Journal: bioRxiv

Article Title: Cell-state specific aggresome formation in fibroblasts is driven by stress-activated MAPK signaling

doi: 10.1101/2021.07.23.453501

Figure Lengend Snippet: p38α MAPK and p38β MAPK inhibition with TAK-715 suppresses aggresome formation in fibroblasts. A-B) Average DESeq2 log2(fold change) values for all MAPK cascade or Regulation of stress-activated MAPK cascade genes as proliferating (purple), senescent (blue), quiescent (red) and immortalized (orange) fibroblasts respond to MG132 treatment. C) Proliferating, senescent, quiescent, and immortalized fibroblasts and HEK293T cells were treated with 0.1% DMSO or 10 µM MG132 for 8 hours and then total protein was analyzed by western blot for actin, p62 and P-p62 T269/S272. D-E) Quiescent fibroblasts were treated with 50 µM TAK-715 or 1% DMSO for 24 hours, then additionally treated with 10 µM MG132 for 7 hours still in the presence of TAK-715 or DMSO prior to being immunostained for K48 polyUb (green) and Hoechst (nucleus; blue). Samples were analyzed for the proportion of cells forming the aggresome (E). F-G) Immortalized fibroblasts were treated with 10 µM MG132 and either 50 µM TAK-715 or 1% DMSO for 7 hours prior to being immunostained for K48 polyUb (green) and Hoechst (nucleus; blue). Samples were analyzed for the proportion of cells forming the aggresome (G). H) Proliferating, senescent, quiescent, and immortalized fibroblasts were treated with 0.1% DMSO or 10 µM MG132 for 8 hours and then total protein was analyzed by western blot for actin, p38 MAPK (p38) and P-p38 MAPK (P-p38; T180/Y182). Aggresomes are denoted by white arrows. Scale bars, 10 µm. **p<0.01, ***p<0.001.

Article Snippet: Western blots probing for p62 (1:1,000; Sigma-Aldrich P0067), P-p62 T269/S272 (1:1,000; PhosphoSolutions p196-269), actin (1:1,000; Bio Rad VMA00048), HSP90β (1:1,000; GeneTex GTX101448), p38 MAPK (1:1,000; Cell Signaling Technology 8690L), P-p38 MAPK (1:1,000; Cell Signaling Technology 4511S) and puromycin (1:1,000; Millipore Sigma MABE343) were performed using the following protocol.

Techniques: Inhibition, Western Blot

Journal: bioRxiv

Article Title: Cell-state specific aggresome formation in fibroblasts is driven by stress-activated MAPK signaling

doi: 10.1101/2021.07.23.453501

Figure Lengend Snippet: TAK1 inhibition supresses aggresome formation in fibroblasts upstream of p38 MAPKs and independent of p62 S351 phosphorylation. A-B) Quiescent fibroblasts were treated with 5 µg/mL 5Z-7-Oxozeaenol or 0.05% DMSO for 24 hours, then additionally treated with 10 µM MG132 for 7 hours still in the presence of TAK-715 or DMSO prior to being immunostained for K48 polyUb (green) and Hoechst (nucleus; blue). Samples were analyzed for the proportion of cells forming the aggresome (B). C-D) Immortalized fibroblasts were treated with 10 µM MG132 and either 5 µg/mL 5Z-7-Oxozeaenol or 0.05% DMSO for 7 hours prior to being immunostained for K48 polyUb (green) and Hoechst (nucleus; blue). Samples were analyzed for the proportion of cells forming the aggresome (D). E) Proliferating, senescent, quiescent, and immortalized fibroblasts were treated with 0.1% DMSO or 10 µM MG132 for 8 hours and then immunostained for P-p62 S351 (green) and stained for nuclei (Hoechst; blue). F-G) Quiescent fibroblasts were transduced with a vector driving expression of either WT or S351A p62-GFP, treated with 10 µM MG132 for 8 hours and then immunostained for GFP (green) and Hoechst (nuclei; blue). Samples were analyzed for the proportion of cells forming the aggresome (G). H-I) Quiescent fibroblasts were treated with 10 µM MG132 and/or 5 µg/mL 5Z-7-Oxozeaenol for 8 hours as indicated in panel H and then analyzed for expression p38 MAPKs, actin and P-p38 MAPKs (T180/Y182) by western blot. J) A working model for the role of MAPK signaling in aggresome formation in adult mouse dermal fibroblasts. Small red circles depict phosphorylations. Aggresomes are denoted by white arrows. Scale bars, 10 µm. *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: Western blots probing for p62 (1:1,000; Sigma-Aldrich P0067), P-p62 T269/S272 (1:1,000; PhosphoSolutions p196-269), actin (1:1,000; Bio Rad VMA00048), HSP90β (1:1,000; GeneTex GTX101448), p38 MAPK (1:1,000; Cell Signaling Technology 8690L), P-p38 MAPK (1:1,000; Cell Signaling Technology 4511S) and puromycin (1:1,000; Millipore Sigma MABE343) were performed using the following protocol.

