Journal: Science advances

Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.

doi: 10.1126/sciadv.adl4043

Figure Lengend Snippet: Fig. 3. Enhancer activity compacts SOX9 promoter-enhancer hub in individual TNBC cells. (A and B) SOX9 promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 cells. Left: Allele percentages with SOX9 promoter interacting with SOX9.EC1, SOX9.EC3, or both (A) and SOX9.EC2, SOX9. EC3, or both (B) in MB157 (n = alleles). Right-top: SOX9 locus schematic, three-color DNA FISH 50-kb probes at SOX9 promoter (green), SOX9.EC3 (magenta), and SOX9.EC1 (A, red) or SOX9.EC2 (B, yellow). Locations per fig. S3A. Right-bottom: Representative cells. Blue: 4′,6-Diamidino-2-phenylindole (DAPI). (C and F) SOX9 enhancers inactiva- tion expands SOX9-EC1-EC3 and SOX9-EC2-EC3 hubs in individual TNBC MB157 cells. Cumulative distribution functions (CDFs) of SOX9-EC1-EC3 (C) and SOX9-EC2-EC3 (F) spatial perimeters in each MB157-dCas9-KRAB expressing control (CTRL), SOX9.EC1, SOX9.EC2, or SOX9.EC3 sgRNA [Kolmogorov-Smirnov (KS) test, n = cells]. Mean (±SD) perimeters (micrometers): (C) Left: CTRL/SOX9.EC1 sgRNA: 3.78 (±2.63)/4.36 (±2.63); middle: CTRL/SOX9.EC2 sgRNA: 3.78 (±2.63)/4.47 (±2.64); right: CTRL/SOX9.EC3 sgRNA: 3.39 (±2.56)/4.28 (±2.65). (G) Left: CTRL/SOX9.EC1 sgRNA: 3.83 (±2.71)/4.45 (±2.72); middle: CTRL/SOX9.EC2 sgRNA: 3.19 (±2.44)/4.26 (±2.61); right: CTRL/SOX9.EC3 sgRNA: 3.83 (±2.71)/4.22 (±2.56). (D and G) Allele percentages with SOX9 promoter interacting with SOX9.EC1, SOX9.EC3, or both (D) and SOX9.EC1, SOX9. EC3, or both (G) in MB157-dCas9-KRAB expressing CTRL, SOX9.EC1, SOX9.EC2, or SOX9.EC3 sgRNA (n = alleles). (E and H) Representative cells of 3C and 3D (E) or 3F and 3G (H). Blue: DAPI. (I) SOX9 promoter inactivation decreases SOX9-EC1-EC3 three-way interaction frequency across individual alleles in TNBC MB157. Top-left: Allele percent- ages with SOX9 promoter interacting with SOX9.EC1, SOX9.EC3, or both in MB157-dCas9-KRAB expressing CTRL or SOX9 promoter sgRNA (SOX9.P sgRNA) (n = alleles). Bottom-left: CDFs of SOX9-EC1-EC3 spatial perimeter in each MB157-dCas9-KRAB cell (KS test, n = cells). CTRL/SOX9.P sgRNA mean (±SD) perimeter: 3.90 (±2.62)/4.44 (±2.66) μm. Right: Representative cells. Blue: DAPI. Scale bars, 3 μm for nuclei and 0.5 μm for alleles.

Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

Techniques: Activity Assay, Expressing, Control

Journal: Science advances

Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.

doi: 10.1126/sciadv.adl4043

Figure Lengend Snippet: Fig. 6. SOX9 regulates oncogene MYC by positioning its enhancers. (A) Genome tracks showing enrichment of pairwise MYC enhancer-enhancer and promoter- enhancer interactions in a population of MB157 cells. From top to bottom: Colored circles marking location of Oligopaint DNA FISH probes labeling 50-kb regions at MYC promoter (green), MYC.EC1 (magenta), MYC.EC2 (red), MYC.EC3 (yellow), and T-ALL-restricted enhancer (black), H3K27ac and SOX9 levels as measured by ChIP-seq, and normalized interaction frequency as measured by SMC1 HiChIP at the MYC locus in MB157. MYC enhancer clusters are marked by grey boxes. (B) A total of 80% of differ- entially expressed genes with SOX9-bound promoter and distal enhancer participate in ensemble hyper-interacting hubs. MB157 hubs plotted in ascending order of their total connectivity as measured by SMC1 HiChIP in TNBC MB157. Hyper-interacting promoter-enhancer hubs are defined as the ones above the elbow of the ranked total connectivity plot. Hyper-interacting ensemble promoter-enhancer hubs containing genes that are significantly down-regulated in MB157-Cas9 cells transfected with SOX9 targeting sgRNA versus control sgRNA for 4 days and have SOX9-bound promoter and distal enhancer are marked in orange. (C to E) SOX9 loss significantly in- creases 3D distances between the MYC promoter and SOX9-bound MYC.EC2 (C) or MYC.EC3 (D) and SOX9-unbound MYC.EC1 (E) in individual cells. CDFs (left) and box and whiskers (middle) of the distances between the MYC promoter and SOX9-bound MYC.EC2 (C) and MYC.EC3 (D) and SOX9-unbound MYC.EC1 (E) in each MB157-Cas9 6 days after transduction with control sgRNA (CTRL) or SOX9-targeting sgRNA (SOX9 KO) (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) distance between MYC promoter and MYC.EC2: 0.389 (±0.358)/0.749 (±0.666) μm; MYC.EC3: 0.447 (±0.457)/0.591 (±0.551) μm; MYC.EC1: 0.494 (±0.447)/0.651 (±0.556) μm. Right: Represen- tative cells. Scale bar per 3A. Blue: DAPI.

Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

Techniques: Labeling, ChIP-sequencing, HiChIP, Transfection, Control, Transduction

Journal: Science advances

Article Title: Oncogenic transcription factors instruct promoter-enhancer hubs in individual triple negative breast cancer cells.

doi: 10.1126/sciadv.adl4043

Figure Lengend Snippet: Fig. 7. SOX9 loss decompacts MYC promoter-enhancer hubs. (A) MYC promoter participates in multiway interactions with its distal enhancer clusters in individual TNBC MB157 and MDA-MB-468 but not ER+ MCF7. Left: Percentage of alleles with MYC promoter interacting (<350 nm) with SOX9-unbound MYC.EC1, SOX9-bound MYC.EC3, or both MYC.EC1 and MYC.EC3 in MB157, MDA-MB-468, and MCF7 as measured by three-color Oligopaint DNA FISH with probes marked in Fig. 6A top genome track (n = alleles). Right: Representative MB157, MDA-MB-468, and MCF7 nuclei and two magnified alleles from three-color DNA FISH. Scale bar per 3A. Blue: DAPI. (B and C) SOX9 loss expands MYC-EC1-EC2 (B) and MYC-EC1-EC3 (C) promoter-enhancer hubs in individual MB157 and decreases three-way interaction frequency across individual alleles. Left: CDFs of MYC-EC1-EC2 (B) and MYC-EC1-EC3 (C) spatial perimeters in each MB157-Cas9 cell expressing CTRL or SOX9 KO sgRNA (KS test, n = cells). Probe locations per 6A. CTRL/SOX9 KO mean (±SD) perimeters MYC-EC1-EC2 (B): 3.84 (±2.70)/4.53 (±2.67) μm; MYC-EC1-EC3 (C): 3.10 (±2.58)/3.90 (±2.64) μm (n = cells). Middle: Allele per- centages with MYC promoter interacting (<350 nm) with MYC.EC1, MYC.EC2, or both MYC.EC1 and MYC.EC2 (B) and MYC.EC1, MYC.EC3, or both MYC.EC1 and MYC.EC3 (C) in CTRL and SOX9 KO MB157-Cas9. Right: Representative cells. Scale bar per 3A. Blue: DAPI.

Article Snippet: For the transcription factor CRISPR screen sgRNA pooled library, lentivirus was produced by transfecting HEK293T cells with helper plasmids (VSVG and psPAX2; Addgene: #12260) using FuGene HD (Promega, catalog no. E2311).

Techniques: Expressing

Journal: Diabetes

Article Title: Applying CRISPR Screen in Diabetes Research

doi: 10.2337/dbi20-0047

Figure Lengend Snippet: Strategies for modifying gene expression with CRISPR/Cas9 construct. A: CRISPR/Cas9 knockout induces a double-stranded DNA break in gDNA. Error-prone nonhomologous end joining (NHEJ) repair causes an indel mutation, causing a reading frameshift in the exon. B: dCas9 fused with VP64 and additional activator domains binds to promoter region and enhances transcription. C: dCas9 fused with KRAB binds to and blocks promoter region, interfering with transcription. Created with BioRender (BioRender.com).

Article Snippet: A large collection of knockout, activation, or inhibition CRISPR libraries, either genome-wide libraries or smaller, more focused, subpool libraries, for both the human and mouse genome, is readily available on Addgene, a nonprofit global repository of plasmids dedicated to academic research.

Techniques: Gene Expression, CRISPR, Construct, Knock-Out, Mutagenesis

Journal: Diabetes

Article Title: Applying CRISPR Screen in Diabetes Research

doi: 10.2337/dbi20-0047

Figure Lengend Snippet: Illustration of a general workflow of a high-throughput CRISPR screen. A: A plasmid library containing CRISPR gRNAs is amplified and used to generate a lentiviral library. B: A cell line or purification of primary cells is infected with the lentiviral library. C: Mutated cells containing the gRNA and selection marker are isolated. D: Cells are selected or sorted based on the phenotype of interest. E: gDNA of the selected cells is isolated. F: The CRISPR gRNA region of the purified gRNA is amplified via PCR and sequenced. G: Enriched and/or depleted gRNAs are analyzed to identify hits related to the phenotype of interest. Created with BioRender (BioRender.com).

Article Snippet: A large collection of knockout, activation, or inhibition CRISPR libraries, either genome-wide libraries or smaller, more focused, subpool libraries, for both the human and mouse genome, is readily available on Addgene, a nonprofit global repository of plasmids dedicated to academic research.

Techniques: High Throughput Screening Assay, CRISPR, Plasmid Preparation, Amplification, Purification, Infection, Selection, Marker, Isolation

Journal: Diabetes

Article Title: Applying CRISPR Screen in Diabetes Research

doi: 10.2337/dbi20-0047

Figure Lengend Snippet: Overview of the design process of a CRISPR screen.

Article Snippet: A large collection of knockout, activation, or inhibition CRISPR libraries, either genome-wide libraries or smaller, more focused, subpool libraries, for both the human and mouse genome, is readily available on Addgene, a nonprofit global repository of plasmids dedicated to academic research.

Techniques: CRISPR

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, b, Centrosome localization of Piezo2 (a) and Piezo1 (b) in C2C12 myoblast cells visualized by IF for Piezo1/2 (green), γ-Tubulin (magenta), and DNA (Hoechst dye, blue) in mitotic and interphase cells. c, d, Centrosome localization of Piezo2 (d) and Piezo1 (c) in IMCD3 and Neuro-2A cells visualized by IF in interphase cells as in (a-b). e-f, STORM imaging performed for fixed unsynchronized C2C12 cells, stained with Piezo2 (green) and γ-Tubulin (magenta). The STORM image (e) showed co-localization of Piezo2 and γ-Tubulin, suggesting Piezo2 localized to the pericentrosomal region, and the insets (f) of the centrosomes are shown with x3 zoom-in. g, Rosa26 (off target control), Piezo1 and 2 CRISPR-Cas9 polyclonal KO (pKO) of C2C12 cells at day 1 post selection imaged by IF as in (a-b), showing supernumerary centrosomes in mitotic cells. h, Quantitative analysis of supernumerary centrosomes in interphase and mitotic C2C12 pKO cells from the IF experiments in (g). i, Quantitative analysis of mitotic cell populations from the IF experiments in . In 3 independent experiments, 190-280 cells were scored for each category based on the mitotic stage. Pro: prometaphase, Meta: metaphase, Ana: anaphase, Telo: telophase, Cyto: cytokinesis, scored as per Methods. j, Piezo2 -/- myoblasts derived from newborn mice imaged by IF as in (a-b), showing supernumerary centrosomes in Piezo2 -/- myoblasts. k, Quantitative analysis of supernumerary centrosomes in interphase and mitotic cells in WT and Piezo2 -/- myoblasts from IF experiments in (j). m, Cell cycle analysis by flow cytometry for WT and Piezo2 -/- myoblasts. All images are maximum intensity Z projections. Centrosomes and centrosome-localized Piezo proteins are marked with white arrowheads. Scale bars are 5 μm for (a-d), 1 μm (e), and 10 μm (g, j). For (h, k), data are represented by mean ± SEM from three independently quantified experiments counting 50-200 cells each. Statistical significance between an experimental group and a control group was assessed by 2-tailed t-test with ***, ** and * denote p < 0.0001, 0.001 and 0.01, respectively.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Imaging, Staining, Control, CRISPR, Selection, Derivative Assay, Cell Cycle Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, b, A field view of interphase C2C12 cells, showing Piezo2 (a) and Piezo 1 (b) localization at the centrosomes. Cells were visualized by IF for Piezo2 (green), γ-Tubulin (magenta), and DNA (Hoechst dye, blue). c, Controls for the fixation method, and primary and secondary antibodies in IF. Top: C2C12 cells were fixed in PFA (instead of methanol) and stained using primary antibodies rabbit anti-Piezo2 and mouse anti-γ-Tubulin, and secondary antibodies goat anti-rabbit Alexa Fluor 488 (green) and goat anti-mouse Alexa Fluor 647 (magenta), as well as Hoechst dye for DNA (blue). Middle: C2C12 cells were stained similarly but with a rabbit IgG replacing rabbit anti-Piezo2. Bottom: C2C12 cells were stained only with secondary antibodies. d, Quantitative analysis of the percentage of C2C12 cells with co-localized centrosome and Piezo1 or 2 from IF images as in (a) and (b). e, f, Centrosome localization of Piezo1 (e) and Piezo2 (f) in IMCD3 cells visualized by IF at different cell cycle stages, imaged by IF as in (a) and (b). g, Snapshots from live imaging of C2C12 cells stably expressing Piezo1-GFP during the cell cycle (Supplementary Video 1). Centrosomes are marked with green arrowheads. h , Co-localization of Piezo1-GFP fluorescence (green) with anti-Piezo1 IF (magenta). Cells were also stained for DNA (Hoechst, blue). i, Localization of Piezo1-GFP fluorescence (green) at the γ-Tubulin (magenta)-marked centrosome by IF. All IF mages are maximum intensity Z projections. Centrosomes and centrosome-localized Piezo1 are marked with white arrowheads. All scale bars are 10 μm, except that the scale bar for (g) is 20 μm.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Staining, Imaging, Stable Transfection, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, Centrosomal localization of Piezo1 ( left) and Piezo2 ( right ) in E14.5 WT mouse forelimb sections, imaged by IF for Piezo1/2 (green), γ-Tubulin (magenta), and DNA (Hoechst dye, blue). b, A thoracic dorsal root ganglion (DRG) section of a WT mouse at E17.5, stained with anti-Piezo2 antibody (green) and Hoechst dye (blue). Right panel shows a 3x zoom-in of the dotted region in the left panel. Piezo2 puncta consistent with a centrosomal localization are labeled with white arrowheads. Of note, Piezo2 positive puncta are often present in DRG neurons whether or not the cell bodies are also Piezo2 positive. c, A thoracic DRG section of a Piezo2 -/- KO mouse at E17.5, stained with anti-Piezo2 antibody (green) and Hoechst dye (blue). Piezo2 expression at DRG neuronal cell body or centrosome is largely undetectable . d, A lung bronchial section of a WT mouse at P0, stained with anti-Piezo2 antibody (green) and Hoechst dye (blue). The cells labeled in green represent scattered pulmonary neuroepithelial cell bodies (NEBs). e, A comparable lung bronchial section of a Piezo2 -/- KO mouse at P0, imaged by IF with anti-Piezo2 antibody and Hoechst dye (blue), showing absence of Piezo2 staining. f, A comparable lung bronchial section of a WT mouse at P0, stained with rabbit IgG (green) as primary antibody and Hoechst dye (blue). g, h, Thoracic DRG sections of WT mice (g) and Piezo2 -/- KO mice, shown at 2 different contrast levels (h) at E17.5, stained with anti-Piezo2 antibody (green), γ-Tubulin (magenta) and Hoechst dye (blue). White arrowheads identify γ-Tubulin-marked centrosomes (magenta) and/or Piezo2. All scale bars are 10 μm.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Staining, Labeling, Expressing

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: iSIM was performed for fixed unsynchronized C2C12 cells, stained with Piezo2 (green) and γ-Tubulin (magenta). The iSIM image (left) shows co-localization of Piezo2 and γ-Tubulin, and the insets of the centrosomes (right) are shown with x4 zoom-in.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Staining

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, Piezo1 and Piezo2 protein levels in Piezo1 pKO, Piezo2 pKO, or Rosa26 pKO (control) of C2C12 cells analyzed by Western blots with GAPDH as the loading control. Densitometric analysis of Western blot bands of Piezo1 or 2 are shown. b, Piezo1 pKO ( left ) and Piezo2 pKO ( right ) interphase C2C12 cells at day 1 post-pKO selection, imaged by IF for Piezo1 or Piezo2 (green), γ-Tubulin (magenta) and DNA (Hoechst, blue). Centrosomes are marked with white arrows, and supernumerary centrosomes are seen in Piezo pKO cells, in comparison with Rosa26 pKO control. c, Piezo1 pKO ( middle row ), Piezo2 pKO ( bottom row ) and Rosa26 pKO ( top row ) of mitotic C2C12 cells at day 1 post-pKO selection, imaged by IF for α-Tubulin (green), γ-Tubulin (magenta) and DNA (Hoechst, blue). Centrosomes and lagging chromatin are marked with white arrows, and misaligned microtubules are seen in Piezo pKO cells, in comparison with the Rosa26 pKO control d, Quantitative real-time RT-PCR (qRT-PCR) of Piezo 1 or 2 transcripts upon small hairpin RNA (shRNA) KD of Piezo1 or Piezo2 in C2C12 cells using two and three different shRNAs for Piezo1 and Piezo2, respectively, in comparison with an off target shRNA against firefly luciferase mRNA as the control. e, Control KD, Piezo1 shRNA KD (one shRNA) or Piezo2 shRNA KD (two shRNAs) C2C12 cells at day 4 post-KD selection. The cells were imaged by IF for γ-Tubulin (red) and DNA (Hoechst, blue), showing supernumerary centrosomes upon Piezo1 or Piezo2 KD. f, Quantitative analysis of C2C12 cells with supernumerary centrosomes upon Piezo1 shRNA KD (one shRNA) or Piezo2 shRNA KD (two shRNAs) at day 4 post-KD selection. All scale bars are 10 μm. g, Off target, Piezo1 and 2 pKO C2C12 cells at day 1 post selection and 45 min after release of G2/M border synchronization with RO-3066. Cells were imaged by IF for α-tubulin (green), γ-tubulin (magenta) and DNA (Hoechst dye, blue), shown at prometaphase and cytokinesis. Quantitative analysis of mitotic cell populations from the IF experiments shown in . Statistical significance between an experimental group and the control group was assessed by 2-tailed t-test. ***, ** and * denote p < 0.0001, 0.001 and 0.01, respectively.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Control, Western Blot, Selection, Comparison, Quantitative RT-PCR, shRNA, Luciferase

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, Validation of the NLS-GCaMP6 reporter stably expressed in C2C12 cells by ionomycin (1 μM), BAPTA-AM (10 μM), or Yoda1 (10 μM) treatment. b , Quantitative analysis of maximal signals for cells imaged in (a). AFU denotes arbitrary fluorescence units. c, Phase and fluorescence snapshots of C2C12 cells expressing GCaMP6 reporter at interphase and different stages of mitosis (green). Concentrated Ca 2+ signals that represent centrosome locations are marked by white arrowheads. d , Phase and fluorescence snapshots of C2C12 cells at interphase and different stages of mitosis, imaged live at 45 min after treatment with the Ca 2+ dye Fluo4-AM (9 μM, green). Concentrated Ca 2+ signals that represent centrosome locations are marked by white arrowheads. e, Quantitative analysis of maximal GCaMP6 signals at centrosomes as shown in , revealing reductions to 43% and 34% in comparison to Rosa26 pKO control values in Piezo1 and 2 pKO cells, respectively. f, GCaMP6 expression by anti-GFP IF (green) in NLS-GCaMP6 expressing C2C12 cells at day 1 post-KO selection of Rosa26 pKO, Piezo1 pKO or Piezo2 pKO. IF for γ-Tubulin (magenta) and Hoechst dye staining of DNA (blue) are also shown, exhibiting equal GCaMP6 expression regardless of Piezo1 or 2 pKO. g , Quantitative analysis of maximal Ca 2+ signal indicated by GCaMP6 fluorescence intensity at centrosomes and in the cytosol as shown in , showing that there was a similar increase or decrease of local Ca 2+ concentration upon Yoda1 activation or GsMTx4 inhibition relative to the untreated control value both in the cytosol and at the centrosomes. Images are maximum intensity Z projections and all scale bars are 10 μm. Data are represented by mean ± SEM from three independently quantified experiments. Statistical significance between an experimental group and the control was assessed by 2-tailed t-test with *** and ** for p < 0.0001 and 0.001, respectively.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Biomarker Discovery, Stable Transfection, Fluorescence, Expressing, Comparison, Control, Selection, Staining, Concentration Assay, Activation Assay, Inhibition

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, HNMR Spectra of caged-Yoda1 and hydroxy-Yoda1 synthesis. The compounds were synthesized by WuXi AppTec with purity determined by LC-MS of 98.2% and 78.6%, respectively. For hydroxy-Yoda1, the compound was clean based on the HNMR spectrum but might not be stable in LC-MS and therefore a lower purity was reported. b, A table describing the top hits from the yeast two-hybrid screen performed for Piezo2 CTD.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Synthesized, Liquid Chromatography with Mass Spectroscopy, Two Hybrid Screening

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a, Validation of the Snx5 interaction with the Piezo2 C-terminal domain (CTD, last 70 aa) using an alternative yeast two-hybrid assay. The Piezo2 CTD was fused to the LexA DNA binding domain and full-length Snx5 was fused to a B42 activation domain. Positivity is indicated by yeast turning dark blue in the presence of the LacZ reporter X-gal (+++) and by yeast growth in the absence of leucine. Negativity is indicated by yeast staying white (-) and no growth in the absence of leucine. Phox homology (PX) domain for lipid binding and Bin/Amphiphysin/Rvs (BAR) domain for membrane-curvature sensing are shown. b, Western blot analysis of Ni-NTA pulldown of human Snx5 by human Piezo2 CTD. Piezo2-CTD-His-Myc was co-expressed with GFP-Snx5 or GFP control in HEK293T cells. The His-tagged protein was precipitated using Ni-NTA beads (lane 1&2) or as a control with NTA beads without Ni (lane 3). Lanes 4 and 5 are cell lysates. Precipitated Piezo2-His-Myc was detected with anti-Myc antibody and GFP-hSnx5 was detected with anti-GFP antibody. c, IF images of C2C12 cells (top panel) and Neuro-2A cells (bottom panel) stained for Piezo2 (green), Snx5 (olive), ɣ-Tubulin (magenta) and DNA (blue). Co-localization among Piezo2, Snx5 and ɣ-Tubulin is shown by arrowheads and all scale bars are 10 μm. d, Western blot analysis of fractionation of isolated centrosomes. Centrosomes isolated from C2C12 cells were fractionated on a sucrose gradient, and blotted with the following antibodies: anti-Piezo1, anti-Piezo2, anti-γ-Tubulin, anti-Pericentrin and anti-Snx5.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Biomarker Discovery, Y2H Assay, Binding Assay, Activation Assay, Membrane, Western Blot, Control, Staining, Fractionation, Isolation

Journal: bioRxiv

Article Title: Piezo mechanosensory channels regulate centrosome integrity

doi: 10.1101/2022.04.12.488050

Figure Lengend Snippet: a-b, IF of C2C12 cells, fixed after 3h cold treatment, 2 h treatment with Parthenolide or Taxol and stained with Piezo1 (a) or Piezo2 (b), ⍺-Tubulin (green) and Hoechst (Blue). Untreated cells were used as a control. Centrosomes are marked with white arrowheads. c , Western blots of C2C12 cells at different cell cycle stages using anti-Piezo1, Piezo2, PLK1 and GAPDH (loading control) antibodies. While Piezo2 is highly expressed at S and G2, Piezo1 expression is relatively constant. d , Cell cycle analysis by PI staining for C2C12 cells treated with PLK1 inhibitor BI-6727. Optimal concentration for BI-6727 treatment in C2C12 was determined as 100 nM. Images are maximum intensity Z projections and all scale bars are 10 μm. Data are represented by mean ± SEM from three independently quantified experiments. Statistical significance between an experimental group and the control was assessed by 2-tailed t-test with ***, ** and * for p < 0.0001, 0.001 and 0.01, respectively.

Article Snippet: The following antibodies were used for Piezo detection: rabbit polyclonal anti-Piezo1 antibody (1:200 Novus Biologicals NBP1-78446) and rabbit polyclonal anti-Piezo2 antibody (1:200 Novus Biologicals NBP1-78624).

Techniques: Staining, Control, Western Blot, Expressing, Cell Cycle Assay, Concentration Assay

Journal: EMBO Reports

Article Title: Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1

doi: 10.15252/embr.202153429

Figure Lengend Snippet: A Schematic representation of the sequence of Rabaptin5. Coiled‐coil (CC) segments are shown in yellow. Colored backgrounds highlight the segments shown to interact with Rab4, Rab5, Rabex5, and the GAE and GAT domains of GGAs (Golgi‐localizing, γ‐adaptin ear homology domain, ARF‐binding proteins). Below, the segments used to test yeast two‐hybrid interaction with residues 257–444 of FIP200 are shown with their number (#) and the observed interaction (+ or –). B Yeast two‐hybrid analysis for interaction between the above‐shown Rabaptin5 segments (Rbpt5#, fused to LexA on the bait plasmid) and residues 257–444 of FIP200 (FIP, fused to the Gal4 activation domain on the prey plasmid) to drive HIS3 expression. Three different clones each were replica‐plated on medium with His or without His, but containing 3‐amino‐1,2,4‐triazole (3AT; an inhibitor of His synthesis to increase stringency) and grown in the absence of Trp and leucine as a control. As negative controls, empty bait or prey plasmids were used. The asterisk indicates a clone invalidated by recombination. C Schematic representation of the sequence of FIP200 with its coiled‐coil segments in yellow. Residues 281–439 (gray) indicate the minimal sequence identified to interact with Rabaptin5 in the yeast two‐hybrid screen. D FIP200 was immunoprecipitated (IP) from lysates of HeLa or HEK293A cells and probed for FIP200, Rabaptin5 (Rbpt5), and EEA1 (early endosome antigen 1) by immunoblotting. Input lysate (10%) was immunoblotted blotted parallel. As a negative control, the immunoprecipitation was performed using an anti‐GAPDH antibody. E–H Lysates of HeLa cells transiently transfected with full‐length FIP200‐mCherry (FIP200‐mCh) or a deletion mutant lacking the segment interacting with Rabaptin5 (∆280–440) were immunoprecipitated with anti‐mCherry (IP FIP200‐mCh) or, as a control, with anti‐FLAG antibodies (IP FLAG). Immunoprecipitates and input lysates (10%) were immunoblotted for mCherry and Rabaptin5 (E), ATG13 (F), or ULK1 (G). Co‐immunoprecipitation of Rabaptin5, ATG13, and ULK1 with FIP200∆280–440 (FIP∆) was quantified in comparison with that with wild‐type FIP200 (H; signals normalized to that of the immunoprecipitated protein; mean ± SD of three independent experiments each).

Article Snippet: The cDNA of human FIP200 was purchased from OriGene (SC114884), pmCherry_Gal3 was a gift from Hemmo Meyer (Addgene plasmid #85662) (Papadopoulos et al , ), mRuby3‐Gal8 (PB‐CAG‐mRuby3‐Gal8‐P2A‐Zeo) was obtained from Addgene (#150815), pSpCas9(BB)‐2A‐GFP (PX458) was a gift from Feng Zhang (Addgene plasmid #48138) (Ran et al , ), and mCherry‐ATG16L1 was a kind gift from Sharon Tooze (Francis Crick Institute).

Techniques: Sequencing, Binding Assay, Plasmid Preparation, Activation Assay, Expressing, Clone Assay, Control, Two Hybrid Screening, Immunoprecipitation, Western Blot, Negative Control, Transfection, Mutagenesis, Comparison

Journal: EMBO Reports

Article Title: Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1

doi: 10.15252/embr.202153429

Figure Lengend Snippet: A–E HeLa cells were transfected with FLAG‐tagged FIP200 alone (A) or together with Rabaptin5 (Rbpt5‐wt) (B), GFP‐Rab4 (C), RFP‐Rab5 (D), or Citrine‐Rab7 (E), fixed after 24 h, and subjected to immunofluorescence microscopy. Scale bar, 10 µm.

Article Snippet: The cDNA of human FIP200 was purchased from OriGene (SC114884), pmCherry_Gal3 was a gift from Hemmo Meyer (Addgene plasmid #85662) (Papadopoulos et al , ), mRuby3‐Gal8 (PB‐CAG‐mRuby3‐Gal8‐P2A‐Zeo) was obtained from Addgene (#150815), pSpCas9(BB)‐2A‐GFP (PX458) was a gift from Feng Zhang (Addgene plasmid #48138) (Ran et al , ), and mCherry‐ATG16L1 was a kind gift from Sharon Tooze (Francis Crick Institute).

Techniques: Transfection, Immunofluorescence, Microscopy

Journal: EMBO Reports

Article Title: Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1

doi: 10.15252/embr.202153429

Figure Lengend Snippet: A–I To more easily visualize Rabaptin5, a stable HEK293A cell line overexpressing Rabaptin5 (HEK +Rbpt5 ) was generated. Immunofluorescence microscopy of Rabaptin5 and transferrin receptor (TfR) showed swelling of early endosomes upon treatment with 60 µM chloroquine for 30 min (+CQ, panel A) compared with untreated cells (–CQ, A'). Rabaptin5 levels were analyzed by immunoblotting in comparison with parental HEK293A cells (panel B). HEK +Rbpt5 cells, untransfected or 24 h after transfection with mCherry‐galectin 3 (mCh‐Gal3), mRuby3‐galectin 8 (mRuby‐Gal8), mCherry‐FIP200, or mCherry‐ATG16L1 were analyzed upon chloroquine treatment by immunofluorescence microscopy for Rabaptin5 and mCherry‐galectin 3 or mRuby3‐galectin 8 (C), ubiquitin (Ub; D), p62 (E), mCherry‐FIP200 (F), WIPI2 (G), mCherry‐ATG16L1 (H), or LC3B (I). Scale bar, 10 µm. In the enlarged insets, arrowheads point out chloroquine‐induced enlarged, ring‐like early endosomes. Rabaptin5‐positive enlarged endosomes positive for mCherry‐galectin 3 (Gal3) or mRuby3‐galectin 8 (Gal8) were quantified (C'; mean ± SD and individual values of three independent experiments counting > 45 structures each). J, K HEK +Rbpt5 cells, untransfected or 24 h after transfection with mCherry‐ATG16L1, were treated with 60 µM chloroquine for 0, 15, and 30 min and stained for Rabaptin5 and either WIPI2 or mCherry‐ATG16L1. Manders’ colocalization coefficients were determined, M1 showing the fraction of Rabaptin5‐positive structures also positive for WIPI2 (J) or mCherry‐ATG16L1 (K), and M2 showing the respective inverse (mean ± SD of three independent experiments; ANOVA: * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The cDNA of human FIP200 was purchased from OriGene (SC114884), pmCherry_Gal3 was a gift from Hemmo Meyer (Addgene plasmid #85662) (Papadopoulos et al , ), mRuby3‐Gal8 (PB‐CAG‐mRuby3‐Gal8‐P2A‐Zeo) was obtained from Addgene (#150815), pSpCas9(BB)‐2A‐GFP (PX458) was a gift from Feng Zhang (Addgene plasmid #48138) (Ran et al , ), and mCherry‐ATG16L1 was a kind gift from Sharon Tooze (Francis Crick Institute).

Techniques: Generated, Immunofluorescence, Microscopy, Western Blot, Comparison, Transfection, Ubiquitin Proteomics, Staining

Journal: EMBO Reports

Article Title: Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1

doi: 10.15252/embr.202153429

Figure Lengend Snippet: HEK293A cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h and treated without (–) or with 60 µM chloroquine (+CQ) or 250 nM Torin1 for 150 min. Scale bar, 10 µm. Below, the efficiency of Rabaptin5 and FIP200 knockdown was assayed by immunoblotting using tubulin (Tub) as a loading control. WIPI2 or LC3B puncta per cell were quantified for each condition (mean ± SD of three independent experiments; ANOVA: * P < 0.05, ** P < 0.01). HEK293A cells were transfected with siCtr or siRbpt5 as in A and incubated without or with 280 µM LLOMe for 150 min to induce lysophagy. Cells were fixed and immunostained for endogenous WIPI2 and LC3B. Scale bar, 10 µm. WIPI2 or LC3B puncta per cell were quantified (mean ± SD of three independent experiments).

Article Snippet: The cDNA of human FIP200 was purchased from OriGene (SC114884), pmCherry_Gal3 was a gift from Hemmo Meyer (Addgene plasmid #85662) (Papadopoulos et al , ), mRuby3‐Gal8 (PB‐CAG‐mRuby3‐Gal8‐P2A‐Zeo) was obtained from Addgene (#150815), pSpCas9(BB)‐2A‐GFP (PX458) was a gift from Feng Zhang (Addgene plasmid #48138) (Ran et al , ), and mCherry‐ATG16L1 was a kind gift from Sharon Tooze (Francis Crick Institute).

Techniques: Transfection, Knockdown, Western Blot, Control, Incubation

Journal: EMBO Reports

Article Title: Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1

doi: 10.15252/embr.202153429

Figure Lengend Snippet: A HeLa cells transiently transfected with full‐length mCherry‐ATG16L1 were treated with 60 µM chloroquine (CQ) for 0, 30, or 120 min, lysed, and immunoprecipitated with anti‐Rabaptin5 (IP: Rbpt5) or, as a control, with anti‐FLAG antibodies (IP FLAG). Immunoprecipitates and input lysates (10%) were immunoblotted for Rabaptin5 and ATG16L1. Signals were quantified and the ratios of mCherry‐ATG16L1/Rabaptin5 normalized to that without (0 min) chloroquine treatment (mean ± S of six independent experiments; two‐tailed Student’s t ‐test: * P < 0.05, ** P < 0.001). B Co‐immunoprecipitation was performed as in panel A using parental HEK293A cells and CRISPR/Cas9 knockout cells lacking FIP200 (FIP‐KO). Anti‐HA antibodies were used as a control (IP HA). On the right, HEK293A‐ and FIP200‐knockout cells were immunoblotted for FIP200 and as a loading control of tubulin (Tub). Signals were quantified and the ratios of mCherry‐ATG16L1/Rabaptin5 normalized to that of HEK293A cells without chloroquine treatment (mean ± SD of four independent experiments; two‐tailed Student’s t ‐test: * P < 0.05, ** P < 0.001). C, D Lysates of HEK293A or HeLa cells transiently transfected with full‐length mCherry‐ATG16L1 (wt) or a mutant lacking the WD domain (∆WD; residues 1–319 of ATG16L1, precisely deleting only the WD40 repeats residues 320–607) were immunoprecipitated with anti‐Rabaptin5 or anti‐FLAG antibodies, and immunoblotted for Rabaptin5 and mCherry‐ATG16L1 (C), or immunoprecipitated with anti‐Rabaptin5 or anti‐HA antibodies, and immunoblotted for Rabaptin5 and mCherry‐ATG16L1 (D). In panel D, cells were incubated with or without 60 µM chloroquine for 30 min before analysis. Co‐immunoprecipitation of ATG16L1∆WD with Rabaptin5 (C) was reduced to 6.6 ± 2.1 and 3.4 ± 2.1% in HEK293A and HeLa cells, respectively, relative to that of full‐length ATG16L1 (signals normalized to the immunoprecipitated protein; mean ± SD deviation of three independent experiments each). E The consensus sequence of the ATG16L1 interaction motifs of TMEM59, NOD2, and TLR2 (above; Boada‐Romero et al, ) is shown together with the matching sequence in Rabaptin5 (below). The three point mutations to alanine to produce the AAA mutant of Rabaptin5 are indicated. F Lysates of HeLa cells transiently transfected with myc‐tagged wild‐type Rabaptin5 (wt) or triple‐alanine mutant (AAA) were immunoprecipitated with anti‐myc (IP myc) or anti‐FLAG antibodies (IP FLAG), and immunoblotted for myc and ATG16L1. Co‐immunoprecipitation of ATG16L1 with Rabaptin5‐AAA triple mutant was reduced to 1.5 ± 1.2% relative to that with wild‐type Rabaptin5 (signals normalized to that of the immunoprecipitated protein; mean ± SD of three independent experiments).

Article Snippet: The cDNA of human FIP200 was purchased from OriGene (SC114884), pmCherry_Gal3 was a gift from Hemmo Meyer (Addgene plasmid #85662) (Papadopoulos et al , ), mRuby3‐Gal8 (PB‐CAG‐mRuby3‐Gal8‐P2A‐Zeo) was obtained from Addgene (#150815), pSpCas9(BB)‐2A‐GFP (PX458) was a gift from Feng Zhang (Addgene plasmid #48138) (Ran et al , ), and mCherry‐ATG16L1 was a kind gift from Sharon Tooze (Francis Crick Institute).

Techniques: Transfection, Immunoprecipitation, Control, Two Tailed Test, CRISPR, Knock-Out, Mutagenesis, Incubation, Sequencing

Journal: EMBO Reports

Article Title: Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1

doi: 10.15252/embr.202153429

Figure Lengend Snippet: HeLa cells were transfected with nontargeting siRNA (siCtr) or siRNAs silencing Rabaptin5 (siRbpt5) or FIP200 (siFIP200) for 72 h. The cells were infected with Salmonella by centrifugation at 500 × g for 5 min at 37°C and incubation for 10 min at 37°C, washed three times, and incubated in fresh culture medium containing gentamicin to prevent growth of extracellular bacteria for 0, 1, 3, or 6 h before lysis of the host cells and plating of the bacteria on LB agar plates at various dilutions to determine the number of live bacteria at the different time points, shown as a percentage of internalized cells after infection (mean ± SD of three independent experiments). The fractions of internalized bacteria alive 1 h after infection are shown separately in the middle (mean ± SD of three independent experiments; ANOVA: * P < 0.05). On the right, the fraction of infected cells was determined for HeLa cells transfected with siRNAs and infected as above with Salmonella expressing GFP, washed, and immediately fixed for fluorescence microscopy and stained with anti‐transferrin receptor and anti‐LC3B as cellular markers. Z ‐stacks for > 5,000 cells/sample were acquired and analyzed in Fiji to determine the fraction of infected cells (mean ± SD of three independent experiments). The average number of bacteria per infected cell was identical (2.16, 2.11, and 2.12 bacteria per cell transfected with siCtr, siRbpt5, and siFIP200, respectively). Wild‐type HEK293A, HEK +Rbpt5 , Rbpt5‐KO, Rbpt5‐KO+wt, Rbpt5‐KO+AAA, and FIP200‐KO cells were infected with Salmonella and treated and analyzed as in panels A (mean ± SD of three independent experiments; ANOVA: * P < 0.05, **** P < 0.0001). Wild‐type HEK293A, HEK +Rbpt5 , Rbpt5‐KO, Rbpt5‐KO+AAA, and FIP200‐KO cells were infected with Salmonella expressing GFP as in panel B, incubated in fresh culture medium containing gentamicin for 0, 5, 15, 30, and 60 min, fixed with methanol, and immunostained for transferrin receptor (TfR) as a marker of early endosomes and for LC3B as a marker of autophagy. Salmonella were classified according to their association with a TfR‐ and/or LC3B‐positive compartment—as illustrated on the top left (scale bar, 2 µm)—during the first hour after infection. In the absence of Rabaptin5, LC3‐positive SCVs with early endosomal characteristics (containing TfR) were strongly reduced. (mean ± SD of three independent experiments, analyzing >50 bacteria for each time point.)

Article Snippet: The cDNA of human FIP200 was purchased from OriGene (SC114884), pmCherry_Gal3 was a gift from Hemmo Meyer (Addgene plasmid #85662) (Papadopoulos et al , ), mRuby3‐Gal8 (PB‐CAG‐mRuby3‐Gal8‐P2A‐Zeo) was obtained from Addgene (#150815), pSpCas9(BB)‐2A‐GFP (PX458) was a gift from Feng Zhang (Addgene plasmid #48138) (Ran et al , ), and mCherry‐ATG16L1 was a kind gift from Sharon Tooze (Francis Crick Institute).

Techniques: Transfection, Infection, Centrifugation, Incubation, Bacteria, Lysis, Expressing, Fluorescence, Microscopy, Staining, Marker

Journal: Cancers

Article Title: Therapeutic Potential of EWSR1–FLI1 Inactivation by CRISPR/Cas9 in Ewing Sarcoma

doi: 10.3390/cancers13153783

Figure Lengend Snippet: Selection of gRNAs targeting EWSR1–FLI1 and analysis of gene editing evolution. ( a ) Experimental design: Ewing sarcoma cell line A673 expressing Cas9 protein (A673/Cas9) were generated by lentiviral infection. After clonal selection, A673/Cas9 cells were infected with a multiplex lentiviral CRISPR library of 18,479 different sgRNAs targeting 1983 transcription factors including ten gRNAs targeting FLI1. After this screening phase, two gRNAs were selected for functional and molecular characterization. A673/Cas9 were infected with lentiviral sgRNAs to generate A673/Cas9/sgRNA cells and then maintained in continuous growth to assess gene editing, cell proliferation, senescence, and studies of mRNA and protein expression at different time points. A673/TR/shEF, which expresses a specific EWSR1–FLI1 shRNA upon doxycycline stimulation, were cultured and analyzed in a similar way. The results obtained upon gene editing and gene silencing were then compared. ( b ) Schematic representation of native FLI1 and EWSR1–FLI1 fusion genes, location of FLI1 gRNAs, and enrichment scores obtained for each gRNA in the CRISPR screening assay (mean ± SD of two independent experiments).

Article Snippet: After, cells were incubated for 1 h in blocking solution (BS; 5% Goat Serum ( v / v ) in PBS 1×), washed with PBS twice, and incubated overnight at 4 °C with anti-FLI1 rabbit monoclonal antibody (1:700 in BS; ab133485, AbCam, Cambridge, UK).

Techniques: Selection, Expressing, Generated, Infection, Multiplex Assay, CRISPR, Functional Assay, shRNA, Cell Culture, Screening Assay

Journal: Cancers

Article Title: Therapeutic Potential of EWSR1–FLI1 Inactivation by CRISPR/Cas9 in Ewing Sarcoma

doi: 10.3390/cancers13153783

Figure Lengend Snippet: Effect of EWSR1–FLI1 gene editing on EWSR1–FLI1 expression and EWSR1–FLI1 target genes. ( a ) EWSR1–FLI1, NR0B1, and CD44 mRNA levels were quantified by RT-qPCR in A673/Cas9 cell lines infected with sgRNA vectors targeting exon 2 and exon 9. The graphs represent the results of two independent experiments performed with MOI = 1 (1) and MOI = 5 (2). A673/sgRNA was used as the control (Ctrl). A673/TR/shEF cells were stimulated with doxycycline (1 µg/mL) for 72 h to induce the expression of the EWSR1–FLI1-specific shRNA. Data shown are the mean ± SD of experiments conducted in triplicate (ns, not significant; ** p < 0.01; *** p < 0.001; Student’s t -test). ( b ) EWSR1–FLI1, NR0B1, and CD44 protein levels were detected by Western blot. β-Tubulin was used as a control for loading and transferring. ( c ) Immunostaining of cells with anti-FLI1 antibody. Intense nuclear staining for EWSR1–FLI1 (red fluorescence colocalized with DAPI staining) was observed in both the A673 control cell line and A673/Cas9/FLI1-EX2, while it was undetectable in the majority of the A673/Cas9/FLI1-EX9 cells.

Article Snippet: After, cells were incubated for 1 h in blocking solution (BS; 5% Goat Serum ( v / v ) in PBS 1×), washed with PBS twice, and incubated overnight at 4 °C with anti-FLI1 rabbit monoclonal antibody (1:700 in BS; ab133485, AbCam, Cambridge, UK).

Techniques: Expressing, Quantitative RT-PCR, Infection, Control, shRNA, Western Blot, Transferring, Immunostaining, Staining, Fluorescence

Journal: Cancers

Article Title: Therapeutic Potential of EWSR1–FLI1 Inactivation by CRISPR/Cas9 in Ewing Sarcoma

doi: 10.3390/cancers13153783

Figure Lengend Snippet: Effect of EWSR1–FLI1 gene editing on cell proliferation. ( a ) A673/Cas9/sgRNA cells were continuously maintained in culture and the cumulative population doubling was recorded. The A673/TR/shEF cells were also continuously maintained in culture in the absence or presence of doxycycline (1 µg/mL). ( b ) Quantification of the population doubling time in each cycle of cell seeding–trypsinization. Mean ± SD of three independent experiments is shown (ns, not significant; ** p < 0.01, *** p < 0.001; two-way ANOVA Tukey’s post hoc vs. control). ( c ) The cell cycle was analyzed by flow cytometry in non-synchronized cells. The percentages of cells in each cell cycle phase are indicated. One representative experiment out of two independent experiments performed with similar results is shown.

Article Snippet: After, cells were incubated for 1 h in blocking solution (BS; 5% Goat Serum ( v / v ) in PBS 1×), washed with PBS twice, and incubated overnight at 4 °C with anti-FLI1 rabbit monoclonal antibody (1:700 in BS; ab133485, AbCam, Cambridge, UK).

Techniques: Control, Flow Cytometry

Journal: Cancers

Article Title: Therapeutic Potential of EWSR1–FLI1 Inactivation by CRISPR/Cas9 in Ewing Sarcoma

doi: 10.3390/cancers13153783

Figure Lengend Snippet: EWSR1–FLI1 gene inactivation induced generalized senescence. ( a ) β-Galactosidase activity was measured in control (Ctrl) cells (A673/sgRNA) and A673/Cas9/sgRNA cells using a β-galactosidase activity assay. β-Galactosidase-positive cells were counted, and the percentage of positive cells were determined. The graphs represent the results of two independent experiments performed with MOI = 1 (1) and MOI = 5 (2). The A673/TR/shEF cells were stimulated with doxycycline (1 µg/mL) for 7 days to induce the expression of the EWSR1–FLI1-specific shRNA (mean ± Scheme 0. Student’s t -test). Representative micrographs of each cell line are also shown. ( b ) Representative micrographs of A673 and A673/Cas9/FLI1-EX9 cells, showing the characteristic appearance of senescent cells in A673/Cas9/FLI1–EX9 cells. ( c ) The A673/Cas9/sgRNAs cells were seeded at a low density and then isolated clones were reseeded independently in 96-well plates. Afterward, Sanger sequencing/ICE CRISPR analysis was performed for each clone to determine the gene edition percentage (% indels). The tables show the phenotype of each picked-up clone, if it expand after reseeding and the percentage of gene edition.

Article Snippet: After, cells were incubated for 1 h in blocking solution (BS; 5% Goat Serum ( v / v ) in PBS 1×), washed with PBS twice, and incubated overnight at 4 °C with anti-FLI1 rabbit monoclonal antibody (1:700 in BS; ab133485, AbCam, Cambridge, UK).

Techniques: Activity Assay, Control, Expressing, shRNA, Isolation, Clone Assay, Sequencing, CRISPR

Journal: Nature communications

Article Title: Identification of regulators of poly-ADP-ribose polymerase inhibitor response through complementary CRISPR knockout and activation screens.

doi: 10.1038/s41467-020-19961-w

Figure Lengend Snippet: Fig. 1 Complementary CRISPR knockout and activation screens identify determinants of PARPi response in parental or BRCA2-knockout HeLa cells. a Schematic representation of the CRISPR knockout screen for olaparib sensitivity in wildtype cells. HeLa cells were infected with the Brunello CRISPR knockout library. Infected cells were divided into PARP inhibitor (olaparib)-treated or control (DMSO) arms. Genomic DNA was extracted from cells surviving the drug treatment and single-guide RNAs (sgRNAs) were identified using Illumina sequencing. b Scatterplot showing the results of this screen. Each gene targeted by the library was ranked based on the MAGeCK negative selection score. Several biologically interesting hits are highlighted. c Schematic representation of the CRISPR knockout screen for olaparib resistance in BRCA2KO cells. HeLa BRCA2KO cells were infected with the Brunello CRISPR knockout library. Infected cells were divided into PARP inhibitor (olaparib)-treated or control (DMSO) arms. d Scatterplot showing the results of this screen, with several biologically interesting hits highlighted. Each gene targeted by the library was ranked based on the MAGeCK positive selection score. e Schematic representation of the CRISPR activation screen for olaparib resistance in BRCA2KO cells. HeLa BRCA2KO cells stably expressing the modified dCas9 enzyme were infected with the Calabrese CRISPR activation library. Infected cells were divided into PARP inhibitor (olaparib)-treated or control (DMSO) arms. f Scatterplot showing the results of this screen, with several biologically interesting hits highlighted. Each gene targeted by the library was ranked based on the MAGeCK positive selection score. Source data are provided as a Source Data file.

Article Snippet: For the CRISPR activation screens, HeLa BRCA2-knockout cells were infected with dCas9 (Addgene, 61425-LV) and selected with blasticidin (3 μg/ml). dCas9expressing cells were then transduced with the Calabrese Human CRISPR Activation Pooled Library (Set A, Addgene, 92379-LV) using enough cells to obtain a library coverage of 500 cells per sgRNA at an MOI of 0.418.

Techniques: CRISPR, Knock-Out, Activation Assay, Infection, Control, Illumina Sequencing, Selection, Stable Transfection, Expressing

Journal: Cancer Discovery

Article Title: Defining the Antitumor Mechanism of Action of a Clinical-stage Compound as a Selective Degrader of the Nuclear Pore Complex

doi: 10.1158/2159-8290.CD-25-0271

Figure Lengend Snippet: TRIM21 promotes PRLX-induced cytotoxicity. A, Chemical structures of PRLX and its parent compound, erastin. B, PRISM pooled barcoded cell line screen overview. C, Scatter plot of erastin vs. PRLX PRISM viability profiles at 2.5 μmol/L demonstrating a lack of correlation. Each point is a single cancer cell line. D, Volcano plot depicting predictive mRNA expression biomarkers of PRLX activity by linear modeling. E, Scatter plot of TRIM21 mRNA expression vs. PRISM viability dose response (AUC). Each point is a single cancer cell line. P values were adjusted by the Benjamini–Hochberg procedure. F, Lineage-enrichment testing for PRLX PRISM activity. Fisher’s exact test was performed to identify cancer types enriched within the most sensitive quartile of cell lines. G, Cell viability of individual cancer cell lines with high or low TRIM21 expression following treatment with PRLX or vehicle for 72 hours ( n = 3–6; error bars, SD). H, Cell proliferation of H661 WT or TRIM21 overexpression (OE) cells treated with PRLX vs. vehicle for 72 hours ( n = 3; error bars, SD). I, CRISPR-Cas9 KO and activation library screening overview. J, Gene KO enrichment in the CRISPR-Cas9 KO screen (21 days; PRLX vs. DMSO). K, Gene activation enrichment in the CRISPR-dCas9 activation screen (13 days; PRLX vs. DMSO). L, Cell proliferation of WT or TRIM21 KO PANC-1 cells treated with PRLX or erastin vs. vehicle for 72 hours ( n = 3; error bars, SD). sgNT, single guide RNA non-targeting control; TPM, transcripts per million.

Article Snippet: For immunoblots, the following primary antibodies were used: TRIM21 (Cell Signaling Technology, cat. # 92043, RRID:AB_2800177), TRIM21 (Santa Cruz Biotechnology, cat. # sc-25351, RRID:AB_628286), IRF1 (Cell Signaling Technology, cat. # 8478s, RRID:AB_10949108), IRF2 (Cell Signaling Technology, cat. # 59452S), IKKα (Cell Signaling Technology, cat. # 2682s, RRID: AB_331626), IKKγ (Cell Signaling Technology, cat. # 2685s, RRID: AB_2124829), NF-κB p65 (Cell Signaling Technology, cat. # 8242s, RRID: AB_10859369), IκBα (Cell Signaling Technology, cat. # 4812T, RRID: AB_10694416), Anti-Nuclear Matrix Protein p84 (Abcam, cat. # ab487, RRID:AB_304696), NUP214 (Abcam, cat. # ab70497, RRID:AB_1269607), NUP98 (Cell Signaling Technology, cat. # 2598T, RRID: AB_2267700), NUP96 (Thermo Fisher Scientific, cat. # A301784A, RRID:AB_1211487), NUP35 (ABclonal, cat. # A12762, RRID: AB_2759608), MCL1 (Cell Signaling Technology, cat. # 94296S, RRID: AB_2722740), GLE1 (Proteintech, cat. # 26466-1-AP, RRID: AB_2880525), BCLXL (Cell Signaling Technology, cat. # 2764T, RRID: AB_2228008), BCL2 (Cell Signaling Technology, cat. # 4223T, RRID: AB_1903909), Cleaved Caspase-3 (Cell Signaling Technology, cat. # 9661s, RRID: AB_2341188), Caspase-3 (Cell Signaling Technology, cat. # 9668s, RRID: AB_2069870), Cleaved Caspase-7 (Cell Signaling Technology, cat. # 8438s, RRID:AB_11178377), Caspase-7 (Cell Signaling Technology, cat. # 9494s, RRID: AB_2068141), PARP (Cell Signaling Technology, cat. # 9532s, RRID:AB_659884), MDM2 (Cell Signaling Technology, cat. # 86934s, RRID:AB_2784534), c-MYC Antibody (Cell Signaling Technology, cat. # 9402s, RRID: AB_2151827), β-actin (Cell Signaling Technology, cat. # 4970s, RRID: AB_2223172), β-actin (Cell Signaling Technology, cat. # 3700S, RRID: AB_2242334), α-tubulin (Cell Signaling Technology, cat. # 2125, RRID: AB_2619646), and GAPDH (Cell Signaling Technology, cat. # 97166, RRID: AB_2756824).

Techniques: Expressing, Activity Assay, Over Expression, CRISPR, Activation Assay, Library Screening, Control

Journal: Cancer Discovery

Article Title: Defining the Antitumor Mechanism of Action of a Clinical-stage Compound as a Selective Degrader of the Nuclear Pore Complex

doi: 10.1158/2159-8290.CD-25-0271

Figure Lengend Snippet: Structure–activity relationship of PRLX analogues targeting TRIM21. A, Chemical structures of PRLX analogues. B, Chemical structure of optimized lead compound JWZ-8-103. C, Cell viability IC 50 values for WT and TRIM21 KO PANC-1 cells treated with each analogue vs. vehicle for 72 hours. D, Cell proliferation of WT or TRIM21 knockout PANC-1 cells treated with each analogue vs. vehicle for 72 hours ( n = 3; error bars, SD). E, Cell proliferation of H661 WT or TRIM21 overexpression (OE) cells treated with each analogue vs. vehicle for 72 hours ( n = 3; error bars, SD). F, Scatter plot of PRISM dose–response viability profiles (AUC) for PRLX vs. JWZ-8-103. Each point is a single cancer cell line. G, Volcano plot depicting predictive mRNA expression biomarkers of PRLX activity by linear modeling. TRIM21 is indicated.

Article Snippet: For immunoblots, the following primary antibodies were used: TRIM21 (Cell Signaling Technology, cat. # 92043, RRID:AB_2800177), TRIM21 (Santa Cruz Biotechnology, cat. # sc-25351, RRID:AB_628286), IRF1 (Cell Signaling Technology, cat. # 8478s, RRID:AB_10949108), IRF2 (Cell Signaling Technology, cat. # 59452S), IKKα (Cell Signaling Technology, cat. # 2682s, RRID: AB_331626), IKKγ (Cell Signaling Technology, cat. # 2685s, RRID: AB_2124829), NF-κB p65 (Cell Signaling Technology, cat. # 8242s, RRID: AB_10859369), IκBα (Cell Signaling Technology, cat. # 4812T, RRID: AB_10694416), Anti-Nuclear Matrix Protein p84 (Abcam, cat. # ab487, RRID:AB_304696), NUP214 (Abcam, cat. # ab70497, RRID:AB_1269607), NUP98 (Cell Signaling Technology, cat. # 2598T, RRID: AB_2267700), NUP96 (Thermo Fisher Scientific, cat. # A301784A, RRID:AB_1211487), NUP35 (ABclonal, cat. # A12762, RRID: AB_2759608), MCL1 (Cell Signaling Technology, cat. # 94296S, RRID: AB_2722740), GLE1 (Proteintech, cat. # 26466-1-AP, RRID: AB_2880525), BCLXL (Cell Signaling Technology, cat. # 2764T, RRID: AB_2228008), BCL2 (Cell Signaling Technology, cat. # 4223T, RRID: AB_1903909), Cleaved Caspase-3 (Cell Signaling Technology, cat. # 9661s, RRID: AB_2341188), Caspase-3 (Cell Signaling Technology, cat. # 9668s, RRID: AB_2069870), Cleaved Caspase-7 (Cell Signaling Technology, cat. # 8438s, RRID:AB_11178377), Caspase-7 (Cell Signaling Technology, cat. # 9494s, RRID: AB_2068141), PARP (Cell Signaling Technology, cat. # 9532s, RRID:AB_659884), MDM2 (Cell Signaling Technology, cat. # 86934s, RRID:AB_2784534), c-MYC Antibody (Cell Signaling Technology, cat. # 9402s, RRID: AB_2151827), β-actin (Cell Signaling Technology, cat. # 4970s, RRID: AB_2223172), β-actin (Cell Signaling Technology, cat. # 3700S, RRID: AB_2242334), α-tubulin (Cell Signaling Technology, cat. # 2125, RRID: AB_2619646), and GAPDH (Cell Signaling Technology, cat. # 97166, RRID: AB_2756824).

