Cdh5 − and FUNDC1 f/Y Cdh5 + endothelial cells. The heat map depicts relative expression values for the 30 discrepant genes ( n = 3 biologically independent samples per group). b HUVECs were transfected with Scr siRNA or FUNDC1 siRNA for 24 h, the protein level of vascular endothelial growth factor receptor 2 (VEGFR2) were determined by western blot assays ( n = 5 independent experiments). c HUVECs were infected with Ad-null or Ad-FUNDC1 for 24 h, and protein level of VEGFR2 were determined by RT-qPCR and western blot assays ( n = 5 independent experiments). d HUVECs were transfected with Scr siRNA or FUNDC1 siRNA for 24 h and then infected with adenovirus encoding control (Ad-Cont) or mitochondrial–endoplasmic reticulum (ER) linker (Ad-Linker) for another 24 h ( n = 5 biologically independent samples per group). Expression of VEGFR2 and FUNDC1 was determined by western blot assay. e HUVECs were transfected with Scr siRNA or FUNDC1 siRNA for 24 h and then infected with adenovirus encoding null or VEGFR2 for another 24 h ( n = 5 independent experiments). Expression of VEGFR2 and FUNDC1 was determined by western blot assay. f Representative images of spheroid-sprouting are shown. Sprout length in ten random microscopic fields per group were measured by NIH ImageJ and statistically analyzed. ( n = 5 independent experiments). Scale bar, 100 µm. g Matrigel containing vascular endothelial growth factor (VEGF) was injected subcutaneously into FUNDC1 f/Y Cdh5 − and FUNDC1 f/Y Cdh5 + mice that had received intravenous injections of VEGFR2 adenoviruses 24 h before. After 10 days, matrigel plugs were removed for analysis of new vessel formation by histological and Hb assays ( n = 5 mice/group) (top). Quantification of Hb extracted from Matrigel plugs from FUNDC1 f/Y Cdh5 − and FUNDC1 f/Y Cdh5 + mice ( n = 5 mice/group) (bottom). h Established LLC tumors approximately 90 mm 3 in size were treated with either intravenous injection of recombinant VEGFR2 adenoviruses or Ad-Null as control (n = 5 mice/group). Scale bar, 100 µm. After 28 days, the tumors were harvested and quantified. Representative images of tumors were shown. i Immunostaining of LLC tumor sections and quantification of relative CD31-positive area. ( n = 5 mice/group). Scale bar, 100 µm. Statistical significance was assessed using two-tailed t -tests for two groups and using one-way ANOVA with post hoc multiple comparisons test for multiple groups. * p < 0.05 vs Scr siRNA; Ad-Null or FUNDC1 f/Y Cdh5 − +Ad-Null. All values are mean ± S.D.

Article Snippet: Antibodies against the following proteins were used as the primary antibodies: CD31 (BD Pharmingen, San Jose, CA; 550274; Cell Signaling Technology, Danvers, MA;77699); β-actin and GAPDH (Santa Cruz Biotechnology, Dallas, Texas; sc-47778 and sc-137179); PCNA, Calreticulin, Cyt C, VDAC1, mitofusin-2 (MFN2), SRF, phosphorylated (p) SRF, VEGFR1, VEGFR2, pVEGFR2, and VEGFR3 (Cell Signaling Technology, Danvers, MA; 13110, 12238, 4280,4661, 9482, 5147,4261, 2893, 2478, 2479, 3408); IP3R1 (Abcam, Cambridge, MA; ab5804; ThermoFisher, Waltham, MA, PA1-901); calnexin (Abcam, Cambridge, MA; ab22595); FUNDC1 (Aviva Systems Biology, San Diego, CA; ARP53281), FUNDC1 (Novus Biologicals, LLC, Littleton CO, NBP1-81063; EMD Millipore Corporation, Temecula, CA, ABC506).

Techniques: Expressing, Transfection, Western Blot, Infection, Quantitative RT-PCR, Control, Injection, Recombinant, Immunostaining, Two Tailed Test

Journal: Nature Communications

Article Title: FUNDC1-dependent mitochondria-associated endoplasmic reticulum membranes are involved in angiogenesis and neoangiogenesis

doi: 10.1038/s41467-021-22771-3

Figure Lengend Snippet: a HUVECs were pre-transfected with or without FUNDC1 siRNA for 24 h, then treated with actinomycin D for different periods of time. RT-qPCR assay analysis of vascular endothelial growth factor receptor 2 (VEGFR2) mRNA levels ( n = 5 independent experiments). b HUVECs were pre-transfected with or without FUNDC1 siRNA for 24 h, then transfected with various VEGFR2 promoter truncation constructs or a mutant (serum response factor [SRF] binding site) construct for 24 h. After this, luciferase activity was measured ( n = 5 independent experiments). c Chromatin immunoprecipitation assays using an anti-SRF antibody to amplify VEGFR2 promoter in HUVECs transfected with or without FUNDC1 siRNA ( n = 5 independent experiments). d HUVECs were pre-transfected with or without FUNDC1 siRNA for 24 h and then treated with VEGF for 30 min. Expression of SRF and phosphorylated SRF at Ser103 (pSRF) was determined by western blot assay ( n = 5 independent experiments). e Cytosolic Ca 2+ levels, as indicated by the fluorescent probe Fluo-4, AM ( n = 5 independent experiments). HUVECs were transfected with Scr siRNA or FUNDC1 siRNA for 24 h and then exposed to VEGF (20 ng/mL) for the indicated times. f HUVECs were infected with adenovirus encoding control (Ad-Cont) or mitochondrial–endoplasmic reticulum (ER) linker (Ad-Linker) for 24 h and then treated with BAPTA-AM (10 µM) or control dimethylsulfoxide (vehicle) for 30 min. Levels of SRF and pSRF (Ser103) were detected by western blot assay ( n = 5 independent experiments). g HUVECs were pre-transfected with or without SRF siRNA for 24 h, then infected with adenovirus encoding control or mitochondria–ER linker for 24 h. VEGFR2 protein expression was detected by western blot assay ( n = 5 independent experiments). Statistical significance was assessed using two-tailed t -tests for two groups and one-way ANOVA with post hoc multiple comparisons test for multiple groups. *p < 0.05 vs Scr siRNA; Scr siRNA+VEGF; Ad-Cont+Vehicle or Ad-Cont+Scr siRNA. All values are mean ± S.D.

