Journal: Cell Death Discovery

Article Title: Coal dust nanoparticles induced pulmonary fibrosis by promoting inflammation and epithelial-mesenchymal transition via the NF-κB/NLRP3 pathway driven by IGF1/ROS-mediated AKT/GSK3β signals

doi: 10.1038/s41420-022-01291-z

Figure Lengend Snippet: A , B The activation level of AKT/GSK3β pathway was detected by western blot in vitro and in vivo. C , D The expression levels of EMT marker molecules (N-cadherin, E-cadherin and TGFβ1), inflammatory pathway protein molecules (NLRP3, Cleaved Caspase1/Caspase1) and fibrosis marker molecules (COL-1, COL-3 and α-SMA) was detected by western blot. E Inflammatory factor IL-1β and inflammatory chemokine CXCL2 the supernatant of alveolar epithelial cells were detected by ELISA. Data were expressed as the mean ± SD, n = 3. ** P < 0.01 and *** P < 0.001. F The migration and invasion abilities of alveolar epithelial cells were detected by scratch healing assay and transwell assay.

Article Snippet: The information of antibodies is as follows: IGF1 (1: 1000, Abclonal, #A11985, China), IGF1R (1: 1000, CST, #9750, USA), p-IGF1R(Tyr1135/1136) (1: 1000, CST, #3024, USA), COL-1 (1: 2000, Abcam, ab260043, UK), COL-3 (1: 1000, Abcam, ab184993, UK), α-SMA (1:1000, CST, #19425, USA), heme oxygenase-1 (HO-1, 1: 1000, CST, #5853, USA), N-cadherin (1: 1000, CST, #49398, USA), E-cadherin (1: 1000, CST, #49398, USA), TGFβ1 (1: 1000, abclonal, #A2124, China), p-NF-kB p65(Ser536) (1: 1000, CST, #3033, USA), NF-kB p65 (1: 1000, CST, #8242, USA), NLRP3 (1: 1000, CST, #15101, USA), Cleaved Caspase1(Asp297) (1: 1000, CST, #4199, USA), Caspase1 (1: 1000, CST, #2225, USA), p-Akt(Ser473) (1: 1000, CST, #4060, USA), Akt (1: 1000, CST, #4691, USA), p-GSK3β(Ser9) (1: 1000, CST, #9322, USA), GSK3β (1: 1000, CST, #9369, USA) and β-actin (1: 1000, CST, #4970, USA).

Techniques: Activation Assay, Western Blot, In Vitro, In Vivo, Expressing, Marker, Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay

Journal: Molecular Cancer

Article Title: Inactivation of MAP kinase signalling in Myc Transformed Cells and Rescue by LiCl inhibition of GSK3

doi: 10.1186/1476-4598-4-13

Figure Lengend Snippet: The effect of LiCl on the levels of GSK3 in non-transformed fibroblasts. (A) A western blot showing the levels of inactive or phosphorylated GSK3 α/β in non-transformed fibroblasts after the addition of Li + or K + salt control for the time points indicated. (B) A western blot showing the levels of phosphorylated and non-phosphorylated GSK3 α/β of the same samples in (A) above after stripping and re-probing of the blot with the appropriate antibody.

Article Snippet: Inactive phosphorylated GSK3 α/β (Ser21/9) protein was detected using a rabbit polyclonal antibody (catalogue number 9331S, Cell Signaling Technology, UK) and phosphorylated and non-phosphorylated GSK3 α/β levels were detected using a rabbit polyclonal antibody raised against GSK3 β but detected both GSK3 α and β (catalogue number 9332, Cell Signaling Technology, UK).

Techniques: Transformation Assay, Western Blot, Stripping Membranes

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Human male gamete endocrinology: 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) regulates different aspects of human sperm biology and metabolism

doi: 10.1186/1477-7827-7-140

Figure Lengend Snippet: 1,25(OH)2D3 induces pERK1/2, AKT and GSK3 phosphorylations in human sperm through VDR . A : Percoll-purified sperm washed spermatozoa were incubated in the unsupplemented Earle's medium for 30 min at 37°C and 5% CO 2 , in the absence (NC) or in the presence of increasing 1,25(OH)2D3 concentrations (0.01 nM, 0.1 nM, 1 nM and 10 nM) or with 0.1 nM 1,25(OH)2D3 combined with anti-VDR Ab (AbVDR). Other samples were incubated in capacitating medium (Cap). B : Time course study (0, 5, 10, 30 and 60 min) of ERK1/2, AKT and GSK3 phosphorylations treated with 0.1 nM 1,25(OH)2D3. Actin was used as loading control. On the side are reported the densitometric evaluations normalised against actin levels. The autoradiographs presented are representative examples of experiments that were performed at least seven times with repetitive results. * P < 0.05 versus control, ** P < 0.01, *** P < 0.002 versus control.

