Journal: Journal of Oncology

Article Title: A Water-Soluble Hydrogen Sulfide Donor Suppresses the Growth of Hepatocellular Carcinoma via Inhibiting the AKT/GSK-3 β / β -Catenin and TGF- β /Smad2/3 Signaling Pathways

doi: 10.1155/2023/8456852

Figure Lengend Snippet: Effects of HA-ADT on the apoptotic level and AKT/GSK-3 β / β -catenin pathway in human HCC cells. (a) TUNEL staining was used to detect the apoptotic level (original magnification, 100x). (b) The apoptotic index was counted as the ratio of TUNEL positive cells to total cells. (c) Flow cytometry assay was adopted to detect apoptosis. (d) Cell apoptosis distribution was analyzed. (e) Western blotting was used to determine the protein levels of AKT, p-AKT, GSK-3 β , p-GSK-3 β , β -catenin, and p- β -catenin. β -actin was adopted as the internal control. (f) The density was analyzed. All data are shown as the mean ± SEM of three independent experiments; ∗ P < 0.05, ∗∗ P < 0.01 vs. control group; △ P < 0.05, △△ P < 0.01 vs. NaHS group; ## P < 0.01 vs. GYY4137 group.

Article Snippet: The primary antibodies include anti-Cyclin E1, anti-Cyclin D1, anti-cyclin-dependent kinase (CDK) 2, anti-CDK4, anti-p27, anti-p21, anti-AKT, anti-phospho (p)-AKT (Ser473), anti-glycogen synthase kinase-3 beta (Gsk‐3 β ), anti-p-Gsk-3 β (Ser9), anti- β -catenin, anti-p- β -catenin (Ser552), anti-beclin-1, anti-p62, anti-LC3A/B, anti-Smad2, anti-p-Smad2 (Ser465/467), anti-Smad3, anti-p-Smad3 (Ser423/425), and anti-transforming growth factor-beta (TGF‐ β ) antibodies, as well as the horseradish peroxidase-conjugated secondary antibody obtained from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: TUNEL Assay, Staining, Flow Cytometry, Western Blot

Journal: The Journal of Cell Biology

Article Title: Lack of Paxillin phosphorylation promotes single-cell migration in vivo

doi: 10.1083/jcb.202206078

Figure Lengend Snippet: Paxillin exhibits reduced phosphorylation on Y118 in migrating cancer cells in vivo as compared to in vitro cell culture conditions in both zebrafish and mouse melanoma models. (A) Schematic of protein structures of human and zebrafish Paxillin (top) and amino acid sequence comparisons of the region encompassing Y118 between zebrafish Paxillin and vertebrate Paxillin (bottom). Red arrowhead and box indicate the conservation of Y118 Paxillin between zebrafish and other vertebrates. (B) Top: Endogenous pY118-Paxillin staining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on 2D in vitro cell culture dishes. White arrowheads mark positive pY118-Paxillin staining. Bottom: Endogenous pY118-Paxillin staining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green, white arrowheads) in larval zebrafish (3 d post-transplantation). Red arrowhead indicates a non-ZMEL cell with positive pY118-Paxillin immunostaining. Zoomed regions reveal pY118-Paxillin immunostaining only. Scale bar is 10 µm. (C) Western blot analysis of mouse melanoma YUMM1.7 cells expressing mammalian WT-Paxillin-T2A-GFP plated on the in vitro cell culture dishes ( n = 3 dishes) and YUMM1.7 melanoma in vivo tumors ( n = 5 tumors). In vitro and in vivo bands are from the same blot—see unmodified Western blot in . GFP was used as the loading control and a control for the number of YUMM1.7 cells in mouse tumors. (D) Quantification of pY118-Paxillin/total Paxillin protein ratio from C. Non-parametric unpaired t -test. (E) Quantification of single cell migration velocity in ZMEL-mCherry cells that exogenously express GFP-tagged zebrafish WT-Paxillin, Y118E-Paxillin, or Y118F-Paxillin in the in vitro cell culture conditions ( n = 64 cells for WT, n = 32 cells for Y118E, and n = 35 cells for Y118F) and in vivo ( n = 8 cells/3 fish for WT, n = 12 cells/3 fish for Y118E, and n = 15 cells/3 fish for Y118F). Larval zebrafish are imaged 1 d post-transplantation. Non-parametric one-way ANOVA, error bars are mean ± SD. (F) Cumulative FRAP recovery curves of WT-Paxillin-EGFP, Y118E-Paxillin-EGFP, or Y118F-Paxillin-EGFP in ZMEL cells in the in vitro cell culture conditions and in vivo after photobleaching. n = 34, 44, and 51 cells for WT, Y118E, Y118F in vitro, and n = 7 cells/6 fish, 6 cells/6 fish, and 6 cells/5 fish for WT, Y118E, Y118F in vivo. (G) Quantification of Paxillin disassembly rates in the WT, Y118E, Y118F-Paxilllin under in vitro cell culture conditions and WT, Y118E, Y118F-Paxilllin under in vivo conditions. n = 13, 13, and 11 cells for WT, Y118E, Y118F in vitro, and n = 8 cells/7 fish, 6 cells/6 fish, 11 cells/10 fish for WT, Y118E, Y118F in vivo. Error bars are mean ± SD. Non-parametric unpaired t test. Source data are available for this figure: .