Article Snippet: Antibodies against the following proteins were used as the primary antibodies: CD31 (BD Pharmingen, San Jose, CA; 550274; Cell Signaling Technology, Danvers, MA;77699); β-actin and GAPDH (Santa Cruz Biotechnology, Dallas, Texas; sc-47778 and sc-137179); PCNA, Calreticulin, Cyt C, VDAC1, mitofusin-2 (MFN2), SRF, phosphorylated (p) SRF, VEGFR1, VEGFR2, pVEGFR2, and VEGFR3 (Cell Signaling Technology, Danvers, MA; 13110, 12238, 4280,4661, 9482, 5147,4261, 2893, 2478, 2479, 3408); IP3R1 (Abcam, Cambridge, MA; ab5804; ThermoFisher, Waltham, MA, PA1-901); calnexin (Abcam, Cambridge, MA; ab22595); FUNDC1 (Aviva Systems Biology, San Diego, CA; ARP53281), FUNDC1 (Novus Biologicals, LLC, Littleton CO, NBP1-81063; EMD Millipore Corporation, Temecula, CA, ABC506).

Techniques: Transfection, Quantitative RT-PCR, Construct, Mutagenesis, Binding Assay, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Expressing, Western Blot, Infection, Control, Two Tailed Test

Journal: Nature Communications

Article Title: FUNDC1-dependent mitochondria-associated endoplasmic reticulum membranes are involved in angiogenesis and neoangiogenesis

doi: 10.1038/s41467-021-22771-3

Figure Lengend Snippet: a Transduction efficiency of peptides 1, 2, and a control peptide in Human umbilical vein endothelial cells (HUVECs) ( n = 5 independent experiments). b HUVECs were treated with different peptides (20 μM) for 24 h, then subjected to immunoprecipitation with antibody against FUNDC1(HA) to quantify the interaction of FUNDC1 and IP3R1 ( n = 3 independent experiments). c HUVECs were treated with different peptides (20 μM) for 24 h, then cytosolic Ca 2+ was detected using the fluorescent probe Fluo-4, AM ( n = 5 independent experiments). d HUVECs were treated with different peptides. After 24 h, the cell lysates were collected and subjected to western blot assays to detect the expression of VEGFR2, IP3R1, SRF, and phosphorylated SRF at Ser103 (pSRF) ( n = 5 independent experiments). e Three-dimensional spheroids and representative images of spheroid-sprouting were analyzed ( n = 5 independent experiments). Scale bar, 100 µm. f Matrigel containing vascular endothelial growth factor (VEGF) was injected subcutaneously into 6-week-old wild-type mice, then the mice received an intravenous injection of the indicated peptides. After 10 days, matrigel plugs were removed for analysis of new vessel formation by histological and Hb assay ( n = 5 mice/group). Quantification of Hb extracted from matrigel plugs of different groups. g C57BL/6 mice with LLC tumors that were approximately 90 mm 3 in size were treated with intravenous injections of different peptides (10 mg/kg/3 days). After 28 days, the tumors were harvested and quantified. Representative images of tumors ( n = 5 mice/group). h Immunostaining of LLC tumor sections and quantification of relative CD31-positive area. ( n = 5 mice/group). Scale bar, 100 µm. Statistical significance was assessed using one-way ANOVA with post hoc multiple comparisons test for multiple groups. *p < 0.05 vs Ctrl peptide. All values are mean ± S.D.

Article Snippet: Antibodies against the following proteins were used as the primary antibodies: CD31 (BD Pharmingen, San Jose, CA; 550274; Cell Signaling Technology, Danvers, MA;77699); β-actin and GAPDH (Santa Cruz Biotechnology, Dallas, Texas; sc-47778 and sc-137179); PCNA, Calreticulin, Cyt C, VDAC1, mitofusin-2 (MFN2), SRF, phosphorylated (p) SRF, VEGFR1, VEGFR2, pVEGFR2, and VEGFR3 (Cell Signaling Technology, Danvers, MA; 13110, 12238, 4280,4661, 9482, 5147,4261, 2893, 2478, 2479, 3408); IP3R1 (Abcam, Cambridge, MA; ab5804; ThermoFisher, Waltham, MA, PA1-901); calnexin (Abcam, Cambridge, MA; ab22595); FUNDC1 (Aviva Systems Biology, San Diego, CA; ARP53281), FUNDC1 (Novus Biologicals, LLC, Littleton CO, NBP1-81063; EMD Millipore Corporation, Temecula, CA, ABC506).

Techniques: Transduction, Control, Immunoprecipitation, Western Blot, Expressing, Injection, Immunostaining