Journal: Journal of neuropathology and experimental neurology

Article Title: The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

doi: 10.1097/01.jnen.0000182979.56612.08

Figure Lengend Snippet: FIGURE 2. Mass spectrometric analysis of the 14-3-3-binding protein in NTera2-N cells. Spot 1 labeled with the rh14-3-3z probe (Fig. 1) was excised from the gel, trypsinized, and processed for nanoESI-MS/MS analysis. (A) The spectra of MS analysis. Each peak indicates individual peptide fragments. The positions of several peaks were automatically numbered on the spectra. The fragments were selected for MS analysis in order of their signal intensity, although autolytic fragments of trypsin (e.g. 412, 421, 523, 737, 762, and 767) were omitted. (B) Amino acid sequence of human Hsp60. Seventeen peptide fragments of spot 1 identified by MS analysis (shadowed) showed a perfect match with the sequence encompassing amino acid residues 38–493 of human Hsp60. The number indicated on each fragment represents the position in the horizontal axis of the spectra.

Article Snippet: After the probes and antibodies were stripped by incubating the membrane at 50 C for 30 minutes in stripping buffer composed of 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol, the membrane was relabeled with goat anti-HSP60 antibody (N-20; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques: Binding Assay, Labeling, Tandem Mass Spectroscopy, Sequencing

Journal: Journal of neuropathology and experimental neurology

Article Title: The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

doi: 10.1097/01.jnen.0000182979.56612.08

Figure Lengend Snippet: FIGURE 3. The interaction of 14-3-3 protein with Hsp60 was not zeta isoform-specific. Two hundred mi- crograms of total cellular protein of NTera2-N was separated on a 2- dimensional PAGE gel ([a, b]: pI 5.3– 6.3 and 4%–12%; [c, d]: pI 4–7 and 4%–12%), followed by silver stain- ing or phosphoprotein staining. The gel was processed for overlay with recombinant human 14-3-3g pro- tein (rh14-3-3g) tagged with Xpress, followed by relabeling with anti- Hsp60 antibody. (a) rh14-3-3z, (b) Hsp60, (c) silver staining, (d) phos- phoprotein staining. Spot 1 is in- dicated by an arrow.

Article Snippet: After the probes and antibodies were stripped by incubating the membrane at 50 C for 30 minutes in stripping buffer composed of 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol, the membrane was relabeled with goat anti-HSP60 antibody (N-20; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques: Silver Staining, Staining, Recombinant

Journal: Journal of neuropathology and experimental neurology

Article Title: The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

doi: 10.1097/01.jnen.0000182979.56612.08

Figure Lengend Snippet: FIGURE 4. The interaction of 14-3-3 protein with Hsp60 was universal in neuronal and nonneuronal cells. Two hundred micrograms of total cellular protein of human cell lines and brain homogenate was separated on a 2-dimensional PAGE gel ([a–l]: pI 5.3–6.3 and 4% to 12%; [m–r]: pI 4-7 and 4% to 12%) followed by silver staining (the left panels). The gel was processed for overlay with the rh14-3-3z probe tagged with Xpress or Myc (the center panels) followed by relabeling with anti-Hsp60 antibody (the right panels). (a–c) Undifferentiated NTera2 teratocarcinoma (NTera2-U), (d–f) SK-N-SH neuroblastoma, (g–i) U-373MG astrocytoma, (j–l) HeLa cervical carcinoma, (m–o) human neuronal progenitor (NP) cells, (p–r) brain homogenate.

Article Snippet: After the probes and antibodies were stripped by incubating the membrane at 50 C for 30 minutes in stripping buffer composed of 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol, the membrane was relabeled with goat anti-HSP60 antibody (N-20; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques: Silver Staining

Journal: Journal of neuropathology and experimental neurology

Article Title: The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

doi: 10.1097/01.jnen.0000182979.56612.08

Figure Lengend Snippet: FIGURE 6. Colocalization of 14-3-3 protein, Hsp60, and PrPC in the mitochondria of human neuronal progenitor cells. Human neuronal progenitor (NP) cells were immunolabeled with 14-3-3z isoform-specific antibody, anti-Hsp60 antibody, anti-prion protein antibody (8G8), MitoTracker Red CMXRos, or DAPI followed by staining with secondary antibodies. (a) 14-3-3z, (b) Hsp60, (c) merge of (a) and (b); (d) 14-3-3z, (e) CMXRos, (f) merge of (d) and (e) labeled with DAPI; (g) PrPC, (h) Hsp60, (i) merge of (g) and (h).