Article Snippet: Primary antibodies used were rabbit anti-Paxillin (1:1,000, STJ94969; Antibodyplus), rabbit anti-pY118-Paxillin (1:1,000, 9369; Cell Signaling Technology), rabbit anti-FAK (1:1,000, 3285; Cell Signaling Technology), rabbit anti-pFAK397 (1:1,000, 3283; Cell Signaling Technology), chicken anti-GFP (1:500, ab13970; Abcam), mouse anti-CrkII (1:1,000610035; BD Bioscience), mouse anti-DOCK180 (1:500, sc-13163; Santa Cruz Biotechnology), mouse anti-C3G (1:250, sc-178403; Santa Cruz Biotechnology), rabbit p-ERK (1:1,000, 9101S; Cell Signaling Technology), and rabbit anti-pY31-Paxillin (1:1,000, 44-720G; Thermo Fisher Scientific).

Techniques: In Vivo, In Vitro, Cell Culture, Sequencing, Staining, Immunostaining, Transplantation Assay, Western Blot, Expressing, Migration

Journal: The Journal of Cell Biology

Article Title: Lack of Paxillin phosphorylation promotes single-cell migration in vivo

doi: 10.1083/jcb.202206078

Figure Lengend Snippet: Y118-Paxillin exhibits distinct phosphorylation status in migrating cancer cells in vivo versus in vitro. (A) Top: pY118-Paxillin immunostaining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on in vitro cell culture dishes. Middle: pY118-Paxillin immunostaining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green) in larval zebrafish (3 d post-transplantation). Bottom: pY118-Paxillin immunostaining (magenta) of the zebrafish developing heart (5 dpf). (B) Western blot showing the specificity of the pY118-Paxillin antibody and that it does not recognize Y118E-Paxillin and Y118F-Paxillin. (C) Representative images of ZMEL-GFP cells plated on 2D surfaces of different stiffnesses (left) and stained for pY118-Paxillin (right). (D) Unmodified Western blot of panels shown in —YUMM1.7 cells plated in culture and YUMM1.7 melanoma tumors in vivo blotted with Paxillin and pY118-Paxillin antibodies. “P” is parental cell line with no GFP expression. GFP was used as the loading control and as a control for the number of YUMM1.7 cells in mouse tumors. Source data are available for this figure: .

Article Snippet: Primary antibodies used were rabbit anti-Paxillin (1:1,000, STJ94969; Antibodyplus), rabbit anti-pY118-Paxillin (1:1,000, 9369; Cell Signaling Technology), rabbit anti-FAK (1:1,000, 3285; Cell Signaling Technology), rabbit anti-pFAK397 (1:1,000, 3283; Cell Signaling Technology), chicken anti-GFP (1:500, ab13970; Abcam), mouse anti-CrkII (1:1,000610035; BD Bioscience), mouse anti-DOCK180 (1:500, sc-13163; Santa Cruz Biotechnology), mouse anti-C3G (1:250, sc-178403; Santa Cruz Biotechnology), rabbit p-ERK (1:1,000, 9101S; Cell Signaling Technology), and rabbit anti-pY31-Paxillin (1:1,000, 44-720G; Thermo Fisher Scientific).

Techniques: In Vivo, In Vitro, Immunostaining, Cell Culture, Transplantation Assay, Western Blot, Staining, Expressing

Journal: The Journal of Cell Biology

Article Title: Lack of Paxillin phosphorylation promotes single-cell migration in vivo

doi: 10.1083/jcb.202206078

Figure Lengend Snippet: Macrophages expressing non-phosphorylatable Y118F-Paxillin exhibit increased motility in vivo. (A) Endogenous pY118 Paxillin immunostaining (magenta) of macrophages (green, white arrowheads) in Tg ( mpeg:Lifeact-GFP ) zj506 larval zebrafish. Red arrowhead marks positive pY118 Paxillin immunostaining of a non-macrophage cell. Zoomed region of macrophage lacking pY118-Paxillin immunostaining. (B) Schematic of zebrafish tail wound transection area and macrophage imaging area for directed cell migration. (C) Still images from zebrafish macrophage tracking timelapse videos in 3 dpf Tg ( mpeg:WT-zebrafish Paxillin- EGFP ) zj503 , Tg ( mpeg:zebrafish Y118E-Paxillin- EGFP ) zj504 , and Tg ( mpeg:zebrafish Y118F-Paxillin- EGFP ) zj505 larvae at timepoint 0 and 10 min. Dotted lines indicate wound sites and arrows show the direction of migration. See also . Scale bar is 10 µm. (D) Quantification of macrophage migration velocities toward the wound in vivo. Non-parametric one-way ANOVA, error bars are mean ± SD. n = 38 cells/6 fish for WT, n = 20 cells/6 fish for Y118E and n = 24 cells/10 fish for Y118F. (E) Cell tracking of macrophage migration trajectories toward the wound in vivo, migration starting points are normalized to 0 in both x and y axes, wound sites are normalized to the positive x axis ( n = 38 cells/6 fish for WT, n = 20 cells/6 fish for Y118E and n = 24 cells/10 fish for Y118F). Arrows show the direction of migration toward the wound.

Article Snippet: Primary antibodies used were rabbit anti-Paxillin (1:1,000, STJ94969; Antibodyplus), rabbit anti-pY118-Paxillin (1:1,000, 9369; Cell Signaling Technology), rabbit anti-FAK (1:1,000, 3285; Cell Signaling Technology), rabbit anti-pFAK397 (1:1,000, 3283; Cell Signaling Technology), chicken anti-GFP (1:500, ab13970; Abcam), mouse anti-CrkII (1:1,000610035; BD Bioscience), mouse anti-DOCK180 (1:500, sc-13163; Santa Cruz Biotechnology), mouse anti-C3G (1:250, sc-178403; Santa Cruz Biotechnology), rabbit p-ERK (1:1,000, 9101S; Cell Signaling Technology), and rabbit anti-pY31-Paxillin (1:1,000, 44-720G; Thermo Fisher Scientific).

Techniques: Expressing, In Vivo, Immunostaining, Imaging, Migration, Cell Tracking Assay

Journal: The Journal of Cell Biology

Article Title: Lack of Paxillin phosphorylation promotes single-cell migration in vivo

doi: 10.1083/jcb.202206078

Figure Lengend Snippet: FAK is downregulated and CRKII-DOCK180/RacGEF exhibits increased interaction with unphosphorylated Y118-Paxillin in vivo compared to in vitro. (A) Schematic of in vitro Paxillin regulation from cell culture studies. Following integrin activation, a tyrosine kinase, FAK, phosphorylates Paxillin. Phosphorylated Paxillin then recruits the adaptor protein CRKII and the Paxillin/CRKII complex further recruits DOCK180/RacGEF, thereby activating downstream Rac-dependent pathways, inducing cell migration. (B) Western blot analysis of FAK levels (FAK) and FAK activation (pY397-FAK) in YUMM1.7 cells expressing mammalian WT-Paxillin-T2A-GFP in culture and YUMM1.7 tumors in vivo. In vitro and in vivo bands are from the same blot. Unmodified Western blot is in . GFP was used as the loading control and as a control for the number of YUMM1.7 cells in mouse tumors. (C and D) Quantification of the pY397-FAK/total FAK ratio (C) and total normalized FAK to GFP expression (D) in the in vitro cell culture and in vivo conditions. n = 3 dishes, 5 tumors for C, n = 5 dishes, 8 tumors for D. Error bars are mean ± SD. Non-parametric unpaired t test. (E) Western blot analysis of pY118-Paxillin levels in YUMM1.7 cells overexpressing GFP-FAK in vitro and in vivo. Actin is used as a loading control. (F) Quantification of pY118-Paxillin/Paxillin levels in E. GFP control tumors are normalized to 1. n = 4 technical replicates. Error bars are mean ± SD. Non-parametric unpaired t test. (G–J) Co-immunoprecipitation analyses of CRKII and Paxillin in YUMM1.7 cell lines that exogenously express mammalian wildtype, Y118E and Y118F Paxillin in vitro (G and H) and in in vivo tumors (I and J). (H and J) Quantification of CRKII/Paxillin ratio from G and I, bands from cells expressing wildtype Paxillin are normalized to 1 both in vitro and in vivo. n = 3 technical replicates. Non-parametric one-way ANOVA, error bars are mean ± SD. (K) Coimmunoprecipitation analyses of DOCK180/RacGEF and Paxillin in YUMM1.7 cell lines that exogenously express mammalian wildtype, Y118E and Y118F Paxillin in vitro and in in vivo tumors. (L–N) Coimmunoprecipitation analyses of CRKII and DOCK180/RacGEF to Paxillin in YUMM1.7 cell lines that exogenously express wildtype Paxillin in in vitro and in in vivo tumors. (M) Quantification of CRKII/Paxillin levels in L. n = 4 tumors. (N) Quantification of DOCK180/Paxillin levels in L. n = 4 tumors. Error bars are mean ± SD. Non-parametric unpaired t test. Source data are available for this figure: .

