Journal: Cell reports

Article Title: Uncovering genetic interactions in the DNA repair network in response to endogenous damage and ionizing radiation

doi: 10.1016/j.celrep.2025.116850

Figure Lengend Snippet: (A) Distribution of the observed phenotypes depleted in the basal screen results, indicating where the MRE11A-UBR5 gene pair falls relative to other depleted interactions. (B) Ranked observed phenotypes from the basal screen for both UBR5 and MRE11A . Blue/red regions highlight significantly enriched/depleted UBR5 -gene and MRE11A -gene interactions, respectively. Blue and red regions highlight gene combinations that are enriched above (blue) or depleted below (red) one standard deviation (+0.6981 and −0.7791) of the mean of all basal interactions and possess a Fisher-combined p < 0.05. (C) Time-lapse images of growth from Dox-induced dual-guide vectors with NT-1 x UBR5 -g2 or NT-1 x NT-2 in nuclear-EGFP-expressing cells co-cultured with nuclear-RFP-expressing cells grown in DMSO or 6.25 μM mirin (+mirin). Images are from one well per condition over the first 72 h of growth. Scale bar: 100 μM. (D) Measurements are calculated as the log 2 fold change (log 2 FC) of Dox induced over uninduced growth for the labeled condition, relative to the growth of co-cultured RFP+ cells, as seen in (C). Data represent mean ± SEM of 3 biological repliactes. The gray bar indicates the 72 h of growth shown in (C), though imaging continued. (E) Comparative growth differences at 96 h of two separate, inducible UBR5 guides from experiments performed similarly to those shown in (C) and (D). Bars are only shown for Dox-treated conditions but are normalized to uninduced, DMSO-treated control wells for each genotype and replicate. Mirin: 5 μM. Blue prediction bars are calculated from an additive model assuming no interaction, with the average effect of mirin on NT cells plus the average effect of UBR5 depletion for each guide and replicate. Data represent mean ± SEM of 2 biological replicates, and statistics represent a two-way ANOVA with Dunnett multiple comparisons test; p = 0.0277. (F) Western blot depicting robust UBR5 KO in two selected HT1080-6TG clones. (G) Dose curve of HT1080-6TG clones shown in (F) grown in increasing doses of mirin in a colony formation assay. IC50 was calculated from a sigmoidal interpolation of all datasets. Error bars: ± SEM from three biological replicates. Statistics match in color the clone they reference, relative to NT cells. Statistics: a two-way ANOVA with Dunnett’s multiple comparisons test between NT-treated cells and UBR5 -KO clones at each concentration; * p < 0.01 and **** p < 0.0001. (H) Western blot showing UBR5 depletion in U2-OS cells electroporated with either an sgNT-RNP or an sg UBR5 -RNP. (I) Log 2 of the relative confluence over time (relative to DMSO) for sgNT-RNP- or sg UBR5 -RNP-treated U2-OS cells from (H), grown with 25 μM mirin over 120 h. The shaded area around each solid line indicates the SEM from three biological replicates. Statistics: a two-way ANOVA at 120 h with Šídák’s multiple comparisons test; p = 0.0317. See also .

Article Snippet: UBR5 (D6O8Z) Rabbit mAb , Cell Signaling Technology , 65344; RRID: AB_2799679.

Techniques: Standard Deviation, Expressing, Cell Culture, Labeling, Imaging, Control, Western Blot, Clone Assay, Colony Assay, Concentration Assay

Journal: Genes & Development

Article Title: Hyd/UBR5 defines a tumor suppressor pathway that links Polycomb repressive complex to regulated protein degradation in tissue growth control and tumorigenesis

doi: 10.1101/gad.351856.124

Figure Lengend Snippet: Identification of Hyd as a critical E3 ubiquitin ligase mediating Lin-induced Bowl degradation. ( A ) Schematic diagram of a pooled RNAi screen for E3 ligases required for Lin-induced Bowl degradation. S2R + cells transfected with HA-Bowl and FLAG-Lin were split into 24 well plates, each well treated with a pool of dsRNAs targeting four different E3 ligases, followed by Western blot analysis of cell lysates. ( B ) Confirmation of Hyd as an E3 ligase required for Lin-induced Bowl degradation. S2R + cells expressing HA-Bowl and FLAG-Lin were treated with different Hyd dsRNAs. (Hyd-1) dsRNA in the original RNAi library (Hyd-2, Hyd-3, and Hyd-4) three synthesized dsRNAs targeting different regions of Hyd. HA-Bowl was normally undetectable under such conditions due to Lin-induced degradation. However, it was stabilized by RNAi against Hyd or Lin. GFP dsRNA was included as a negative control. ( C ) S2R + cells expressing the indicated constructs were subjected to coimmunoprecipitation (co-IP) assay as indicated. Cells were treated with 10 μM PS-341 for 4 h before harvesting. Interaction was readily detected between FLAG-Lin and HA-Bowl (lane 3 ) and between FLAG-Lin and Hyd-HA (lane 4 ). (Lane 5 ) Neither pairwise interaction was affected by coexpression of the third protein. ( D ) S2R + cells expressing the indicated constructs were subjected to co-IP assay as indicated. Cells were treated with PS-341 as in C . Co-IP between FLAG-Bowl and Hyd-HA was detected only in the presence of Myc-Lin (cf. lanes 2 and 5 ). ( E ) S2R + cells expressing the indicated constructs were subjected to co-IP assay as indicated. (HA-Bowl 122–373K137R ) The K137R mutant form of Bowl fragment 122–373 as described in C. Note that the Lin–Bowl co-IP was severely impaired by lin A25 , but not lin A24 , mutation. ( F ) S2R + cells expressing the indicated constructs were subjected to co-IP assay as indicated. Note that the Lin–Hyd co-IP was impaired by both lin A24 and lin A25 mutations. ( G ) S2R + cells expressing the indicated constructs were subjected to co-IP assay as indicated. Cells were treated with PS-341 as in C . Note the disruption of the Lin–Bowl interaction by Drm (cf. lanes 2 and 3 ). ( H ) S2R + cells expressing the indicated constructs were treated with PS-341 before IP with FLAG antibody. The IP product was probed with antiubiquitin antibody to detect Bowl ubiquitination. Lin-induced Bowl ubiquitination was suppressed by Drm. ( I ) Schematic model for the regulation of Bowl degradation. In the absence of Drm, Lin promotes Bowl degradation by functioning as a substrate adaptor protein recruiting Bowl to Hyd. Drm competes with Bowl for Lin binding, thus releasing Bowl from Lin–Hyd, resulting in Bowl stabilization.

Article Snippet: The following antibodies were used for Western blot assay: rabbit polyclonal anti-UBR5 antibody (1:1000; ABclonal A13816), mouse anti-Myc antibody (1:1000; Millipore Sigma 05-419), mouse anti-FLAG antibody (1:1000; Sigma-Aldrich A8592), mouse anti-HA antibody (1:1000; Sigma-Aldrich 11583816001), mouse antiubiquitin antibody (1:100; Santa Cruz Biotechnology sc-8017), mouse antiactin (1:10,000; Millipore Sigma MAB1501R), rabbit antitubulin (Cell Signaling Technology 2148S), and rabbit anti-GFP (1:1000; Cell Signaling Technology 2555S).

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Expressing, Synthesized, Negative Control, Construct, Co-Immunoprecipitation Assay, Mutagenesis, Disruption, Binding Assay