Article Snippet: Rabbit anti-p-extracellular signal-regulated kinase (ERK 1/2) and anti-p-GSK3 Abs were from Cell Signalling (Milan, Italy).

Techniques: Purification, Incubation

Journal: bioRxiv

Article Title: Focal adhesion-based cell migration is differentially regulated in vivo versus in vitro by Paxillin phosphorylation

doi: 10.1101/2022.03.02.482703

Figure Lengend Snippet: (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) Upper panel: 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. Lower panel: pY118-Paxillin staining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green, white arrowheads) in larval zebrafish (3 days post-transplantation). Red arrowhead indicates a non-ZMEL cell with positive pY118-Paxillin staining. Scale bar is 10 µm. (C) Western blot analysis of mouse melanoma YUMM1.7 cells expressing WT-Paxillin-T2A-GFP plated on the in vitro cell culture dishes and YUMM1.7 melanoma in vivo tumors. In vitro and in vivo bands are from the same blot – see entire blot in . GFP was used as the loading control and a control for number of YUMM1.7 cells in mouse tumors. (D) Quantification of pY118-Paxillin/total Paxillin protein ratio from (C). Non-parametric unpaired t test, *p<0.05. n=3 individual experiments. (E) Quantification of single cell migration velocity in ZMEL-mCherry cells that exogenously express GFP-tagged 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 at 1 day post-transplantation. Non-parametric one-way ANOVA, *p<0.05, ****p<0.0001. Error bars are mean ± SD.

Article Snippet: Primary antibodies used were Rabbit anti-Paxillin (Antibodyplus, STJ94969, 1:1000), Rabbit anti-pY118-Paxillin (Cell Signaling Technology, 9369, 1:1000), Rabbit anti-FAK (Cell Signaling Technology, 3285, 1:1000), Rabbit anti-pFAK397 (Cell Signaling Technology, 3283, 1:1000), Chicken anti-GFP (Abcam, ab13970, 1:500), Mouse anti-CrkII (BD Bioscience, 610035, 1:1000).

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

Journal: bioRxiv

Article Title: Focal adhesion-based cell migration is differentially regulated in vivo versus in vitro by Paxillin phosphorylation

doi: 10.1101/2022.03.02.482703

Figure Lengend Snippet: (A) Upper panel: pY118-Paxillin staining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on in vitro cell culture dishes. Middle panel: pY118-Paxillin staining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green) in larval zebrafish (3 days post-transplantation). Lower panel: pY118-Paxillin staining (magenta) of zebrafish developing heart (5dpf). (B) pY118-Paxillin staining of ZMEL cells expressing wildtype Paxillin plated on collagen coated dishes with different stiffness: 0.5kPa (upper panel), 50kPa (middle panel), and glass bottom dishes (lower panel). Scale bar is 10 µm. (C) Entire 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. GFP was used as the loading control, and as a control for the number of YUMM1.7 cells in mouse tumors.

Article Snippet: Primary antibodies used were Rabbit anti-Paxillin (Antibodyplus, STJ94969, 1:1000), Rabbit anti-pY118-Paxillin (Cell Signaling Technology, 9369, 1:1000), Rabbit anti-FAK (Cell Signaling Technology, 3285, 1:1000), Rabbit anti-pFAK397 (Cell Signaling Technology, 3283, 1:1000), Chicken anti-GFP (Abcam, ab13970, 1:500), Mouse anti-CrkII (BD Bioscience, 610035, 1:1000).

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

Journal: bioRxiv

Article Title: Focal adhesion-based cell migration is differentially regulated in vivo versus in vitro by Paxillin phosphorylation

doi: 10.1101/2022.03.02.482703

Figure Lengend Snippet: (A) pY118 Paxillin immunostaining (magenta) of macrophages (green, white arrowheads) in Tg(mpeg:Lifeact-GFP) zj506 larval zebrafish. Red arrowhead marks positive pY118 Paxillin staining of a non-macrophage cell. (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-Paxillin-EGFP) zj503 , Tg(mpeg: Y118E-Paxillin-EGFP) zj504 and Tg(mpeg:Y118F-Paxillin-EGFP) zj505 embryos at timepoint 0 and 10 mins. Dotted lines indicate wound sites and arrows show the direction of migration. See also supplemental video S7. Scale bar is 10 µm. (D) Quantification of macrophage migration velocities toward the wound in vivo . Error bars are mean ± SD. Non-parametric one-way ANOVA, ****p<0.0001. (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 (Antibodyplus, STJ94969, 1:1000), Rabbit anti-pY118-Paxillin (Cell Signaling Technology, 9369, 1:1000), Rabbit anti-FAK (Cell Signaling Technology, 3285, 1:1000), Rabbit anti-pFAK397 (Cell Signaling Technology, 3283, 1:1000), Chicken anti-GFP (Abcam, ab13970, 1:500), Mouse anti-CrkII (BD Bioscience, 610035, 1:1000).

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