Journal: bioRxiv

Article Title: Colorimetric and fluorescent TRAP assays for visualising and quantifying fish osteoclast activity

doi: 10.1101/2021.12.10.472045

Figure Lengend Snippet: A-M: Confocal images of dissected 5 th ceratobranchial arch with pharyngeal teeth of a ctsk:Dsred; sp7:egfp double transgenic adult which had been processed for ELF97 TRAP staining (C, H) and immunostained with antibodies against EGFP (A, G) and DsRed (B, I). Both wholemount (A-F) and cryosectioned (G-M) samples are presented. Co-localisation demonstrated ELF97 signal associated with DSRed positive osteoclasts found at the base of the teeth (D, E, K, L), whilst eGFP positive osteoblasts are found within the teeth (F, J, L). Fluorescent channels are also shown with DIC image (F, M). Scale bars: (F) = 200μm, (M) = 50μm.

Article Snippet: Primary antibodies used were chicken anti-GFP (Abcam: Ab16901) at 1:500 and rabbit anti-Dsred (BD Biosciences: #632496) at 1:200.

Techniques: Transgenic Assay, Staining

Journal: PLoS ONE

Article Title: Arsenite-Activated JNK Signaling Enhances CPEB4-Vinexin Interaction to Facilitate Stress Granule Assembly and Cell Survival

doi: 10.1371/journal.pone.0107961

Figure Lengend Snippet: (A) U2OS cells were treated without (control) or with arsenite ± cycloheximide (CHX) for 30 min prior to immunostaining of Vinexin, Vinculin (FA marker) and TIA-1 (SG marker). Arrow heads and arrows indicate FAs and SGs, respectively. One hundred FAs were randomly selected from ten cell images in each group to quantify the fluorescence intensities of Vinexin and Vinculin. Similarly, a hundred SGs from ten arsenite-treated cells were analyzed for the signals of Vinexin and TIA-1. The top scatter plot shows the reduction of Vinexin signal in FAs after the addition of arsenite. (B) Distribution of Vinexin and CPEB4 in arsenite or heat shock (42°C, 20 min)-treated HeLa cells. Arrows denote colocalization of CPEB4 and Vinexin in SGs. (C) Live imaging of EGFP-Vinexin β and RFP-TIA-1 distribution in HeLa cells treated with arsenite (see for the entire cell images). Arrow heads and arrows indicate FAs and SGs, respectively. Scale: 10 µm.

Article Snippet: Antibodies used in the study are β-actin (AC-15), flag epitope (F1804), myc (M4439), PABP (P6246) and Vinculin (V9264) from Sigma-Aldrich; TIA-1 (sc-1751) and p-c-Jun (Ser63/73, sc-16312) from Santa Cruz Biotechnology; RFP antibody (GTX59862) and Vinexin (GTX115362) from GeneTex; GFP (#29779) from AnaSpec; c-Jun (#9165) from Cell Signaling Technology and AlexaFluor 488, 594 or 647-conjugated secondary antibodies from Invitrogen.

Techniques: Control, Immunostaining, Marker, Fluorescence, Imaging

Journal: PLoS ONE

Article Title: Arsenite-Activated JNK Signaling Enhances CPEB4-Vinexin Interaction to Facilitate Stress Granule Assembly and Cell Survival

doi: 10.1371/journal.pone.0107961

Figure Lengend Snippet: (A) The domain organization of CPEB4, showing the N-terminal four PRDs and the C-terminal RNA-binding domain composed of two RNA recognition motifs (RRM) and zinc fingers (Zif). The various CPEB4 mutants were illustrated. (B) The 293T lysates containing flag-Vinexin β along with myc-tagged wt or mutant (mut) CPEB4 were precipitated by myc IgG, followed by immunoblotting with myc and flag antibodies. (C) FRET analysis. The plasmids encoding the FRET donor EGFP-Vxn β and acceptor RFP-CPEB4 (CP4) or mutants (CP4mut7 and ΔPRD) were co-transfected to COS-7 cells. The formaldehyde-fixed samples were used for FRET analysis to detect CPEB4-Vineixn interaction in SGs. The example images and the line graph show that the fluorescent signal of EGFP-Vxn β increases after photobleaching the acceptor RFP-CP4 in the selected SG (red circle). In contrast, no increasing change in EGFP signal if RFP-CP4mut7 was used as the acceptor. (D) The changes in fluorescence intensity of EGFP-Vxn β right before and after photobleaching RFP were calculated for FRET efficiency. The FRET interaction between EGFP-Vxn β and RFP-CP4 wt, mut7 or ΔPRD with ± arsenite was determined. Similarly, the FRET interaction of EGFP-Vxn β and RFP-CP4 was also measured in the presence of JNK inhibitor, SP600125 (SP). All of the data were expressed as the mean ± s.e.m. n: the number of SGs in each group (one SG per cell was analyzed by FRET acceptor bleaching). The significant difference between wt and CPEB4 mutants as well as between mock and arsenite (Ars) ± SP treatments was analyzed with Student’s t -test. One and two asterisks denote * P <0.05 and ** P <0.01. (E) Schematic model of arsenite-induced redistribution of CPEB4 and Vinexin to SGs. Arsenite stress activates JNK signaling, which somehow promotes the association of CPEB4 and Vinexin to recruit Vinexin in SGs. Once translocated to SGs, Vinexin plays an active role in facilitating SG assembly, most likely recruiting additional factors (e.g., RTKN: Rhotekin) through its third SH3 motif. Meanwhile, the dissociation of Vinexin from cytoskeletal proteins, such as Vinculin, weakens focal adhesions and promotes Vinexin translocation from FAs to SGs. ECM, extracellular matrix.

Article Snippet: Antibodies used in the study are β-actin (AC-15), flag epitope (F1804), myc (M4439), PABP (P6246) and Vinculin (V9264) from Sigma-Aldrich; TIA-1 (sc-1751) and p-c-Jun (Ser63/73, sc-16312) from Santa Cruz Biotechnology; RFP antibody (GTX59862) and Vinexin (GTX115362) from GeneTex; GFP (#29779) from AnaSpec; c-Jun (#9165) from Cell Signaling Technology and AlexaFluor 488, 594 or 647-conjugated secondary antibodies from Invitrogen.

Techniques: RNA Binding Assay, Zinc-Fingers, Mutagenesis, Western Blot, Transfection, Fluorescence, Translocation Assay