Journal: Cells

Article Title: Exploring the Interplay of RUNX2 and CXCR4 in Melanoma Progression

doi: 10.3390/cells13050408

Figure Lengend Snippet: Impact of RUNX2 expression on autophagy markers in melanoma cells. ( a ) Western blots of 1A/1B-light chain 3 (LC3) and p62 in MELHO and in the 1E7, 1B3, and 1F5 RUNX2 KO melanoma cell lines. ( b ) Western blots ( left ) and densitometry analyses ( right ) of LC3 and p62 protein levels in MELHO and in the 1F5 RUNX2 KO cell line mock-treated or treated with bafilomycin A1 or 3-methyladenine. ( c ) Western blots ( left ) and densitometry analyses ( right ) of RUNX2 and LC3 in the 1F5 cell line, in the absence or presence of transfection with the pcDNA3/RUNX2 plasmid at 1.3 µg/mL (RUNX2 ++) or 2.6 µg/mL (RUNX2 +++). ( d ) Western blots ( left ) and densitometry analyses ( right ) of beclin in MELHO and in 1F5 cells without or with transfection with the pcDNA3/RUNX2 plasmid. β-Actin was used as a loading control. Western blot images are representative of three independent experiments. Asterisks indicate statistically significant differences relative to the control (( b , c ), right ) or 1F5 RUNX2 KO melanoma cells ((( d ), right ); *: p < 0.05; **: p < 0.005; ***: p < 0.001). Original blots are presented in .

Article Snippet: The PVDF membranes were probed with specific primary antibodies, to detect RUNX2 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA, reference: 8486), CXCR4 (1:100 dilution; Abcam, Cambridge, UK, reference: ab124824), MMP13 (1:500 dilution; GeneTex, Irvine, CA, USA, reference: GTX 100665), RANKL (1:500 dilution; Santa Cruz Biotechnology, Dallas, TX, USA, reference: sc377079), LC3B (1:1000 dilution; Invitrogen, Waltham, MA, USA, reference: PA5-22939), p62/SQSTM1 (1:1000 dilution; Rockland, Baltimore, MD, USA, reference: 600-401-HB8), beclin 1 (1:1000 dilution; GeneTex, Irvine, CA, USA, reference: GTX133555), mTOR (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA, reference: 2983), P-mTOR (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA, reference: 5536), p70-S6 (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA, reference: 9202), P-p70-S6 (1:500 dilution; Cell Signaling Technology, Danvers, MA, USA, reference: 9205), and β-actin (1:10,000 dilution; Thermo Scientific, Waltham, MA, USA, reference: BA3R).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/mcb.01383-13

Figure Lengend Snippet: FIG. 3. Cdk inhibition down-regulates SQSTM1/p62 but fails to affect LC3 processing during

Article Snippet: The SDS-insoluble aggregates trapped on the filter were probed with anti-ubiquitin (Cell Signaling, Beverly, MA), anti-p62 (Santa Cruz Biotech, Santa Cruz, CA), or anti-Bik (ProSci, Poway, CA), respectively.

Techniques: Inhibition

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/mcb.01383-13

Figure Lengend Snippet: FIG. 4. SQSTM1/p62 down-regulation results in cargo loading failure and inefficient autophagy.

Article Snippet: The SDS-insoluble aggregates trapped on the filter were probed with anti-ubiquitin (Cell Signaling, Beverly, MA), anti-p62 (Santa Cruz Biotech, Santa Cruz, CA), or anti-Bik (ProSci, Poway, CA), respectively.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/mcb.01383-13

Figure Lengend Snippet: FIG. 5. Expression of SQSTM1/p62 diminishes the increased lethality of BH3-mimetics in p62-

Article Snippet: The SDS-insoluble aggregates trapped on the filter were probed with anti-ubiquitin (Cell Signaling, Beverly, MA), anti-p62 (Santa Cruz Biotech, Santa Cruz, CA), or anti-Bik (ProSci, Poway, CA), respectively.

Techniques: Expressing

Journal: Molecular and Cellular Biology

Article Title: Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik

doi: 10.1128/mcb.01383-13

Figure Lengend Snippet: FIG. 9. A mechanistic model of SQSTM1/p62 and NBK/Bik acting as novel molecular switches