Article Snippet: After the probes and antibodies were stripped by incubating the membrane at 50 C for 30 minutes in stripping buffer composed of 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol, the membrane was relabeled with goat anti-HSP60 antibody (N-20; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques: Immunolabeling, Staining, Labeling

Journal: Journal of neuropathology and experimental neurology

Article Title: The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

doi: 10.1097/01.jnen.0000182979.56612.08

Figure Lengend Snippet: FIGURE 8. Coexpression of 14-3-3 protein, Hsp60, and PrPC in reactive astrocytes in the human brain. Serial sections derived from autopsied brains of multiple sclerosis were processed for immunohistochemis- try using 14-3-3z isoform-specific antibody, anti-Hsp60 antibody, an- ti-prion protein antibody (8G8), or anti-GFAP antibody. (a–d) Chronic active demyelinating lesions in the subcortical white matter of the frontal lobe of 744 multiple sclerosis: (a) 14-3-3z, (b) Hsp60, (c) PrPC, (d) GFAP.

Article Snippet: After the probes and antibodies were stripped by incubating the membrane at 50 C for 30 minutes in stripping buffer composed of 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol, the membrane was relabeled with goat anti-HSP60 antibody (N-20; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques: Derivative Assay

Journal: Journal of neuropathology and experimental neurology

Article Title: The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

doi: 10.1097/01.jnen.0000182979.56612.08

Figure Lengend Snippet: FIGURE 7. Coexpression of 14-3-3 protein, Hsp60, and PrPC in neurons in the human brain. Serial sections derived from autopsied brains of multiple sclerosis were processed for immunohistochemistry using 14-3-3z isoform-specific antibody, anti-Hsp60 antibody, anti-prion pro- tein antibody (8G8), or anti-neuron- specific enolase (NSE) antibody. (a– d) The cerebral cortex of the frontal lobe of #791 MS: (a) 14-3-3z, (b) Hsp60, (c) PrPC, (d) NSE.

Article Snippet: After the probes and antibodies were stripped by incubating the membrane at 50 C for 30 minutes in stripping buffer composed of 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol, the membrane was relabeled with goat anti-HSP60 antibody (N-20; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques: Derivative Assay, Immunohistochemistry

Journal: Journal of neuropathology and experimental neurology

Article Title: The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

doi: 10.1097/01.jnen.0000182979.56612.08

Figure Lengend Snippet: FIGURE 9. A possible mechanism for elevation of 14-3-3 protein in the cerebrospinal fluid of prion diseases. When affected with the pathoge- netic prion, the molecular complex composed of 14-3-3, Hsp60, and PrPC becomes disintegrated during the conversion of PrPC into PrPSc aggregates, which displace the 14- 3-3 protein from the complex, resulting in the release of 14-3-3 from degenerating neurons into the cerebrospinal fluid.

Article Snippet: After the probes and antibodies were stripped by incubating the membrane at 50 C for 30 minutes in stripping buffer composed of 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol, the membrane was relabeled with goat anti-HSP60 antibody (N-20; Santa Cruz Biotechnology) followed by incubation with HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques:

Journal: bioRxiv

Article Title: Unraveling mechanisms of iron acquisition in malaria parasites

doi: 10.1101/2024.05.10.587216

Figure Lengend Snippet: (A) Western blot analysis of endogenously tagged PfDMT1-5HA ± aTC. ACPL-GFP present in the NF54 PfMev parasite line was used as a loading control . (B) Western blot of episomally expressed PfDMT1-GFP and parental Dd2 parasites. Hsp60 was used as a loading control.

Article Snippet: P. falciparum HSP60 was used as a loading control in PfDMT1-GFP parasites using a rabbit HSP60 antibody (Novus NBP2-12734) at 1:1000 dilution and then probed with anti-rabbit IRDye680 (Licor 926-68023).

