skbr3  (ATCC)

Journal: Nature Communications

Article Title: Cysteine availability tunes ubiquitin signaling via inverse stability of LRRC58 E3 ligase and its substrate CDO1

doi: 10.1038/s41467-026-72524-3

Figure Lengend Snippet: a Volcano plot presenting RNA-seq analysis of HEK293T cells cultured for 24 h in complete or cysteine-free media ( n = 3 independent replicates). Differential expression was assessed using DESeq2 by fitting gene-wise negative binomial generalized linear models; statistical significance was evaluated using two-sided Wald tests, with p -values adjusted for multiple testing using the Benjamini-Hochberg procedure (adj. p ≤ 0.01). LRRC58 transcript levels show no significant change. Dashed lines indicate thresholds at 2-fold change and p -value = 0.05. b Average LRRC58 intensities in HEK293T proteomes ( n = 4 independent replicates) after 24 h treatment with proteasome (MG132) or neddylation inhibitors (MLN4924) in complete or cysteine-free media. c Average intensities of proteins identified in HEK293T proteomes ( n = 4 independent replicates), which were detectable when cultured in 10-fold excess cysteine but undetectable in cysteine-free media. d Average intensities of LRRC58 and CDO1 in HEK293T proteomes after 24 h culture in cysteine-free media, complete media (control), or with 10-fold excess cysteine ( n = 4 independent replicates). CDO1 was undetectable after culture in cysteine-free media; LRRC58 was undetectable in the control. e Average IPTM and PEAK scores from HT-Colabfold analysis of interactions between LRRC58-EloB/C and all proteins absent in cysteine starvation proteomes, but detected with 10-fold excess cysteine for the cell lines; HEK293T, HepG2, Jurkat, HeLa, and SKBR3. f Western blot analysis of CDO1 levels in WT-HEK293T and LRRC58 CRISPR-Cas9 knockout (KO) lysates after 24 h culture in cysteine-free media. g Average intensities of LRRC58 and CDO1 in WT-HEK293T and LRRC58-KO proteomes after 24 h culture in cysteine-free media ( n = 4 independent replicates). LRRC58-KO is confirmed by the absence of LRRC58; CDO1 is detectable only in KO. h WT-HEK293T and LRRC58-KO cells were transiently transfected with Flag-LRRC58 (WT, or A266F variant (disrupts EloB/C binding)). Western blot of CDO1 levels 24 h post-transfection, followed by 24 h culture in cysteine-free or complete media. All blots are representative of n = 2 technical replicates. Flag and vinculin serve as transfection and loading controls, respectively. All error bars report standard deviation. Purple and orange asterisks indicate samples where LRRC58 or CDO1, respectively, were undetectable. Source data provided as Source Data file.

Article Snippet: Jurkat (TIB-152), HepG2 (HB-8065), and SKBR3 (HTB-30) cells were purchased from the American Type Culture Collection (ATCC).

Techniques: RNA Sequencing, Cell Culture, Quantitative Proteomics, Control, Western Blot, CRISPR, Knock-Out, Transfection, Variant Assay, Binding Assay, Standard Deviation

Journal: Science Advances

Article Title: IRS4 is a PI3K-activating cancer dependency up-regulated through DNA rearrangements or epigenetic mechanisms in multiple solid tumors

doi: 10.1126/sciadv.aeb3503

Figure Lengend Snippet: ( A ) Diagram of IRS family protein signaling based largely on IRS1 and IRS2 studies. PH, PTB, and tail represent IRS4 domains. Red “P”, phosphotyrosine. The bottom shows the structure of the IRS1 PH and PTB domains (1QQG), with a small-molecule binding pocket indicated. ( B ) Left: IRS4 domain variants with deleted regions shown as absent or dotted lines. Right: Western blot of BT474 cells transduced with IRS4 domain variants (C-terminally FLAG tagged). The IRS4 antibody binding location is indicated. ( C ) MTT assay after 4 days of lapatinib treatment in BT474 or SKBR3 cells expressing indicated domain variants (performed in two batches; fig. S18). ( D ) Western blot of BT474 cells expressing indicated domain variants after 4 hours of lapatinib treatment. “Short” and “long” indicate exposure times. ( E ) Top: siRNA binding locations in IRS4 mRNA, indicating domain variants affected. HCC2429 cells were transduced with indicated IRS4 domain variants, followed by siRNA transfection. Proliferation was measured by the MTT assay (middle) 4 days after transfection (which occurred on days 0 and 2), and Western blotting (bottom) was performed 3 days after transfection. The bar plot shows the means and standard deviation of 10 technical replicates, which are shown as individual points. P values, two-sided t test. ( F ) CellTiter-Glo assay in TTC1240 IRS4-dTAG knockin cells after 4 days of dTAG V -1 treatment, after transduction with indicated IRS4 domain variants. Means and standard deviations are shown. ( G ) IP-Western blot of BT474 cells transduced as indicated. IP was performed using an α-IRS4 antibody (binding to the IRS4 C terminus) or IgG control. Left three lanes show input protein. ( H ) BT474 cells were stably transduced with GFP, IRS4 WT-GFP, or IRS4 ΔPH/PTB-GFP (latter two with GFP as a C-terminal tag). Cells were stained with NucRed Live 647, and live cell imaging was then performed using a Zeiss Apotome microscope.

Article Snippet: G401, RPMI2650, BT474, and SKBR3 cells were obtained from American Type Culture Collection.

Techniques: Binding Assay, Western Blot, Transduction, MTT Assay, Expressing, Transfection, Standard Deviation, Glo Assay, Knock-In, Control, Stable Transfection, Staining, Live Cell Imaging, Microscopy