Article Snippet: The SDS-insoluble aggregates trapped on the filter were probed with anti-ubiquitin (Cell Signaling, Beverly, MA), anti-p62 (Santa Cruz Biotech, Santa Cruz, CA), or anti-Bik (ProSci, Poway, CA), respectively.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Loss of the Essential Autophagy Regulators FIP200 or Atg5 Leads to Distinct Effects on Focal Adhesion Composition and Organization

doi: 10.3389/fcell.2020.00733

Figure Lengend Snippet: Loss of the autophagy regulators FIP200 or Atg5 results in inhibition of directional cell motility. (A) Wild-type and FIP200 knockout (KO) MCF10A cells processed for immunofluorescence microscopy and immunostained for LC3 (green). Nuclei are labeled with Hoechst (blue). Scale bar = 10 μm. (B) Western blot analysis on lysates harvested from wild-type and FIP200 KO MCF10A cells either left untreated or treated with Bafilomycin A1 (BfnA1) for 2 and 4 hours. Immunoblotting was performed against the indicated proteins. (C) Quantitation of western blot data illustrating the fold change in p62 and LC3-II levels in untreated and Bafilomycin treated cells. Levels of p62 and LC3-II were normalized to actin and represented as fold change from untreated wild-type control cells. Results represent at least 3 independent experiments and error bars are SEM. (D) MCF10A wild-type and FIP200 KO cells were subjected to a scratch wound assay and brightfield images were taken at initial wounding (0 h) and at 6, 12, and 24 hours post-wounding. Dotted white lines highlight wound edge. Scale bar = 200 μm. (E) Quantitation of scratch wound assay was performed and represented as % of wound closure, calculated as a percentage of total area of initial wound at 0 h. Results represent at least 3 independent experiments and errors bars illustrate SEM. (F) Western blot analysis on lysates harvested from wild-type and two clones of Atg5 KO MCF10A cells either left untreated or treated with BfnA1 for 2 and 4 hours. Immunoblotting was performed against the indicated proteins. (G) MCF10A wild-type and Atg5 KO cells were subjected to a scratch wound assay and brightfield images were taken at initial wounding (0 h) and at 6, 12, and 24 hours post-wounding. Dotted white lines highlight wound edge. Scale bar = 200 μm. (H) Quantitation of scratch wound assay was performed and represented as % of wound closure, calculated as a percentage of total area of initial wound at 0 h. Results represent at least 3 independent experiments and errors bars illustrate SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The antibodies used and their dilutions for western blot (WB) and immunofluorescence microscopy (IF) were as follows: FIP200/RB1CC1 rabbit polyclonal (Proteintech; 17250-1-Ab) WB 1:1000, IF 1:200; LC3B/MAP1LC3B rabbit polyclonal (Novus Biologicals; NB100-2220) WB 1:2000, IF 1:200; p62/SQSTM1 rabbit polyclonal (ProSci; 5449) WB 1:2000, IF 1:300; actin mouse monoclonal (612656) WB 1:3000, p-Akt ser473 rabbit monoclonal (560378) WB 1:800, fibronectin mouse monoclonal (610077) WB 1:2000, IF1:200; E-cadherin mouse monoclonal (610181) WB 1:4000, paxillin mouse monoclonal (610619) WB 1:2000, and FAK pY397 (61172) WB 1:1000, IF 1:200 were purchased from BD Transduction Laboratories; phospho-p44/42 MAPK (Erk1/2) (137F5) rabbit monoclonal (Cell Signalling Technology) WB 1:1000; FAK mouse polyclonal (BioLegend; 603801) WB 1:1000; vinculin mouse monoclonal (EMD Millipore; MAB3574) WB 1:2000, IF 1:200; Atg5 rabbit polyclonal (Cell Signalling; 2630S) WB 1:3000; GAPDH rabbit polyclonal (ProSci; 3781) WB 1:1000.

Techniques: Inhibition, Knock-Out, Immunofluorescence, Microscopy, Labeling, Western Blot, Quantitation Assay, Control, Scratch Wound Assay Assay, Clone Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Loss of the Essential Autophagy Regulators FIP200 or Atg5 Leads to Distinct Effects on Focal Adhesion Composition and Organization