Techniques: Western Blot, Control

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Antioxidant removal damages mitochondria and activates mitophagy in iPSC-derived i3Neurons (A) WT i3Neurons expressing mito-EBFP2 were untreated or cultured in antioxidant (AOx) removal media for 3-4 h and stained with TMRM and SiR-tubulin. White arrowheads indicate mitochondria lacking membrane potential. (B) WT i3Neurons expressing mito-Keima (mt-Keima) were untreated, cultured in AOx removal media for 4-5 h or 6-7 h and stained with SiR-Tubulin. (C and D) WT i3Neurons were untreated or cultured in AOx removal media for 4 h and immunostained for phospho-Serine 65 ubiquitin (S65 UB), HSP60 and tubulin (C) and the percentage of mitochondrial volume positive for S65 UB was quantified (D). S65 UB is a product of PINK1/Parkin activity. White arrowheads point to S65 UB located on mitochondria. (E and F) WT i3Neurons were cultured in AOx removal media for 4 h and immunostained for ATG13, HSP60 and Tubulin (E) and the percentage of mitochondrial volume positive for ATG13 was quantified (F). Untreated samples shown in . White arrowheads indicate ATG13 puncta localised to mitochondria. (G and H) WT i3Neurons were untreated or cultured in AOx removal media for 4 h and immunostained for ATG9A, HSP60 and Tubulin (G) and the ATG9A intensity (Arbitrary unit (A.U.)) at axon was quantified (H). Data in (D), (F) and (H) are mean ± SD from three independent experiments. Two-way ANOVA. **p<0.005, ***p<0.001. n.s., not significant. Scale bars, 15 µm.

Article Snippet: Primaries were anti-mouse WIPI2 (Abcam; ab105459), anti-chicken HSP60 (Novus; NBP3-05536), and anti-rabbit Synapsin I (Novus; NB300-104).

Techniques: Derivative Assay, Expressing, Cell Culture, Staining, Membrane, Ubiquitin Proteomics, Activity Assay

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Antioxidant removal damages mitochondria and promotes mitophagy in iPSC-derived i3Neurons (A) Representative confocal images of untreated WT i3Neurons immunostained for S65 UB, HSP60 and Tubulin. Related to and . (B) Representative confocal images of untreated WT i3Neurons immunostained for ATG13, HSP60 and Tubulin. Related to and . Scale bars, 15 µm.

Article Snippet: Primaries were anti-mouse WIPI2 (Abcam; ab105459), anti-chicken HSP60 (Novus; NBP3-05536), and anti-rabbit Synapsin I (Novus; NB300-104).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Presynapse is a hotspot for axonal PINK1/Parkin dependent mitophagosome formation (A and B) WT i3Neurons were immunostained for Synapsin I (A) or Synaptophysin (B) and Tubulin. The Synapsin I and Synaptophysin stainings were displayed as RGB thermal spectrum. (C) Immunoblot (IB) for autophagy adaptors including OPTN, NDP52, TAX1BP1, NBR1 and p62 in iPSCs, day 3 pre-differentiated (Pre-Diff) iPSCs, i3Neurons and WT Hela cells. kDa, kilodaltons. (D and E) WT i3Neurons expressing EGFP-OPTN were cultured in AOx removal media for 4 h and immunostained for Synapsin I, EGFP, HSP60 and Tubulin (D) and the percentage of mitochondrial volume positive for OPTN was quantified (E). Untreated samples are shown in . The Synapsin I staining were displayed as RGB thermal spectrum. White arrowheads indicate OPTN puncta localised to mitochondria. (F and G) WT and PINK1 KO iNeurons were cultured in AOx removal media for 6 h and immunostained for ATG13, HSP60 and Tubulin (F) and the percentage of mitochondrial volume positive for ATG13 was quantified (G). Untreated samples shown in . White arrowheads point to ATG13 puncta localised to mitochondria. Data in (E) and (G) are mean ± SD from three independent experiments. Two-way ANOVA. **p<0.005, ***p<0.001. n.s., not significant. Scale bars, 15 µm.

Article Snippet: Primaries were anti-mouse WIPI2 (Abcam; ab105459), anti-chicken HSP60 (Novus; NBP3-05536), and anti-rabbit Synapsin I (Novus; NB300-104).