Techniques: Inhibition, Phospho-proteomics, Staining, Transduction, Plasmid Preparation, Expressing, Western Blot

Journal: Frontiers in immunology

Article Title: Palmatine treats urticaria by reducing inflammation and increasing autophagy.

doi: 10.3389/fimmu.2023.1268467

Figure Lengend Snippet: FIGURE 6 Immunofluorescence staining (A) and fluorescence intensity (B) of P62. Beclin-1, LC3-I, LC3-II, P62, and GAPDH protein bands in skin samples from the Control, OVA, OVA+LOR and OVA+PAL groups of rats (C). Graphs showing the levels of expression of Beclin-1 (D), P62 (E), LC3-I (F), and LC3-II (G) and LC3-II/LC3-I (H) in the four rat groups. Data are presented as mean ± standard deviation (SD). #P<0.05, ##P<0.01, ###P<0.001 vs. Control group. *P<0.05, ***P<0.001 vs. OVA group. OVA, ovalbumin; LOR, loratadine; PAL, palmatine.

Article Snippet: The membranes were stained with Rexchip Red S dye for 5 min, washed twice with TBST, permeated with TBS, transferred to a sealing solution containing 5% skimmed milk powder in TBST, and sealed by shaking at room temperature for 1 h. The membrane was washed with TBST, sealed, and incubated overnight at 4°C with primary antibody to beclin-1 (1:1000; 66665-1- Ig, Prointech), LC3 A/B (1:1000; 4108, Prointech), P62 (1:5000; 18420- 1-AP, Prointech), LKB1 (1:1000; 3047, Prointech), AMPK (1:1000; 5831, Proteintech), p-AMPK (1:1000), p70S6K1 (1:5000 ratio), pp70S6K1 (1:500; ab32529, Abcam, Cambridge, UK), Atg5-Atg12 complex (1:1000; AAM79 Bio-Rad, CA, USA), or GAPDH (1:1000). frontiersin.org The membranes were washed three times with TBST, followed by incubation at room temperature for 1 h with goat anti-rat IgG H&L (HRP; 1:10,000, ab150157, Abcam) or goat anti-rabbit IgGH&L (HRP; 1:10,000, ab6721, Abcam).

Techniques: Staining, Control, Expressing, Standard Deviation

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: Nature Communications

Article Title: The P-type ATPase transporter ATP7A promotes angiogenesis by limiting autophagic degradation of VEGFR2

doi: 10.1038/s41467-021-23408-1

Figure Lengend Snippet: a Immunofluorescence analysis of LC3 (Microtubule-associated protein 1 A/1B-light chain 3)-RFP and GFP puncta (mature and immature LC3 puncta, respectively), human umbilical vein endothelial cells (HUVECs) transfected with LC3-RFP-GFP plasmids in the presence of either ATP7A or control siRNAs were treated with vascular endothelial growth factor (VEGF) (20 ng/ml) for 30 min. Scale, 10 μm. The bar graph represents averaged number of RFP positive and GFP negative puncta per cell. n = 6, ** p = 0.0033, ** p = 0.0022 (two-tailed unpaired t -test). b . ATP7A mut mice crossed with RFP-EGFP-LC3 transgenic mice to generate ATP7A mut /RFP-EGFP-LC3 transgenic reporter mice. Wild Type (WT) and ATP7A mut reporter mice were subjected to hind limb ischemia. Gastrocnemius muscles were harvested at 3 days after ischemia and examined for RFP and GFP expression. The scale bar; 10 µm. Bar graph represents averaged number of RFP positive and GFP negative puncta per fiber. n = 5, # p = 0.0005 (two-tailed unpaired t -test). c Representative western analysis of ischemic gastrocnemius muscles of WT and ATP7A mut mice at 3 days after ischemia. The bar graph represents averaged fold change over control and tubulin as a loading control. n = 3, * p = 0.0196 (two-tailed unpaired t -test). d HUVECs transfected with control or ATP7A siRNAs were stimulated with VEGF (20 ng/ml) for 30 min and were analyzed by transmission electron microscopy. Double membrane indicates autophagosome (AP) and black vesicle included AP indicated autolysosome (AL). ( n = 3). e , f HUVECs transfected with LC3-GFP plasmid in the presence of either ATP7A or control siRNAs were stimulated with VEGF (20 ng/ml) for 30 min. Cells were stained with anti p62 or VEGFR2 antibodies. The right panels depict magnified images of the boxed areas seen in the left panels. The scale bar: 10 µm. Bar graph represents averaged number of p62 or VEGFR2 and LC3 colocalized puncta per cell. e n = 4, ** p = 0.0016; f n = 4 ** p = 0.0036 (two-tailed unpaired t -test). Data are mean ± SEM.

Article Snippet: Goat anti-VEGFR2 (R&D System), Mouse anti-p62 (BD), and Mouse anti-α-tubulin (Santa Cruz) were incubated on nitrocellulose membranes containing protein with 1:1000 dilution.

Techniques: Immunofluorescence, Transfection, Two Tailed Test, Transgenic Assay, Expressing, Western Blot, Transmission Assay, Electron Microscopy, Plasmid Preparation, Staining

Journal: Nature Communications

Article Title: The P-type ATPase transporter ATP7A promotes angiogenesis by limiting autophagic degradation of VEGFR2

doi: 10.1038/s41467-021-23408-1

Figure Lengend Snippet: a Human umbilical vein endothelial cells (HUVECs) transfected with control or ATP7A siRNAs were stimulated with vascular endothelial growth factor (VEGF) (20 ng/ml) for 30 min in the presence of N-ethylmaleimide (5 mM). Lysates were IP with VEGFR2 followed by immunoblotting (IB) with anti Ubiquitin antibody (Ab) to detect VEGFR2 ubiquitination. The bar graph represents averaged fold change over the control. n = 3, ** p = 0.0031, # p = 0.0002, ** p = 0.0023 (two-tailed unpaired t -test). b HUVECs transfected with ATP7A or control siRNAs were stimulated with VEGF (20 ng/ml) for 30 min and analyzed by immunofluorescence using VEGFR2 and p62 Abs. The scale bar = 10 µm. Bar graph represents averaged number of VEGFR2 and p62 colocalized dots per cell. n = 5, # p = 0.0007 (two-tailed unpaired t -test). c HUVECs transfected with control or ATP7A siRNAs were stimulated with VEGF (20 ng/ml) for 15 and 30 min. Lysates were immunoprecipitated (IP) with VEGFR2 Ab followed by IB with p62 or VEGFR2 Abs. n = 3 independent experiments, # p = 0.0007, * p = 0.0232, * p = 0.0294 (two-tailed unpaired t -test). d Bovine aortic endothelial cells (BAEC) transfected with bovine control or ATP7A siRNAs along with empty vector or ATP7A-Myc plasmid and cells were stimulated with VEGF (20 ng/ml) for 30 min. Lysates were IP with VEGFR2 Ab followed by IB with p62 or VEGFR2 Abs. ( n = 3). e HUVECs transfected with Adenovirus expressing p62 WT or p62 lacking ubiquitin-associated (UBA) domain were treated with VEGF (20 ng/ml) for 60 min. Lysates were used for IB with VEGFR2, HA-p62. n = 3 independent experiments,* p = 0.0145, ** p = 0.0082 (two-tailed unpaired t -test). f HUVECs transfected with empty vector or HA-p62 were used to measure basal and VEGF-induced endothelial cell (EC) migration using the modified Boyden chamber method. Bar graph represents averaged fold change over control. Scale bars = 100 μm. n = 3, ** p = 0.0059, * p = 0.0499 (two-tailed unpaired t-test). Data are mean ± SEM.

Article Snippet: Goat anti-VEGFR2 (R&D System), Mouse anti-p62 (BD), and Mouse anti-α-tubulin (Santa Cruz) were incubated on nitrocellulose membranes containing protein with 1:1000 dilution.

Techniques: Transfection, Western Blot, Two Tailed Test, Immunofluorescence, Immunoprecipitation, Plasmid Preparation, Expressing, Migration, Modification

Journal: Nature Communications

Article Title: The P-type ATPase transporter ATP7A promotes angiogenesis by limiting autophagic degradation of VEGFR2

doi: 10.1038/s41467-021-23408-1

Figure Lengend Snippet: Proposed model showing that ATP7A binds to VEGFR2 in endothelial cells in response to VEGF, which protects against p62/SQSTM1-mediated autophagic and lysosomal degradation of VEGFR2. This in turn promotes VEGFR2 signaling, thereby promoting angiogenesis and ischemic neovascularization.

Article Snippet: Goat anti-VEGFR2 (R&D System), Mouse anti-p62 (BD), and Mouse anti-α-tubulin (Santa Cruz) were incubated on nitrocellulose membranes containing protein with 1:1000 dilution.

Techniques: