Journal: Cellular oncology (Dordrecht)

Article Title: Single-cell RNA profiling identifies diverse cellular responses to EWSR1/FLI1 downregulation in Ewing sarcoma cells

doi: 10.1007/s13402-021-00640-x

Figure Lengend Snippet: A, Subpopulation studies reveal down-regulation of FAM134B and up-regulation of LC-3-like modifiers (MAPLC3B, GABARAPL2) and ER stress response proteins in cluster C2 (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA, error bars indicate S.D.); B, Fluorescence images of EW-8 cells transfected with si-Control or si-EWSR1/FLI1. EW-8 cells were fixed in D1, immunostained and subsequently imaged via fluorescent microscopy to study the induction of the autophagy marker MAPLC3B and the lysosomal marker CD63; C, Quantification of FAM134B co-localization with LCB3, presented as Pearson’s correlation coefficient (r); **** p < 0.0001, t-test, n = 15 fields (~150 cells) in the dormant cells shown in A using the colocalization tool in imageJ; D, EW-8 cells were treated with si-EWSR1/FLI1 or si-Control 48 h before being stained for the ER resident protein SEC61B. The expansion of ER is shown following EWSR1/FLI1 downregulation (boxed areas). E, Quantification of cells with expanded ER after masking nuclei. **** p < 0.0001, t-test, error bars indicate S.D., n = 150 cells.

Article Snippet: As secondary antibody Alexa Fluor 488 (ab150077; Abcam) against FAM134B, SEC61B (sc-393633; Santa Cruz) was used.

Techniques: Fluorescence, Transfection, Control, Microscopy, Marker, Staining

Journal: PLoS ONE

Article Title: Involvement of Endoplasmic Reticulum Stress in TULP1 Induced Retinal Degeneration

doi: 10.1371/journal.pone.0151806

Figure Lengend Snippet: P1 WT mice retinas were co-transfected with GFP-fused (green) plasmids and a mCherry tagged ER reporter (mCh-Sec61b: red). Retinas were harvested and sectioned at P30 (A) Confocal images of retinas show co-localization (merge: yellow) of GFP-fused mutant D94Y-TULP1 and F491L-TULP1 with mCh-Sec61b, the ER resident protein, in the inner segments. Scale bar = 10μM. (B-C) Activation of the ER-UPR stress markers in transgenic mice expressing mutant TULP1 protein. qRT-PCR indicates a 1.3–5 fold increase of multiple ER-UPR stress markers in mutant D94Y-TULP1 (B) or mutant F491L-TULP1 (C) retinas vs WT-TULP1 injected retinas. qRT-PCR analysis was normalized to GAPDH (as a mRNA quantity control) and GFP (as a transfection efficiency control). The ΔΔCt method was employed to calculate fold changes. Samples were assayed in triplicates for each ER-UPR marker and qRT-PCR experiments were repeated twice. Statistical significance was assessed by using the two-tailed Student’s t -test. * = p<0.001; n.s. not statistically significant.

Article Snippet: For co-localization studies, WT or mutant TULP1 plasmids were co-electroporated with the mCherry-tagged ER-marker Sec61b plasmid DNA (mCherry-Sec61b, a gift from Dr. Gia Voeltz (Addgene #49155)).

Techniques: Transfection, Mutagenesis, Activation Assay, Transgenic Assay, Expressing, Quantitative RT-PCR, Injection, Marker, Two Tailed Test

Journal: eLife

Article Title: Human VPS13A is associated with multiple organelles and influences mitochondrial morphology and lipid droplet motility

doi: 10.7554/eLife.43561

Figure Lengend Snippet:

Article Snippet: BFP-Sec61B (Addgene plasmid #49154) and mCherry-Sec61B (Addgene plasmid #49155) were kind gifts from Gia Voeltz ( ).

Techniques: Recombinant, Mutagenesis