Techniques: Western Blot, Expressing, Cell Culture, Staining

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: The initiation of PINK1/Parkin dependent mitophagosome formation primarily occurs in presynapses (A) The expression of presynaptic marker Synapsin I and postsynaptic marker PSD95 were examined in iPSCs, iPSC-derived i3Neurons, ESCs and ESC-derived iNeurons using immunoblotting (IB). (B) Immunoblot for indicated autophagy adaptors and regulators in iPSCs, day 3 pre-differentiated (Pre-Diff) iPSCs, iPSC-derived i3Neurons, ESCs, day 7 Pre-Diff ESCs, ESC-derived iNeurons and WT Hela cells. Second from the bottom actin panel is the loading control for p62, FIP200, ULK1, TBK1 and GABARAPL1. The VCP panel is the loading control for NAP1, SINTBAD and LC3A. kDa, kilodaltons. Related to . (C) The expression of EGFP-OPTN in WT i3Neurons was confirmed by IB for OPTN and EGFP. (D) Representative images of untreated WT i3Neurons expressing EGFP-OPTN that were immunostained for Synapsin I, EGFP, HSP60 and Tubulin. The Synapsin I staining is displayed as RGB thermal spectrum. (E) Confirmation of PINK1 KO ESC-derived iNeurons by IB. (F) WT ESC-derived iNeurons were immunostained for Synapsin I. (G) Untreated WT and PINK1 KO iNeurons were immunostained for ATG13, HSP60 and Tubulin. Related to and . Scale bars, 15 µm.

Article Snippet: Primaries were anti-mouse WIPI2 (Abcam; ab105459), anti-chicken HSP60 (Novus; NBP3-05536), and anti-rabbit Synapsin I (Novus; NB300-104).

Techniques: Expressing, Marker, Derivative Assay, Western Blot, Control, Staining

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Confirmation that AOx removal-induced PINK1/Parkin mitophagy is OPTN dependent, and PINK1/Parkin mitophagy prevents axon degeneration. (A) Confirmation of OPTN KO ESC-derived iNeurons by immunoblotting. (B) Representative confocal images of WT and OPTN KO iNeurons with or without EGFP-OPTN expression cultured in AOx removal media for 6 h and immunostained for ATG13, HSP60 and TUJ1. White arrowheads point to ATG13 puncta localised to mitochondria. (C) Representative confocal images of untreated WT, PINK1 KO and OPTN KO iNeurons stained with MitoTracker Green (MTG), MitoSOX red (MSR) and SiR-Tubulin. Related to and . (D) Representative confocal images of untreated WT, PINK1 KO and OPTN KO iNeurons immunostained for Cleaved Caspase 3 (CC3), HSP60 and Tubulin. Related to . Scale bars, 15 µm.

Article Snippet: Primaries were anti-mouse WIPI2 (Abcam; ab105459), anti-chicken HSP60 (Novus; NBP3-05536), and anti-rabbit Synapsin I (Novus; NB300-104).

Techniques: Derivative Assay, Western Blot, Expressing, Cell Culture, Staining

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Evidence of presynaptic mitophagy in highly active and mature iPSC-derived midbrain neurons and human brain (A) Representative images (left) and quantification (right) of DIV90-DIV120 human iPSC-derived neuronal cultures expressing dopaminergic (first and third row) and substantia nigra pars compacta (second row) and neurons (MAP2+) versus astrocyte (GFP+; bottom row) identity markers (after 90-120 days in BrainPhys maturation medium. Means ± SEM. Each data point represents one replicate well containing an average of 5600 neurons (MAP2+) across 25 fields of view. (B-D) Whole-cell patch-clamping of healthy human iPSC-derived midbrain neurons after 142-164 days in BrainPhys maturation medium. Mean ± SEM. Each data point represents a single neuron patched. (B) Example of a live human neuron that has been filled with rhodamine from the patch pipette. Midbrain iPSC-derived neurons develop functional voltage-gated sodium channels. (C) Midbrain iPSC-derived neurons develop mature, repetitive action potentials. (D) Midbrain iPSC-derived neurons develop functional excitatory (AMPA) and inhibitory (GABA) postsynaptic circuits. (E) Representative images of DIV157 iPSC-derived midbrain neurons cultured in basal (left) or 6 h of AOx removal condition (middle and right) and immunostained for Synapsin I, WIPI2, HSP60 and Tubulin. White arrowheads indicate WIPI2 puncta localised to mitochondria. (F) Electron microscopy (EM) and the 3D rendered structures images were obtained from the open access dataset generated by Shapson-Coe et al., featuring high-resolution serial section EM of 1 mm of human temporal cortex . The presynapses were identified by the distinct bouton morphology along the axon (shown in 3D rendered panel) and the presence of presynaptic density (shown in EM overview or insets, indicated by orange arrowhead). Several examples of mitophagy events were observed in the presynapses, indicated by the autophagosomal membrane forming around the mitochondrion (white arrowhead). Different Z-plane of the EM images (bottom right panel) were examined to identify the completion of the autophagosomal membrane structure.

