Journal: Journal of Cell Science

Article Title: Redefining colocalization analysis with a novel phasor mixing coefficient

doi: 10.1242/jcs.264388

Figure Lengend Snippet: The PMC recapitulates the expected quantification across a wide range of biomolecular association. (A–C) Examples illustrating how the PMC quantifies known high (A), moderate (B) and low (C) biomolecular associations. (A) Color mixing analysis of U2OS cells with mitochondria double-labeled by using an anti-TOMM20 antibody with both AF488-conjugated and AF555-conjugated secondary antibodies. The merged pseudo-color image (left; AF488 in cyan; AF555 in magenta) was extracted from the multispectral image. Scale bar: 10 µm. The phasor plot (middle) exhibits a single narrow cluster near the midpoint of the mixing band. The CMI (right) highlights essentially all mitochondria. The brighter regions near the nucleus reflect a slight difference in depth throughout the cell. (B) Color mixing analysis of a PtK2 cell with MHC labeled with AF488 and phosphorylated RLC labeled with AF568. The merged pseudo-color image (left; AF488 in cyan, AF568 in magenta) was extracted from the multispectral image. Scale bars: 20 µm. The phasor plot (middle) exhibits a single broad cluster centered near the midpoint of the mixing band. The cluster near the origin corresponds to the significant number of background pixels that are excluded from the mixing band. The CMI (right) highlights regions with significant mixing (solid white arrows) and minimal mixing (dashed white arrows). (C) Color mixing analysis of U2OS cells with mitochondria labeled with anti-TOMM20 antibody and an AF488-conjugated secondary antibody, and with nuclei labeled with Hoechst–JF549. The merged pseudo-color image (left; AF488 in cyan, JF549 in magenta) was extracted from the multispectral image. Scale bar: 10 µm. The phasor plot (middle) exhibits a largely bimodal cluster in which the two lobes sit near the two PCs. The mixing image (right) is largely uniformly cold, with only a region near the edge of the nucleus displaying any mixing. This small region reflects areas where the mitochondria extend over (or under) the nucleus but are still captured in the optical section. In the phasor plots, dashed gray lines indicate the bounds of the mixing band. The overlaid histograms display the S coordinate of the underling phasor cluster defined within the mixing band. (D) PCC, Manders' coefficients (M 1 and M 2 ) and the PMC (PMC 1 and PMC 2 ) for each example in A–C (from top to bottom). PCC and M 1,2 were calculated after applying an intensity threshold determined by the Otsu method (see Fig. S4 ). Importantly, PMC 1,2 capture the known association moving from high (top, A) to low (bottom, C) mixing. Individual N =3 points correspond to individual samples; line marker indicates the mean value.

Article Snippet: Rabbit IgG anti-TOMM20 (eLabScience, E-AB-81488) was diluted to 300 ng/ml in blocking buffer, and slides were incubated at 4°C in a humid, dark chamber overnight.

Techniques: Labeling, Marker

Journal: Journal of Cell Science

Article Title: Redefining colocalization analysis with a novel phasor mixing coefficient

doi: 10.1242/jcs.264388

Figure Lengend Snippet: The PMC recapitulates the expected quantification across a wide range of biomolecular association. (A–C) Examples illustrating how the PMC quantifies known high (A), moderate (B) and low (C) biomolecular associations. (A) Color mixing analysis of U2OS cells with mitochondria double-labeled by using an anti-TOMM20 antibody with both AF488-conjugated and AF555-conjugated secondary antibodies. The merged pseudo-color image (left; AF488 in cyan; AF555 in magenta) was extracted from the multispectral image. Scale bar: 10 µm. The phasor plot (middle) exhibits a single narrow cluster near the midpoint of the mixing band. The CMI (right) highlights essentially all mitochondria. The brighter regions near the nucleus reflect a slight difference in depth throughout the cell. (B) Color mixing analysis of a PtK2 cell with MHC labeled with AF488 and phosphorylated RLC labeled with AF568. The merged pseudo-color image (left; AF488 in cyan, AF568 in magenta) was extracted from the multispectral image. Scale bars: 20 µm. The phasor plot (middle) exhibits a single broad cluster centered near the midpoint of the mixing band. The cluster near the origin corresponds to the significant number of background pixels that are excluded from the mixing band. The CMI (right) highlights regions with significant mixing (solid white arrows) and minimal mixing (dashed white arrows). (C) Color mixing analysis of U2OS cells with mitochondria labeled with anti-TOMM20 antibody and an AF488-conjugated secondary antibody, and with nuclei labeled with Hoechst–JF549. The merged pseudo-color image (left; AF488 in cyan, JF549 in magenta) was extracted from the multispectral image. Scale bar: 10 µm. The phasor plot (middle) exhibits a largely bimodal cluster in which the two lobes sit near the two PCs. The mixing image (right) is largely uniformly cold, with only a region near the edge of the nucleus displaying any mixing. This small region reflects areas where the mitochondria extend over (or under) the nucleus but are still captured in the optical section. In the phasor plots, dashed gray lines indicate the bounds of the mixing band. The overlaid histograms display the S coordinate of the underling phasor cluster defined within the mixing band. (D) PCC, Manders' coefficients (M 1 and M 2 ) and the PMC (PMC 1 and PMC 2 ) for each example in A–C (from top to bottom). PCC and M 1,2 were calculated after applying an intensity threshold determined by the Otsu method (see Fig. S4 ). Importantly, PMC 1,2 capture the known association moving from high (top, A) to low (bottom, C) mixing. Individual N =3 points correspond to individual samples; line marker indicates the mean value.

Article Snippet: Cells were stained with rabbit anti-TOMM20 (eLabScience, E-AB-81488) at 1:500 and mouse anti-LAMP1 (eBioScience, 14-1079-80) at 1:200 in PBS including 0.1 mg/ml IgG-free bovine serum albumin.

Techniques: Labeling, Marker

Journal: bioRxiv

Article Title: A genome-wide atlas of human cell morphology

doi: 10.1101/2023.08.06.552164

Figure Lengend Snippet: Dilution factors of phenotypic probes

Article Snippet: For mitochondria labeling, the secondary anti-TOMM20 antibody, F(ab’)2-goat-anti-rabbit IgG (H+L) (Thermo Fisher #31239) was conjugated to Alexa Fluor 594-Azide (Thermo Fisher #A10270).

Techniques: Staining, Concentration Assay

Journal: Autophagy

Article Title: Impaired TFEB activation and mitophagy as a cause of PPP3/calcineurin inhibitor-induced pancreatic β-cell dysfunction.

doi: 10.1080/15548627.2022.2132686

Figure Lengend Snippet: Figure 1. Effect of FK506, a PPP3/calcineurin inhibitor, on mitophagy of INS-1 insulinoma cells after treatment with mitochondrial stressors. (A) INS-1 cells transfected with pMito-Keima plasmid were incubated with rotenone (Rot) or oligomycin+antimycin A (O/A) for 18 h in the presence or absence of FK506 pretreatment. Fluorescent microscopy was performed at excitation wavelengths of 440 and 590 nm to visualize fluorescence from mitochondrial Keima and that from acidic Mito- Keima delivered to lysosome, thus from mitophagy, respectively (left). The number of acidic Mito-Keima puncta per cell (middle) and Mito-Keima red:green ratio representing mitophagy (right) were calculated. Scale bar: 5 μm. n = 30 (Veh, vehicle; Rot, rotenone; O/A, oligomycin+antimycin A). (B) INS-1 cells transfected with mRFP-LC3 were treated for 24 h and stained with Ab to TOMM20, a mitochondrial outer membrane protein, to visualize autophagosomes colocalized with TOMM20, thus the occurrence of mitophagy (left). The number of RFP puncta colocalized with TOMM20 was counted (right). Scale bar: 5 μm. n = 20. (C) INS-1 cells transfected with pMito-Keima were cultured without mitochondrial stressors in the presence or absence of FK506 for 72 h. Fluorescent microscopy was performed as in (A) (left). The number of acidic Mito-Keima puncta per cell (middle) and Mito-Keima red:green ratio representing mitophagy (right) were calculated. Scale bar: 10 μm. n = 30. (D) INS-1 cells transfected with pMito-Keima were incubated in hypoxic chamber (1% O2) for 24 h with or without FK506. Fluorescence microscopy was performed as in (A) (left). The number of acidic Mito-Keima puncta per cell (right upper) and Mito-Keima red:green ratio representing mitophagy (right lower) were calculated. Scale bar: 5 μm. n = 20. (E and F) After O/A treatment of INS-1 cells for 1 (E) or 24 h (F) with or without FK506 pretreatment for 1 h, cells were stained with MitoTracker Green to visualize mitochondria (left). Average length of mitochondria was measured to estimate mitochondrial fission (right). Scale bar: 10 μm. n = 8. Cells in the rectangles were magnified. All data in this figure are the means ± SEM from more than three independent experiments. **P < 0.01, ***P < 0.001 by one- way ANOVA with Tukey’s test (A,B,D,E,F) or two-tailed t-test (C). ns: not significant.

Article Snippet: To visualize mitophagy in another way, cells transfected with the mRFPLC3 construct were treated with mitochondrial stressors and then incubated with anti-TOMM20 Ab (Cell Signaling Technology, 42406S; 1:200).

Techniques: Transfection, Plasmid Preparation, Incubation, Microscopy, Fluorescence, Staining, Membrane, Cell Culture, Two Tailed Test

Journal: Autophagy

Article Title: Impaired TFEB activation and mitophagy as a cause of PPP3/calcineurin inhibitor-induced pancreatic β-cell dysfunction.

doi: 10.1080/15548627.2022.2132686

Figure Lengend Snippet: Figure 2. Effect of FK506 on TFEB nuclear translocation. (A) After treatment of TFEB-GFP- transfected INS-1 cells with rotenone or O/A for 4 h with or without FK506 pretreatment for 1 h, fluorescent microscopy was conducted (left). TFEB nucleus:cytosol fluorescence ratio was determined using ImageJ software (right). Scale bar: 50 μm. n = 50. (B) After rotenone or O/A treatment of TFE3-GFP-transfected INS-1 cells with or without FK506 pretreatment as in (A), fluorescent microscopy was conducted (left). TFE3 nucleus:cytosol fluorescence ratio was determined (right). Scale bar: 50 μm. n = 50. (C) After treatment of INS-1 cells with rotenone or O/A with or without pretreatment with FK506, cells were fractionated by centrifugation. Cytoplasmic and nuclear fractions were subjected to immunoblot analysis using the indicated Abs. Densitometric values after ImageJ analysis of the band intensity normalized to that of GAPDH (middle) or LMNA (lamin A) band (right) were compared between groups. Representative immunoblots are presented (left). n = 3. (D) After transfection of INS-1 cells with TFEB-GFP or constitutively active TFEB∆N30-GFP together with mRFP-LC3, cells were treated with rotenone or O/A for 4 h with or without FK506 pretreatment for 1 h. After immunostaining with anti-TOMM20 Ab, mRFP-LC3 puncta colocalized with TOMM20 were visualized by confocal microscopy as in Figure 1B (left). The number of mRFP-LC3 puncta colocalized with TOMM20 was counted (right). Scale bar: 5 μm. n = 20. All data in this figure are the means ± SEM from more than three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Tukey’s test.

Article Snippet: To visualize mitophagy in another way, cells transfected with the mRFPLC3 construct were treated with mitochondrial stressors and then incubated with anti-TOMM20 Ab (Cell Signaling Technology, 42406S; 1:200).

Techniques: Translocation Assay, Transfection, Microscopy, Fluorescence, Software, Centrifugation, Western Blot, Immunostaining, Confocal Microscopy

Journal: Autophagy

Article Title: Impaired TFEB activation and mitophagy as a cause of PPP3/calcineurin inhibitor-induced pancreatic β-cell dysfunction.

doi: 10.1080/15548627.2022.2132686

Figure Lengend Snippet: Figure 5. Effect of FK506 on glucose profile and β-cell function in vivo. (A) C57BL/6 mice were treated with daily administration of 10 mg/kg FK506 for 8 weeks with or without combined administration of 50 mg/kg MSL-7 treatment three times a week. Non-fasting blood glucose (left) and body weight (right) were monitored. n = 5. (B) After treatment of C57BL/6 for 8 weeks as in (A), intraperitoneal GTT was performed (left). Area under the curve (AUC) was calculated (right). n = 11. (C) In mice treated with FK506 as in (A), insulinogenic index was calculated as described in the Materials and methods. n = 9–15. (D) Mitochondrial COX activity in pancreatic islets of mice treated as in (B) was determined as described in the Materials and methods, and expressed as the mean pixel intensity per islet area (right). Representative DAB images are presented (left). Scale bar: 50 μm. n = 5. (E) GFP-RFP-LC3-transgenic mice were treated with daily administration of 10 mg/kg FK506 for 8 weeks with or without combined administration of 50 mg/kg MSL-7 treatment three times a week for 8 weeks. The number of RFP puncta colocalized with TOMM20 in pancreatic islets was counted (middle). Representative fluorescence images of RFP puncta colocalized with TOMM20 are presented (left). The number of red puncta representing autolysosome or autophagic flux and that of yellow puncta representing autophagosome was counted (right). Arrows indicate RFP puncta colocalized with TOMM20. Scale bar: 5 μm. n = 8–10. (F and G) In pancreatic sections of the mice of (E), colocalization of LAMP2 spot, a lysosomal marker, with TOMM20 was estimated by Pearson’s correlation analysis (F). Representative fluorescence images are presented (G). Arrows indicate LAMP2 spots colocalized with TOMM20. Scale bar: 10 μm. n = 8. (H and I) %mitophagy level in pancreatic islets of FK506-injected Mito-Keima-transgenic mice with or without combined administration of MSL-7 was calculated as described in the Materials and method (H). Representative Mito-Keima red fluorescence images in pancreatic islets identified by insulin immunofluorescence are presented (I). Scale bar: 10 (boxed areas) or 20 µm. n = 6–7. Cells in the rectangles were magnified. All data in this figure are the means ± SEM from more than three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Tukey’s test (B-F) or two- way ANOVA with Bonferroni’s test (A,B). ns: not significant.

Article Snippet: To visualize mitophagy in another way, cells transfected with the mRFPLC3 construct were treated with mitochondrial stressors and then incubated with anti-TOMM20 Ab (Cell Signaling Technology, 42406S; 1:200).

Techniques: Cell Function Assay, In Vivo, Activity Assay, Transgenic Assay, Fluorescence, Marker, Injection, Immunofluorescence