Article Snippet: Primary antibodies used were rabbit anti-Paxillin (1:1,000, STJ94969; Antibodyplus), rabbit anti-pY118-Paxillin (1:1,000, 9369; Cell Signaling Technology), rabbit anti-FAK (1:1,000, 3285; Cell Signaling Technology), rabbit anti-pFAK397 (1:1,000, 3283; Cell Signaling Technology), chicken anti-GFP (1:500, ab13970; Abcam), mouse anti-CrkII (1:1,000610035; BD Bioscience), mouse anti-DOCK180 (1:500, sc-13163; Santa Cruz Biotechnology), mouse anti-C3G (1:250, sc-178403; Santa Cruz Biotechnology), rabbit p-ERK (1:1,000, 9101S; Cell Signaling Technology), and rabbit anti-pY31-Paxillin (1:1,000, 44-720G; Thermo Fisher Scientific).

Techniques: In Vivo, In Vitro, Cell Culture, Activation Assay, Migration, Western Blot, Expressing, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Selective PPARγ modulator diosmin improves insulin sensitivity and promotes browning of white fat

doi: 10.1016/j.jbc.2023.103059

Figure Lengend Snippet: Acute diosmin (Dios) administration improves diabetic gene programs in iWAT of mice. A , experimental model of acute control (Con) or Dios administration in mice with iWAT unilateral injection (n = 4). B , protein levels of S273 p-PPARγ, ( C ) p-IRβ, p-AKT, and p-GSK3β, ( D ) expression of gene set regulated by PPARγ S273 phosphorylation in iWAT of mice after acute Dios administration. Data are presented as mean ± SEM and ∗ p < 0.05, ∗∗ p < 0.01 compared with control group. iWAT, inguinal white adipose tissue; PPARγ, peroxisome proliferator–activated receptor γ.

Article Snippet: Membranes were incubated in 5% bovine serum albumin for 2 h and with primary antibodies overnight at 4 °C, including anti-p-PPARγ (1:2000 dilution) (catalog no.: bs-4888R; Bioss biotech), anti-PPARγ (1:1000 dilution) (catalog no.: sc-7273; Santa Cruz), anti-p-IRβ (1:2000 dilution) (catalog no.: 3025; Cell Signaling Technology), anti-p-AKT (1:2000 dilution) (catalog no.: 13038; Cell Signaling Technology), anti-p-GSK3β (1:2000 dilution) (catalog no.: 9322; Cell Signaling Technology) (1:2000 dilution), anti-UCP1 (catalog no.: Ab10983; Abcam), or β-actin (1:2000 dilution) (catalog no.: sc-47778; Santa Biotechnology).

Techniques: Injection, Expressing

Journal: Cancers

Article Title: P38 Mediates Tumor Suppression through Reduced Autophagy and Actin Cytoskeleton Changes in NRAS-Mutant Melanoma

doi: 10.3390/cancers15030877

Figure Lengend Snippet: List of primary antibodies.

Article Snippet: Phospho GSK-ß 9322 , 1:1000 , Cell Signaling , LC3 ab51520 , 1:3000 , Abcam.

Techniques: