Journal: Signal Transduction and Targeted Therapy

Article Title: Cancer cell employs a microenvironmental neural signal trans -activating nucleus-mitochondria coordination to acquire stemness

doi: 10.1038/s41392-023-01487-4

Figure Lengend Snippet: ATF1 depletion impairs mitochondrial rejuvenation. a Representative images of the mitochondria in iDox-shATF1 MDA-MB-231 (Dox −/+) and iDox-shATF1-ATF1 rescued cells under the electron microscope. Bar graphs show the results from the morphometric analysis of cristae number or cristae length/mitochondrial area in cells ( n = 20 mitochondria/group; mean ± SD). Scale bar, 0.2 μm. b Mitochondrial ROS as indicated by flow cytometry analysis of MitoSOX Red. Parental and iDox-shATF1#1/#4 MDA-MB-231 cells were treated with Dox for 3 days or 6 days ( n = 3; mean ± SD). c Mitochondrial turnover determined by flow cytometry analysis of cells 48 hr after transiently transfected with mitoTimer (left panel. n = 3; mean ± SD). d Mitochondrial turnover as determined by flow cytometry analysis of cells 48 hr after transiently transfected with mitoTimer in iDox-shATF1 H460, A549 and T47D cells ( n = 3; mean ± SD). e Percentages of cells with damaged mitochondria as indicated by MitoTracker Deep-Red low Green high cells ( n = 3; mean ± SD). For a – e , P values, Tukey’s multiple comparisons after 1-way ANOVA. f Representative plots showing mitochondrial damage as determined by flow cytometry analysis of MitoTracker Deep-Red and MitoTracker Green. The iDox-shATF1#1/#4 MDA-MB-231 cells were treated with Dox for 4 days, challenged with CCCP for 6 h before MitoTracker staining. g Percentages of cells with damaged mitochondria as indicated by MitoTracker Deep-Red low Green high cells treated with CCCP in combination with dynein inhibitor (DynI). n = 3; mean ± SD; P values, Tukey’s multiple comparisons after 1-way ANOVA. h Immunofluorescence of Tom20 and alpha tubulin in iDox-shATF1#1 MDA-MB-231 cells treated with Dox for 4 days. Scale bars, 20 μm. i Left panel, mitochondria localization determined by confocal microscopy using MitoTracker Red staining. Right panel, MitoTracker Red intensity (mean ± SD) as a function of distance to nuclei was analyzed ( n = 28 for CT group, n = 52 for Dox group and n = 20 for wild-type rescued group, respectively). Scale bar, 20 μm. j Sphere formation of iDox-shATF1 MDA-MB-231 cells with/without Dox treatment, cells were rescued with MitoTempo (MitoT, 10, 20, 30 μM), and collected for sphere forming assays. k Sphere formation of iDox-shATF1 MDA-MB-231 cells with/without Dox treatment, cells were rescued with MitoQ (0.05 μM), and analyzed for sphere forming capacity (for j – k , n = 3; mean ± SD; P values, Tukey’s multiple comparisons after 1-way ANOVA). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns not significant

Article Snippet: Scale bar, 20 μm. j Sphere formation of iDox-shATF1 MDA-MB-231 cells with/without Dox treatment, cells were rescued with MitoTempo (MitoT, 10, 20, 30 μM), and collected for sphere forming assays. k Sphere formation of iDox-shATF1 MDA-MB-231 cells with/without Dox treatment, cells were rescued with MitoQ (0.05 μM), and analyzed for sphere forming capacity (for j – k , n = 3; mean ± SD; P values, Tukey’s multiple comparisons after 1-way ANOVA).

Techniques: Microscopy, Flow Cytometry, Transfection, Staining, Immunofluorescence, Confocal Microscopy

Journal:

Article Title: Schistosomiasis-Induced Experimental Pulmonary Hypertension

doi: 10.2353/ajpath.2010.100063

Figure Lengend Snippet: Vascular remodeling and PH after S. mansoni infection. A: Arterial remodeling was observed by H&E staining, as was a perivascular inflammatory cell infiltrate (arrowheads; Scale bars: 50 μm). B: With S. mansoni infection, thickening of the medial (representative images of wild-type (WT), IL-13Rα1−/−, and IL-13Rα2−/− mouse lungs) and intimal (seen predominantly in IL-13Rα2−/− mouse lungs) layers was observed based on immunofluorescence for α-smooth muscle actin (SMA), identifying vascular media, and thrombomodulin (TM), identifying vascular intima (Scale bars: 50 μm). C: Quantification of vascular remodeling confirmed increased media thickness in the wild-type and IL-13Rα2−/− infected mice (three to five animals per group; analysis of variance, P < 0.001 for media comparison; for full posthoc analysis results see Supplemental Table 1 at http://ajp.amjpathol.org). D: Right ventricular catheterization demonstrated increased pressure in the infected groups (four to five animals per group; P = 0.018 by Kruskal-Wallis one-way analysis of variance on ranks, *P < 0.005 by posthoc pairwise multiple comparison by Dunn’s method; for full posthoc analysis results see Supplemental Table 3 at http://ajp.amjpathol.org) E: Quantification of intima thickness demonstrated an increase only in the IL-13Rα2−/− infected mice (three to five animals per group; analysis of variance P = 0.003; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 by posthoc Tukey test for both media and intima analysis; for full posthoc analysis results see Supplemental Table 2 at http://ajp.amjpathol.org).

Article Snippet: Human Tissue Immunostaining Immunohistochemistry and immunofluorescence staining on formalin-fixed and paraffin embedded samples of lung tissue obtained at autopsy from two patients who died of schistosomiasis-associated PAH in Brazil was performed using the reagents listed in the . table ft1 table-wrap mode="anchored" t5 caption a7 Immunostain Antigen retrieval Block Primary antibody Secondary antibody Tertiary reagent p-Smad2/3 Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300) 5% Horse serum in PBS 1 hour at room temperature 1/2000 overnight at 4°C (Cell Signaling Technology 3101) 1/100 AF488 Donkey anti-Rabbit (Invitrogen A21206) 1 hour at room temperature None α-Smooth muscle actin Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300) 5% Goat serum in PBS 1 hour at room temperature 1/200 1 hour at room temperature (DakoCytomation M0851) 1/100 AF594 Goat anti-mouse (Invitrogen A11005) 1 hour at room temperature None CD31 Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300) 1.5% Rabbit serum in PBS 1 hour at room temperature 1:500 1 hour at room temperature (antibody courtesy of Lee Lab, Mayo Clinic) 1/100 Biotin-bound rabbit anti-rat (DakoCytomation E0468) 1 hour at room temperature Strepavidin-horseradish peroxidase (Vector Laboratories SA-5704), then DAB 5 minutes, then hematoxylin Open in a separate window Reagents for Immunostains on Human Tissue Statistics Analysis of variance was used to compare multiple groups (Kruskal-Wallis one-way analysis of variance on ranks for nonnormally distributed data), and the posthoc Tukey test (for normally distributed data) or the posthoc pairwise multiple comparison by Dunn’s method (for nonnormally distributed data) was used to identify individual statistical differences within the groups.

Techniques: Infection, Staining, Immunofluorescence

Journal:

Article Title: Schistosomiasis-Induced Experimental Pulmonary Hypertension

doi: 10.2353/ajpath.2010.100063

Figure Lengend Snippet: Vascular and inflammatory cell composition and evidence of cell proliferation in pulmonary peri-egg granulomas in infected mice. Granulomas in wild-type (upper panels), IL-13Rα1−/− (middle panels), and IL-13Rα2−/− (lower panels) infected mice are composed of smooth muscle cells or myofibroblasts (αSM-actin, α-smooth muscle actin), macrophages (MAC-3) and eosinophils (MBP, major basic protein). Considerable proliferation was also detected (PCNA, proliferating cell nuclear antigen) (Scale bars: 100 μm).

Article Snippet: Human Tissue Immunostaining Immunohistochemistry and immunofluorescence staining on formalin-fixed and paraffin embedded samples of lung tissue obtained at autopsy from two patients who died of schistosomiasis-associated PAH in Brazil was performed using the reagents listed in the . table ft1 table-wrap mode="anchored" t5 caption a7 Immunostain Antigen retrieval Block Primary antibody Secondary antibody Tertiary reagent p-Smad2/3 Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300) 5% Horse serum in PBS 1 hour at room temperature 1/2000 overnight at 4°C (Cell Signaling Technology 3101) 1/100 AF488 Donkey anti-Rabbit (Invitrogen A21206) 1 hour at room temperature None α-Smooth muscle actin Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300) 5% Goat serum in PBS 1 hour at room temperature 1/200 1 hour at room temperature (DakoCytomation M0851) 1/100 AF594 Goat anti-mouse (Invitrogen A11005) 1 hour at room temperature None CD31 Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300) 1.5% Rabbit serum in PBS 1 hour at room temperature 1:500 1 hour at room temperature (antibody courtesy of Lee Lab, Mayo Clinic) 1/100 Biotin-bound rabbit anti-rat (DakoCytomation E0468) 1 hour at room temperature Strepavidin-horseradish peroxidase (Vector Laboratories SA-5704), then DAB 5 minutes, then hematoxylin Open in a separate window Reagents for Immunostains on Human Tissue Statistics Analysis of variance was used to compare multiple groups (Kruskal-Wallis one-way analysis of variance on ranks for nonnormally distributed data), and the posthoc Tukey test (for normally distributed data) or the posthoc pairwise multiple comparison by Dunn’s method (for nonnormally distributed data) was used to identify individual statistical differences within the groups.

Techniques: Infection

Journal:

Article Title: Schistosomiasis-Induced Experimental Pulmonary Hypertension

doi: 10.2353/ajpath.2010.100063

Figure Lengend Snippet: p-Smad2/3 signaling is present in vascular lesions of patients who died of schistosomiasis-associated PAH. Tissue from two patients was immunostained. A: Costaining for α-smooth muscle actin (αSM-actin) and p-Smad2/3 reveals nuclear activity in the media of vessels with thickened media (arrowheads mark representative positive cells). B: Costaining for CD31 and p-Smad2/3 reveals nuclear activity in many cells present within plexiform lesions (arrowheads mark representative positive cells) (Scale bars: 50 μm.)

Article Snippet: Human Tissue Immunostaining Immunohistochemistry and immunofluorescence staining on formalin-fixed and paraffin embedded samples of lung tissue obtained at autopsy from two patients who died of schistosomiasis-associated PAH in Brazil was performed using the reagents listed in the . table ft1 table-wrap mode="anchored" t5 caption a7 Immunostain Antigen retrieval Block Primary antibody Secondary antibody Tertiary reagent p-Smad2/3 Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300) 5% Horse serum in PBS 1 hour at room temperature 1/2000 overnight at 4°C (Cell Signaling Technology 3101) 1/100 AF488 Donkey anti-Rabbit (Invitrogen A21206) 1 hour at room temperature None α-Smooth muscle actin Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300) 5% Goat serum in PBS 1 hour at room temperature 1/200 1 hour at room temperature (DakoCytomation M0851) 1/100 AF594 Goat anti-mouse (Invitrogen A11005) 1 hour at room temperature None CD31 Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300) 1.5% Rabbit serum in PBS 1 hour at room temperature 1:500 1 hour at room temperature (antibody courtesy of Lee Lab, Mayo Clinic) 1/100 Biotin-bound rabbit anti-rat (DakoCytomation E0468) 1 hour at room temperature Strepavidin-horseradish peroxidase (Vector Laboratories SA-5704), then DAB 5 minutes, then hematoxylin Open in a separate window Reagents for Immunostains on Human Tissue Statistics Analysis of variance was used to compare multiple groups (Kruskal-Wallis one-way analysis of variance on ranks for nonnormally distributed data), and the posthoc Tukey test (for normally distributed data) or the posthoc pairwise multiple comparison by Dunn’s method (for nonnormally distributed data) was used to identify individual statistical differences within the groups.

Techniques: Activity Assay

Journal:

Article Title: Schistosomiasis-Induced Experimental Pulmonary Hypertension

doi: 10.2353/ajpath.2010.100063

Figure Lengend Snippet: Reagents for Immunostains on Mouse Tissue

Article Snippet: Human Tissue Immunostaining Immunohistochemistry and immunofluorescence staining on formalin-fixed and paraffin embedded samples of lung tissue obtained at autopsy from two patients who died of schistosomiasis-associated PAH in Brazil was performed using the reagents listed in the . table ft1 table-wrap mode="anchored" t5 caption a7 Immunostain Antigen retrieval Block Primary antibody Secondary antibody Tertiary reagent p-Smad2/3 Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300) 5% Horse serum in PBS 1 hour at room temperature 1/2000 overnight at 4°C (Cell Signaling Technology 3101) 1/100 AF488 Donkey anti-Rabbit (Invitrogen A21206) 1 hour at room temperature None α-Smooth muscle actin Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300) 5% Goat serum in PBS 1 hour at room temperature 1/200 1 hour at room temperature (DakoCytomation M0851) 1/100 AF594 Goat anti-mouse (Invitrogen A11005) 1 hour at room temperature None CD31 Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300) 1.5% Rabbit serum in PBS 1 hour at room temperature 1:500 1 hour at room temperature (antibody courtesy of Lee Lab, Mayo Clinic) 1/100 Biotin-bound rabbit anti-rat (DakoCytomation E0468) 1 hour at room temperature Strepavidin-horseradish peroxidase (Vector Laboratories SA-5704), then DAB 5 minutes, then hematoxylin Open in a separate window Reagents for Immunostains on Human Tissue Statistics Analysis of variance was used to compare multiple groups (Kruskal-Wallis one-way analysis of variance on ranks for nonnormally distributed data), and the posthoc Tukey test (for normally distributed data) or the posthoc pairwise multiple comparison by Dunn’s method (for nonnormally distributed data) was used to identify individual statistical differences within the groups.

Techniques: Blocking Assay, Plasmid Preparation, Avidin-Biotin Assay, TUNEL Assay

Journal:

Article Title: Schistosomiasis-Induced Experimental Pulmonary Hypertension

doi: 10.2353/ajpath.2010.100063

Figure Lengend Snippet: Reagents for Immunostains on Human Tissue

Article Snippet: Human Tissue Immunostaining Immunohistochemistry and immunofluorescence staining on formalin-fixed and paraffin embedded samples of lung tissue obtained at autopsy from two patients who died of schistosomiasis-associated PAH in Brazil was performed using the reagents listed in the . table ft1 table-wrap mode="anchored" t5 caption a7 Immunostain Antigen retrieval Block Primary antibody Secondary antibody Tertiary reagent p-Smad2/3 Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300) 5% Horse serum in PBS 1 hour at room temperature 1/2000 overnight at 4°C (Cell Signaling Technology 3101) 1/100 AF488 Donkey anti-Rabbit (Invitrogen A21206) 1 hour at room temperature None α-Smooth muscle actin Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300) 5% Goat serum in PBS 1 hour at room temperature 1/200 1 hour at room temperature (DakoCytomation M0851) 1/100 AF594 Goat anti-mouse (Invitrogen A11005) 1 hour at room temperature None CD31 Citrate buffer 30 minutes in steamer (Vector Laboratories H-3300) 1.5% Rabbit serum in PBS 1 hour at room temperature 1:500 1 hour at room temperature (antibody courtesy of Lee Lab, Mayo Clinic) 1/100 Biotin-bound rabbit anti-rat (DakoCytomation E0468) 1 hour at room temperature Strepavidin-horseradish peroxidase (Vector Laboratories SA-5704), then DAB 5 minutes, then hematoxylin Open in a separate window Reagents for Immunostains on Human Tissue Statistics Analysis of variance was used to compare multiple groups (Kruskal-Wallis one-way analysis of variance on ranks for nonnormally distributed data), and the posthoc Tukey test (for normally distributed data) or the posthoc pairwise multiple comparison by Dunn’s method (for nonnormally distributed data) was used to identify individual statistical differences within the groups.

Techniques: Blocking Assay, Plasmid Preparation