Techniques: Activity Assay, Analogues, Knock-Out, Over Expression, Expressing

Journal: Cancer Discovery

Article Title: Defining the Antitumor Mechanism of Action of a Clinical-stage Compound as a Selective Degrader of the Nuclear Pore Complex

doi: 10.1158/2159-8290.CD-25-0271

Figure Lengend Snippet: PRLX and related analogs bind TRIM21. A, TRIM21 primary structure showing RING, B-box, coiled-coil, and PRYSPRY domains. B, Predicted structure of the TRIM21 dimer with bound E2 and ubiquitin. A single protomer is shown in color. Mutated residues conferring resistance in the base editor screen are shown as spheres (yellow, carbon; blue, nitrogen; red, oxygen). C, SPR sensorgrams showing PRLX, JWZ-8-103, or JWZ-8-60 binding to immobilized TRIM21 PRYSPRY when tested at 32 μmol/L. D–F, Crystal structure of TRIM21 PRYSPRY bound to JWZ-8-103. Close-up views show key contacts. Fo – Fc compound omit map contoured at 3.5 σ is shown G, TRIM21 complementation experiments: cell proliferation of TRIM21 KO PANC-1 cells expressing the indicated TRIM21-mutant cDNAs treated with the indicated PRLX analogue vs. vehicle for 72 hours ( n = 3; error bars, SD).

Article Snippet: For immunoblots, the following primary antibodies were used: TRIM21 (Cell Signaling Technology, cat. # 92043, RRID:AB_2800177), TRIM21 (Santa Cruz Biotechnology, cat. # sc-25351, RRID:AB_628286), IRF1 (Cell Signaling Technology, cat. # 8478s, RRID:AB_10949108), IRF2 (Cell Signaling Technology, cat. # 59452S), IKKα (Cell Signaling Technology, cat. # 2682s, RRID: AB_331626), IKKγ (Cell Signaling Technology, cat. # 2685s, RRID: AB_2124829), NF-κB p65 (Cell Signaling Technology, cat. # 8242s, RRID: AB_10859369), IκBα (Cell Signaling Technology, cat. # 4812T, RRID: AB_10694416), Anti-Nuclear Matrix Protein p84 (Abcam, cat. # ab487, RRID:AB_304696), NUP214 (Abcam, cat. # ab70497, RRID:AB_1269607), NUP98 (Cell Signaling Technology, cat. # 2598T, RRID: AB_2267700), NUP96 (Thermo Fisher Scientific, cat. # A301784A, RRID:AB_1211487), NUP35 (ABclonal, cat. # A12762, RRID: AB_2759608), MCL1 (Cell Signaling Technology, cat. # 94296S, RRID: AB_2722740), GLE1 (Proteintech, cat. # 26466-1-AP, RRID: AB_2880525), BCLXL (Cell Signaling Technology, cat. # 2764T, RRID: AB_2228008), BCL2 (Cell Signaling Technology, cat. # 4223T, RRID: AB_1903909), Cleaved Caspase-3 (Cell Signaling Technology, cat. # 9661s, RRID: AB_2341188), Caspase-3 (Cell Signaling Technology, cat. # 9668s, RRID: AB_2069870), Cleaved Caspase-7 (Cell Signaling Technology, cat. # 8438s, RRID:AB_11178377), Caspase-7 (Cell Signaling Technology, cat. # 9494s, RRID: AB_2068141), PARP (Cell Signaling Technology, cat. # 9532s, RRID:AB_659884), MDM2 (Cell Signaling Technology, cat. # 86934s, RRID:AB_2784534), c-MYC Antibody (Cell Signaling Technology, cat. # 9402s, RRID: AB_2151827), β-actin (Cell Signaling Technology, cat. # 4970s, RRID: AB_2223172), β-actin (Cell Signaling Technology, cat. # 3700S, RRID: AB_2242334), α-tubulin (Cell Signaling Technology, cat. # 2125, RRID: AB_2619646), and GAPDH (Cell Signaling Technology, cat. # 97166, RRID: AB_2756824).

Techniques: Ubiquitin Proteomics, Binding Assay, Expressing, Mutagenesis

Journal: Cancer Discovery

Article Title: Defining the Antitumor Mechanism of Action of a Clinical-stage Compound as a Selective Degrader of the Nuclear Pore Complex

doi: 10.1158/2159-8290.CD-25-0271

Figure Lengend Snippet: PRLX induces TRIM21-mediated ubiquitylation and degradation of the NPC. A and B, Global proteome profiling of WT PANC-1 cells ( A ) or TRIM21 KO PANC-1 cells ( B ) treated with 500 nmol/L PRLX for 6 hours, showing differential statistics of LC-MS/MS quantified proteins ( n = 3–4 biological replicates). Nuclear pore proteins are colored blue. C, Immunoblotting of key proteins in WT or TRIM21 KO PANC-1 cells treated with 500 nmol/L PRLX (6 hours). D, Ubiquitylation proteomics of WT PANC-1 cells pretreated with 1 μmol/L MG132 for 1 hour before adding 500 nmol/L PRLX for 0.5 hours showing differential statistics of LC-MS/MS quantified ubiquitylation sites ( n = 3 biological replicates). Nuclear pore protein ubiquitylated peptides are colored purple. E, Proximity proteomics in TRIM21-TurboID–expressing PANC-1 cells following 1 μmol/L MG132 (0.5 hours) pretreatment before adding 500 nmol/L PRLX (0.5 hours) or DMSO, followed by 100 nmol/L biotin (0.5 hours), showing differential statistics of LC-MS/MS quantified proteins ( n = 3 biological replicates). Nuclear pore protein-labeled proteins are colored green. F, Immunoblotting validation of TurboID-based proximity enrichment following 500 nmol/L PRLX and 100 nmol/L biotin treatment of PANC-1 cells containing TRIM21-TurboID (C-terminal fusion), TurboID-TRIM21 (N-terminal fusion), or TRIM21 KO. G, Heatmap of nuclear pore proteins organized by annotated structural location, showing log 2 fold changes in global (blue), ubiquitylation (purple), or proximity (green) proteomics in PANC-1 cells treated with 500 nmol/L PRLX (*, Benjamini–Hochberg adjusted P value < 0.01). H, Schematic of the NPC with structural features labeled. All displayed adjusted P values ( A , B , D , E , and G ) were calculated using a LIMMA-based moderated t test with Benjamini–Hochberg adjustment. [ H, Created in BioRender. Dwyer, B. (2025) https://BioRender.com/wdnlhkr ]. TM, transmembrane.

Article Snippet: For immunoblots, the following primary antibodies were used: TRIM21 (Cell Signaling Technology, cat. # 92043, RRID:AB_2800177), TRIM21 (Santa Cruz Biotechnology, cat. # sc-25351, RRID:AB_628286), IRF1 (Cell Signaling Technology, cat. # 8478s, RRID:AB_10949108), IRF2 (Cell Signaling Technology, cat. # 59452S), IKKα (Cell Signaling Technology, cat. # 2682s, RRID: AB_331626), IKKγ (Cell Signaling Technology, cat. # 2685s, RRID: AB_2124829), NF-κB p65 (Cell Signaling Technology, cat. # 8242s, RRID: AB_10859369), IκBα (Cell Signaling Technology, cat. # 4812T, RRID: AB_10694416), Anti-Nuclear Matrix Protein p84 (Abcam, cat. # ab487, RRID:AB_304696), NUP214 (Abcam, cat. # ab70497, RRID:AB_1269607), NUP98 (Cell Signaling Technology, cat. # 2598T, RRID: AB_2267700), NUP96 (Thermo Fisher Scientific, cat. # A301784A, RRID:AB_1211487), NUP35 (ABclonal, cat. # A12762, RRID: AB_2759608), MCL1 (Cell Signaling Technology, cat. # 94296S, RRID: AB_2722740), GLE1 (Proteintech, cat. # 26466-1-AP, RRID: AB_2880525), BCLXL (Cell Signaling Technology, cat. # 2764T, RRID: AB_2228008), BCL2 (Cell Signaling Technology, cat. # 4223T, RRID: AB_1903909), Cleaved Caspase-3 (Cell Signaling Technology, cat. # 9661s, RRID: AB_2341188), Caspase-3 (Cell Signaling Technology, cat. # 9668s, RRID: AB_2069870), Cleaved Caspase-7 (Cell Signaling Technology, cat. # 8438s, RRID:AB_11178377), Caspase-7 (Cell Signaling Technology, cat. # 9494s, RRID: AB_2068141), PARP (Cell Signaling Technology, cat. # 9532s, RRID:AB_659884), MDM2 (Cell Signaling Technology, cat. # 86934s, RRID:AB_2784534), c-MYC Antibody (Cell Signaling Technology, cat. # 9402s, RRID: AB_2151827), β-actin (Cell Signaling Technology, cat. # 4970s, RRID: AB_2223172), β-actin (Cell Signaling Technology, cat. # 3700S, RRID: AB_2242334), α-tubulin (Cell Signaling Technology, cat. # 2125, RRID: AB_2619646), and GAPDH (Cell Signaling Technology, cat. # 97166, RRID: AB_2756824).

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Expressing, Labeling, Biomarker Discovery

Journal: Cancer Discovery

Article Title: Defining the Antitumor Mechanism of Action of a Clinical-stage Compound as a Selective Degrader of the Nuclear Pore Complex

doi: 10.1158/2159-8290.CD-25-0271

Figure Lengend Snippet: NUP98 is the nuclear pore receptor for TRIM21. A, Schematic of NUP98–NUP96 proteins (NCBI gene 4928). The site disrupted by NUP98 sgRNA3 in the CRISPR-Cas9 KO screen is marked. B, Enrichment of NUP98 sgRNA3 reads in the CRISPR screen. C, Cell viability of WT or sgRNA3 PANC-1 cells treated with the indicated compounds ( n = 4; error bars, SD). D, Immunoblots of indicated proteins in PANC-1 cells (WT, TRIM21 KO, or sgRNA3) treated with PRLX or JWZ-8-103 for 6 hours. E, Schematic for the TRIM21-NUP98 NanoBiT split luciferase assay (top). NanoBiT assay showing dose-dependent induced proximity between TRIM21 and NUP98 (bottom, isoform 2/3; n = 3, error bars, SD). F, NUP98 APD mutations (top) and induced proximity measurements as in ( E ) between TRIM21 and NUP98 ( n = 3, area fill, SD). G, Coimmunoprecipitation (IP) assay in TRIM21 KO PANC-1 cells expressing 3× Flag-tagged TRIM21 treated with JWZ-8-103. H, SPR sensorgrams showing immobilized NUP98 APD exposed to the indicated analytes: JWZ-8-103 alone (left), TRIM21 PRYSPRY (center left), TRIM21 PRYSPRY with increasing JWZ-8-103 (center right), or JWZ-8-103 with increasing TRIM21 PRYSPRY (right). I, TR-FRET assay measuring ternary complex formation between the NUP98 APD and full-length TRIM21.