Article Snippet: Primaries were anti-mouse WIPI2 (Abcam; ab105459), anti-chicken HSP60 (Novus; NBP3-05536), and anti-rabbit Synapsin I (Novus; NB300-104).

Techniques: Derivative Assay, Expressing, Transferring, Functional Assay, Cell Culture, Electron Microscopy, Generated, Membrane

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Evidence of presynaptic mitophagy in highly active and mature iPSC-derived midbrain neurons and human brain (A) Representative images of DIV157 midbrain iPSC-derived neurons cultured in basal condition and immunostained for Synapsin I, WIPI2, HSP60 and Tubulin. White arrowhead indicates WIPI2 puncta localised to mitochondria. Related to . (B) Another example electron microscopy (EM) images of presynaptic mitophagy shown in the open access dataset generated by Shapson-Coe et al., featuring high-resolution serial section EM of 1 mm of human temporal cortex. The presynapses were identified by the distinct bouton morphology along axon (shown in 3D rendered panel) and the presence of presynaptic density (shown in EM overview or insets, indicated by orange arrowhead). Mitophagy was indicated by the autophagosomal membrane forming around the mitochondrion (white arrowhead). Different Z-plane of the EM images were examined to identify the completion of the autophagosomal membrane structure. Related to .

Article Snippet: Primaries were anti-mouse WIPI2 (Abcam; ab105459), anti-chicken HSP60 (Novus; NBP3-05536), and anti-rabbit Synapsin I (Novus; NB300-104).

Techniques: Derivative Assay, Cell Culture, Electron Microscopy, Generated, Membrane

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: PINK1/Parkin mitophagy protects neurons from oxidative stress-induced mitochondrial collapse and neuronal degeneration (A and B) WT, PINK1 KO and OPTN KO iNeurons were cultured in AOx removal media for 5-6 h and stained with MitoTracker Green (MTG), MitoSOX red (MSR) and SiR-Tubulin (A) and the percentage change in MSR to MTG ratio compared to untreated control was quantified (B). Untreated samples are shown in . (C-E, F-G) WT, PINK1 KO and OPTN KO iNeurons were treated with pan-caspase inhibitor QVD-OPh (QVD) and cultured in untreated or AOx removal conditions for 4 days, and were immunostained for Synapsin I, TOM20, Cytochrome c (Cyt c ) and Tubulin (C, F). The percentage of mitochondrial volume lacking Cyt c (D), the percentage of the number of presynapse with damaged mitochondria lacking Cyt c (E) and the average density of total mitochondria per presynapse (G) were quantified. (H) Schematic of the mechanism of reactive oxygen species (ROS) activating apoptosis. (I and J) WT, PINK1 KO and OPTN KO iNeurons were cultured in AOx removal media for 4 days and were immunostained for Cleaved Caspase 3 (CC3), HSP60 and Tubulin (I). The fold change of CC3 intensity per axon volume (J) was quantified. Untreated samples are shown in . White arrowheads point to axons with caspase 3 activation. Data in (B), (D), (E), (G) and (J) are mean ± SD from three independent experiments. Two-way ANOVA. p*<0.05, **p<0.005, ***p<0.001, ****p<0.0001. n.s., not significant. Scale bars, 15 µm.

Article Snippet: Primaries were anti-mouse WIPI2 (Abcam; ab105459), anti-chicken HSP60 (Novus; NBP3-05536), and anti-rabbit Synapsin I (Novus; NB300-104).