doi: 10.3389/fcell.2020.00733

Figure Lengend Snippet: FIP200 or Atg5 depletion increases fibronectin-induced cell adhesion and spreading. (A) Western blot analysis of cell lysates harvested along the time course of fibronectin-induced MCF10A wild-type cell spreading. Immunoblotting was performed against the indicated proteins. Quantitation of p62 expression was normalized to actin and represented as the fold change from the zero time point (cells in suspension). Results represent at least 3 independent experiments. (B) Immunofluorescence microscopy of MCF10A cells following attachment and spreading on fibronectin for indicated time points. Cells were immunostained for p62 and LC3. Scale bar = 10 μm. Quantitation was performed on multiple images from random fields of view. The number of p62 or LC3 puncta (indicated with arrowheads) were quantified from 100 cells for p62 and 13–15 cells for LC3 across 3 independent experiments. Results represent the average number of p62 or LC3 puncta per cell. Error bars represent SEM. (C) Brightfield images across the time course of adhesion to fibronectin of MCF10A wild-type, FIP200 KO, and Atg5 KO cells. Higher magnification regions illustrate phenotypic differences and dotted line represents representative cell diameter. Scale bar = 100 μm. (D) Quantitation of individual cell area represented as square pixels were plotted across the time course of fibronectin adhesion. Results represent 50–100 cells per experiment, repeated at least 3 independent times. Black bar represents the mean and error bars are the SEM. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The antibodies used and their dilutions for western blot (WB) and immunofluorescence microscopy (IF) were as follows: FIP200/RB1CC1 rabbit polyclonal (Proteintech; 17250-1-Ab) WB 1:1000, IF 1:200; LC3B/MAP1LC3B rabbit polyclonal (Novus Biologicals; NB100-2220) WB 1:2000, IF 1:200; p62/SQSTM1 rabbit polyclonal (ProSci; 5449) WB 1:2000, IF 1:300; actin mouse monoclonal (612656) WB 1:3000, p-Akt ser473 rabbit monoclonal (560378) WB 1:800, fibronectin mouse monoclonal (610077) WB 1:2000, IF1:200; E-cadherin mouse monoclonal (610181) WB 1:4000, paxillin mouse monoclonal (610619) WB 1:2000, and FAK pY397 (61172) WB 1:1000, IF 1:200 were purchased from BD Transduction Laboratories; phospho-p44/42 MAPK (Erk1/2) (137F5) rabbit monoclonal (Cell Signalling Technology) WB 1:1000; FAK mouse polyclonal (BioLegend; 603801) WB 1:1000; vinculin mouse monoclonal (EMD Millipore; MAB3574) WB 1:2000, IF 1:200; Atg5 rabbit polyclonal (Cell Signalling; 2630S) WB 1:3000; GAPDH rabbit polyclonal (ProSci; 3781) WB 1:1000.

Techniques: Western Blot, Quantitation Assay, Expressing, Suspension, Immunofluorescence, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Loss of the Essential Autophagy Regulators FIP200 or Atg5 Leads to Distinct Effects on Focal Adhesion Composition and Organization

doi: 10.3389/fcell.2020.00733

Figure Lengend Snippet: Differential dysregulation of FAK pY397 trafficking in FIP200 and Atg5 KO cells. (A) MCF10A wild-type and FIP200 KO cells were subjected to fibronectin induced cell adhesion and fixed at indicated time points of 120 and 240 minutes post-plating. Immunostaining was performed against FAK pY397 (green) and p62 (red). Nuclei were labeled with Hoechst (blue). Arrowheads indicate FAK pY397 spots and arrows indicate FAK pY397 and p62 colocalization. (B) Quantitation of immunofluorescence images taken from cells subjected to fibronectin induced adhesion. The number of FAK pY397 puncta per cell were quantified from between 30 and 34 cells per group across random fields of view from 2 independent experiments. Each data point indicates a cell, the red bar indicates mean value and error bars represent SEM. **** p < 0.0001. Immunofluorescence microscopy performed on wild-type (WT) and Atg5 KO MCF10A cells following attachment to fibronectin. Cells were fixed at 120 min post-plating and immunostained in (C) for FAK pY397 (red) and FIP200 (green), and in (D) for FAK pY397 (red) and LC3 (green). Nuclei are labeled with Hoechst (blue). Arrowheads indicate areas of colocalization and arrows indicate FA-like immunostaining. Scale bars = 10 μm.

Article Snippet: The antibodies used and their dilutions for western blot (WB) and immunofluorescence microscopy (IF) were as follows: FIP200/RB1CC1 rabbit polyclonal (Proteintech; 17250-1-Ab) WB 1:1000, IF 1:200; LC3B/MAP1LC3B rabbit polyclonal (Novus Biologicals; NB100-2220) WB 1:2000, IF 1:200; p62/SQSTM1 rabbit polyclonal (ProSci; 5449) WB 1:2000, IF 1:300; actin mouse monoclonal (612656) WB 1:3000, p-Akt ser473 rabbit monoclonal (560378) WB 1:800, fibronectin mouse monoclonal (610077) WB 1:2000, IF1:200; E-cadherin mouse monoclonal (610181) WB 1:4000, paxillin mouse monoclonal (610619) WB 1:2000, and FAK pY397 (61172) WB 1:1000, IF 1:200 were purchased from BD Transduction Laboratories; phospho-p44/42 MAPK (Erk1/2) (137F5) rabbit monoclonal (Cell Signalling Technology) WB 1:1000; FAK mouse polyclonal (BioLegend; 603801) WB 1:1000; vinculin mouse monoclonal (EMD Millipore; MAB3574) WB 1:2000, IF 1:200; Atg5 rabbit polyclonal (Cell Signalling; 2630S) WB 1:3000; GAPDH rabbit polyclonal (ProSci; 3781) WB 1:1000.

Techniques: Immunostaining, Labeling, Quantitation Assay, Immunofluorescence, Microscopy