Journal: Cell reports
Article Title: HRS mediates tumor immune evasion by regulating proteostasis-associated interferon pathway activation
doi: 10.1016/j.celrep.2023.113352
Figure Lengend Snippet: (A and B) Control or HRS KO B16F10 (A) and Braf Pten (B) cells were treated with thapsigargin (Tg). Cell lysates were fractionated into soluble (‘‘S’’) and precipitated (‘‘P’’) fractions. Ubiquitinated proteins were probed with anti-ubiquitin antibodies. Quantification of ubiquitinated proteins is shown to the right. The blots of soluble and precipitated fractions in control groups were normalized to 1, respectively. (C) Immunofluorescence of ubiquitin in control and HRS KO B16 cells treated with Tg, MG132, and VER-155008, respectively. Cell nuclei were stained with DAPI. Scale bar, 20 μm. (D) Quantification of the ubiquitin-positive puncta in control and HRS KO B16 cells with indicated treatment as shown in (C). (E) Ubiquitinated proteins in the same amounts of exosomes from control or HRS KO B16 and Braf Pten cells were analyzed by immunoblotting. CD63 was used as an exosome marker. (F) Quantification of ubiquitinated proteins in exosomes from B16 (left) and Braf Pten (right). The levels of ubiquitinated proteins in exosomes from control groups were normalized to 1. (G) The human A375 melanoma cells were transfected with firefly luciferase (Fluc)-GFP or the mutant FlucDM-GFP. The co-localization of Fluc-GFP or FlucDM-GFP (green) with HRS (red) in A375 cells with MG132 treatment was analyzed. Nuclei were stained with DAPI. Scale bar, 20 μm. (H) HRS was knocked down in A375 cells with stable expression of FlucDM-GFP. The levels of soluble and precipitated FlucDM-GFP were analyzed. (I) Quantification of Fluc-GFP or FlucDM-GFP in the soluble (left) and precipitated (right) fractions from (H). GADPH was used as a control for soluble fraction loading. The blots of soluble and precipitated fractions in control groups were normalized to 1, respectively. (J) Specific activities of Fluc in the soluble extracts from A375 cells that were stably transfected with Fluc-GFP or its mutant. Activities of Fluc in control A375 cells were set as 100%. Data represent the mean ± SD from three or four independent experiments. The p values were determined by one-way ANOVA with Sidak’s multiple comparison tests (A and B), one-sample t test (F and I), and unpaired Student’s t test (D and J). See also and .
Article Snippet: Plasmid: pCI-neo Fluc EGFP (Fluc-EGFP) , Addgene , #90170; RRID:Addgene_90170.
Techniques: Control, Ubiquitin Proteomics, Immunofluorescence, Staining, Western Blot, Marker, Transfection, Luciferase, Mutagenesis, Expressing, Stable Transfection, Comparison
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