Techniques: Cell Culture, Staining, Control, Activation Assay

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Roxadustat protects PINK1/Parkin-mitophagy defective neurons from axon degeneration by restoring mitochondrial health. (A) Representative live cell confocal images of QVD treated control WT, PINK1 KO and OPTN KO iNeurons expressing mt-mKeima XL stained with SiR-Tubulin and Hoechst33342. Related to and . (B) Representative confocal images of untreated and 48 h 40 µM Roxadustat treated WT, PINK1 KO and OPTN KO iNeurons immunostained for Cleaved Caspase 3 (CC3), HSP60 and Tubulin. Related to and Scale bars, 15 µm. (C) Experimental overview of how the 4 days AOx removal, a combination of 4 days AOx removal with 40 µM Roxadustat treatment in the final 48 h and the 48 h 40 µM Roxadustat treatment were performed.

Article Snippet: Primaries were anti-mouse WIPI2 (Abcam; ab105459), anti-chicken HSP60 (Novus; NBP3-05536), and anti-rabbit Synapsin I (Novus; NB300-104).

Techniques: Control, Expressing, Staining

Journal: bioRxiv

Article Title: Presynapses are mitophagy pit stops that prevent axon degeneration

doi: 10.1101/2024.09.09.611943

Figure Lengend Snippet: Roxadustat restores mitochondrial health in PINK1/Parkin-mitophagy defective neurons and prevents axon degeneration. (A and B) WT, PINK1 KO and OPTN KO iNeurons were treated with either AOx removal media for 4 days or a combination of AOx removal for 4 days with 40 µM Roxadustat treatment in the final 48 h. The neurons were immunostained for Cleaved Caspase 3 (CC3), HSP60 and Tubulin (A). The percentage change of CC3 intensity per axon volume (B) was quantified. Untreated and 48 h 40 µM Roxadustat treated samples shown in . (C and D) The activation of Caspase-3, levels of synaptic proteins Synapsin I and PSD95, and the expression of PINK1/Parkin-independent mitophagy adaptor proteins BNIP3 and NIX were analysed through immunoblotting in WT, PINK1 KO and OPTN KO iNeurons that were untreated, treated with either AOx removal condition for 4 days, 40 µM Roxadustat for 48 h, a combination of AOx removal for 4 days with 40 µM Roxadustat treatment in the final 48 h, or QVD. The ratio of Cleaved Caspase-3 to total full length Caspase-3 were quantified (D). Data in (B) and (D) were mean ± SD from three independent experiments. Two-way ANOVA. ***p<0.001, ****p<0.0001. n.s., not significant. Scale bars, 15 µm.

Article Snippet: Primaries were anti-mouse WIPI2 (Abcam; ab105459), anti-chicken HSP60 (Novus; NBP3-05536), and anti-rabbit Synapsin I (Novus; NB300-104).

Techniques: Activation Assay, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Differential Subcellular Distribution and Translocation of Seven 14-3-3 Isoforms in Response to EGF and During the Cell Cycle

doi: 10.3390/ijms21010318

Figure Lengend Snippet: Subcellular localization of the pan14-3-3 protein in Cos-7 cells by double indirect immunofluorescence. ( A ) Co-localization of pan14-3-3 (red) and actin (green); ( B ) co-localization of pan14-3-3 protein (red) and tubulin (green); ( C ) co-localization of pan14-3-3 protein (red) and HSP60 (green); ( D ) co-localization of pan14-3-3 protein (red) and calnexin (green). For ( A – D ), the zoomed-in areas were used to calculate Mander’s coefficients. ( E ) Subcellular localization of pan14-3-3 protein during the cell cycle and in response to EGF. With or without EGF stimulation, pan14-3-3 proteins (red) and pEGFR (green) were revealed by double indirect immunofluorescence. Mitotic cells are labeled by * and endosomes are marked by arrows. Scale bar = 10 μm.

Article Snippet: Rabbit polyclonal anti-HSP60 antibody was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Immunofluorescence, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Differential Subcellular Distribution and Translocation of Seven 14-3-3 Isoforms in Response to EGF and During the Cell Cycle

doi: 10.3390/ijms21010318

Figure Lengend Snippet: Subcellular localization of 14-3-3β in Cos-7 cells by double indirect immunofluorescence. ( A ) Co-localization of 14-3-3β (red) and actin (green); ( B ) co-localization of 14-3-3β (red) and tubulin (green); ( C ) co-localization of 14-3-3β (red) and HSP60 (green); ( D ) co-localization of 14-3-3β (red) and calnexin (green). For ( A – D ), the zoomed-in areas were used to calculate Mander’s coefficients. ( E ) Subcellular localization of 14-3-3β during the cell cycle and in response to EGF. With or without EGF stimulation, 14-3-3β (red) and pEGFR (green) were revealed by double indirect immunofluorescence. Mitotic cells are labeled by * and endosomes are marked by arrows. Scale bar = 10 μm.

Article Snippet: Rabbit polyclonal anti-HSP60 antibody was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Immunofluorescence, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Differential Subcellular Distribution and Translocation of Seven 14-3-3 Isoforms in Response to EGF and During the Cell Cycle

doi: 10.3390/ijms21010318

Figure Lengend Snippet: Subcellular localization of 14-3-3η in Cos-7 cells by double indirect immunofluorescence. ( A ) Co-localization of 14-3-3η (red) and HSP60 (green). The zoomed-in areas were used to calculate Mander’s coefficients. ( B ) Subcellular localization of 14-3-3η during the cell cycle and in response to EGF. With or without EGF stimulation, 14-3-3η (red) and pEGFR (green) were revealed by double indirect immunofluorescence. Mitotic cells are labeled by * and endosomes are marked by arrows. Scale bar = 10 μm.

Article Snippet: Rabbit polyclonal anti-HSP60 antibody was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Immunofluorescence, Labeling

Journal: Clinical Cancer Research

Article Title: Inhibition of Melanoma Growth by Small Molecules That Promote the Mitochondrial Localization of ATF2

doi: 10.1158/1078-0432.ccr-12-2689

Figure Lengend Snippet: Figure 3. SBI-0089410 and SBI- 0087702 promote the translocation of endogenous ATF2 to the cytoplasm/mitochondria. A, representative images of WM793 melanoma cells following treatment with DMSO, SBI-0089410, or SBI- 0087702, showing increased localization of ATF2 to the cytoplasm and mitochondria (arrowheads). WM793 cells were cultured on glass coverslips and incubated with DMSO (0.2%, 24 hours), SBI-0089410 (10 mmol/L, 6 hours), or SBI-0087702 (10 mmol/L, 24 hours). The cells were then fixed and stained with specific anti-ATF2 antibodies (green channel), anti-HSP60 antibodies to label mitochondria (red channel), and DAPI to label nuclei (blue channel). The cells were imaged using a FluoView1000 confocal microscope with an 100 oil immersion objective. Scale bar, 10 mm. Similar results were obtained with UACC903 cells (data not shown). B, to measure ATF2 translocation, either the GFP fluorescence intensity of GFP-ATF2- expressing UACC903 (graph on left) or the ATF2 immunofluorescence intensity in WM793 and UACC903 cells (graphs on right) in the nucleus and cytoplasm were quantified using ImageJ software. Between 6 and 17 cells were analyzed for each condition. Quantification reveals a reduction of approximately 40% to 50% in the nuclear-to-cytoplasmic ATF2 ratio.

Article Snippet: The antibodies used were purchased as follows: ATF2 (#20F1), Stat3 (#9132), Cox IV (3E11), pATF2-T69/71 (#9225), pan-pPKC T514 (#9379), P38 (#9216), pP38 (#9212), pErk1/2 (#9106), Erk1/2 (#9102), PKC isoform sampler kit (#9960), pAkt (#9271), and Akt (#9272) from Cell Signaling Technologies; PKCe (C15), ATF2 (N96 & C19), b-tubulin (G8), p53 (FL393), pan-PKC (H300), and GFP (B2) from Santa Cruz Biotechnology, Inc.; pS729 PKCe (44977G) from Invitrogen; HSP60 (clone 24) and b-catenin (clone 14) from BD Pharmingen; Actin (ACTN05) from Thermo-Fisher; pT52 ATF2 antibodies were produced by PhosphoSolutions Inc..

Techniques: Translocation Assay, Cell Culture, Incubation, Staining, Microscopy, Expressing, Software