Article Snippet: For immunoblots, the following primary antibodies were used: TRIM21 (Cell Signaling Technology, cat. # 92043, RRID:AB_2800177), TRIM21 (Santa Cruz Biotechnology, cat. # sc-25351, RRID:AB_628286), IRF1 (Cell Signaling Technology, cat. # 8478s, RRID:AB_10949108), IRF2 (Cell Signaling Technology, cat. # 59452S), IKKα (Cell Signaling Technology, cat. # 2682s, RRID: AB_331626), IKKγ (Cell Signaling Technology, cat. # 2685s, RRID: AB_2124829), NF-κB p65 (Cell Signaling Technology, cat. # 8242s, RRID: AB_10859369), IκBα (Cell Signaling Technology, cat. # 4812T, RRID: AB_10694416), Anti-Nuclear Matrix Protein p84 (Abcam, cat. # ab487, RRID:AB_304696), NUP214 (Abcam, cat. # ab70497, RRID:AB_1269607), NUP98 (Cell Signaling Technology, cat. # 2598T, RRID: AB_2267700), NUP96 (Thermo Fisher Scientific, cat. # A301784A, RRID:AB_1211487), NUP35 (ABclonal, cat. # A12762, RRID: AB_2759608), MCL1 (Cell Signaling Technology, cat. # 94296S, RRID: AB_2722740), GLE1 (Proteintech, cat. # 26466-1-AP, RRID: AB_2880525), BCLXL (Cell Signaling Technology, cat. # 2764T, RRID: AB_2228008), BCL2 (Cell Signaling Technology, cat. # 4223T, RRID: AB_1903909), Cleaved Caspase-3 (Cell Signaling Technology, cat. # 9661s, RRID: AB_2341188), Caspase-3 (Cell Signaling Technology, cat. # 9668s, RRID: AB_2069870), Cleaved Caspase-7 (Cell Signaling Technology, cat. # 8438s, RRID:AB_11178377), Caspase-7 (Cell Signaling Technology, cat. # 9494s, RRID: AB_2068141), PARP (Cell Signaling Technology, cat. # 9532s, RRID:AB_659884), MDM2 (Cell Signaling Technology, cat. # 86934s, RRID:AB_2784534), c-MYC Antibody (Cell Signaling Technology, cat. # 9402s, RRID: AB_2151827), β-actin (Cell Signaling Technology, cat. # 4970s, RRID: AB_2223172), β-actin (Cell Signaling Technology, cat. # 3700S, RRID: AB_2242334), α-tubulin (Cell Signaling Technology, cat. # 2125, RRID: AB_2619646), and GAPDH (Cell Signaling Technology, cat. # 97166, RRID: AB_2756824).

Techniques: CRISPR, Western Blot, Luciferase, Expressing

Journal: Cancer Discovery

Article Title: Defining the Antitumor Mechanism of Action of a Clinical-stage Compound as a Selective Degrader of the Nuclear Pore Complex

doi: 10.1158/2159-8290.CD-25-0271

Figure Lengend Snippet: NPC destruction impairs mRNA export and induces cancer cell death. A, Caspase-3/7 activation in WT and TRIM21 KO PANC-1 cells treated with PRLX for 6, 12, or 24 hours ( n = 3; error bars, SD). B, Immunoblots of indicated proteins in WT or TRIM21 KO PANC-1 cells treated with PRLX for 6 hours. C, qRT-PCR analysis of cytoplasmic and nuclear MCL-1 and NUP98 mRNA. PANC-1 cells were treated with PRLX for 4 or 6 hours and fractionated as described in the methods section ( n = 3–4). Target gene transcript abundance in each fraction was normalized to GAPDH using the 2 (−ΔΔCt) method and analyzed by one-way ANOVA, comparing with the DMSO control (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; error bars, SD). D–E, Immunoblots of indicated proteins in KP3 cells (WT, TRIM21 KO) or H661 (WT, TRIM21 OE) treated with PRLX, JWZ-8-103, or JWZ-8-60 for 6 hours. F, smFISH for MCL1 (red puncta) and c-MYC (yellow puncta) transcripts in PANC-1 cells (box, enlarged at the bottom) treated with DMSO, PRLX, JWZ-8-103, or JWZ-8-60 for 6 hours to assess mRNA expression and localization. Nuclei are outlined with dashed circles. Arrowheads denote PANC-1 cells in which MCL1 and c-MYC transcripts were primarily localized in the nucleus. Scale bars, 50 μm. OE, overexpression.

Article Snippet: For immunoblots, the following primary antibodies were used: TRIM21 (Cell Signaling Technology, cat. # 92043, RRID:AB_2800177), TRIM21 (Santa Cruz Biotechnology, cat. # sc-25351, RRID:AB_628286), IRF1 (Cell Signaling Technology, cat. # 8478s, RRID:AB_10949108), IRF2 (Cell Signaling Technology, cat. # 59452S), IKKα (Cell Signaling Technology, cat. # 2682s, RRID: AB_331626), IKKγ (Cell Signaling Technology, cat. # 2685s, RRID: AB_2124829), NF-κB p65 (Cell Signaling Technology, cat. # 8242s, RRID: AB_10859369), IκBα (Cell Signaling Technology, cat. # 4812T, RRID: AB_10694416), Anti-Nuclear Matrix Protein p84 (Abcam, cat. # ab487, RRID:AB_304696), NUP214 (Abcam, cat. # ab70497, RRID:AB_1269607), NUP98 (Cell Signaling Technology, cat. # 2598T, RRID: AB_2267700), NUP96 (Thermo Fisher Scientific, cat. # A301784A, RRID:AB_1211487), NUP35 (ABclonal, cat. # A12762, RRID: AB_2759608), MCL1 (Cell Signaling Technology, cat. # 94296S, RRID: AB_2722740), GLE1 (Proteintech, cat. # 26466-1-AP, RRID: AB_2880525), BCLXL (Cell Signaling Technology, cat. # 2764T, RRID: AB_2228008), BCL2 (Cell Signaling Technology, cat. # 4223T, RRID: AB_1903909), Cleaved Caspase-3 (Cell Signaling Technology, cat. # 9661s, RRID: AB_2341188), Caspase-3 (Cell Signaling Technology, cat. # 9668s, RRID: AB_2069870), Cleaved Caspase-7 (Cell Signaling Technology, cat. # 8438s, RRID:AB_11178377), Caspase-7 (Cell Signaling Technology, cat. # 9494s, RRID: AB_2068141), PARP (Cell Signaling Technology, cat. # 9532s, RRID:AB_659884), MDM2 (Cell Signaling Technology, cat. # 86934s, RRID:AB_2784534), c-MYC Antibody (Cell Signaling Technology, cat. # 9402s, RRID: AB_2151827), β-actin (Cell Signaling Technology, cat. # 4970s, RRID: AB_2223172), β-actin (Cell Signaling Technology, cat. # 3700S, RRID: AB_2242334), α-tubulin (Cell Signaling Technology, cat. # 2125, RRID: AB_2619646), and GAPDH (Cell Signaling Technology, cat. # 97166, RRID: AB_2756824).

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Control, Expressing, Over Expression

Journal: Cancer Discovery

Article Title: Defining the Antitumor Mechanism of Action of a Clinical-stage Compound as a Selective Degrader of the Nuclear Pore Complex

doi: 10.1158/2159-8290.CD-25-0271

Figure Lengend Snippet: Efficacy of TRIM21 molecular glues against PDO and in vivo tumor models. A, TRIM21 expression distribution across HCMI pancreaticobiliary organoids by mRNAseq ( n = 65). Models selected for testing are shown in orange. B, Immunoblot to compare TRIM21 protein expression levels between organoids selected for compound testing. C, Cell proliferation of selected HCMI organoids treated with the indicated compounds ( n = 3–6; error bars, SEM). D, Immunoblots for nuclear pore proteins and MCL1 in HCMI organoid models. E, Plasma concentrations of PRLX and JWZ-8-103 following a single 3 mg/kg IP injection ( n = 3; 8 hours of experiment shown; error bars, SD). F, Immunoblot analysis of indicated PANC-1 xenograft tumor tissue collected 2 hours after the second daily dose of 50 mg/kg PRLX, 15 mg/kg JWZ-8-103, 50 mg/kg JWZ-8-103, or vehicle. G and H, Tumor growth in NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ mice injected intraperitoneally with compound (50 mg/kg PRLX, 15 mg/kg JWZ-8-103, 50 mg/kg JWZ-8-103, or vehicle) daily for 3 weeks. Compound treatments started on day 21 ( G ) or day 19 ( H ) after tumor inoculation ( n = 9 or 10). Tumor volumes at the end of the experiment were analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test, comparing with the vehicle (*, P < 0.05; ****, P < 0.0001; error bars, SEM).

Article Snippet: For immunoblots, the following primary antibodies were used: TRIM21 (Cell Signaling Technology, cat. # 92043, RRID:AB_2800177), TRIM21 (Santa Cruz Biotechnology, cat. # sc-25351, RRID:AB_628286), IRF1 (Cell Signaling Technology, cat. # 8478s, RRID:AB_10949108), IRF2 (Cell Signaling Technology, cat. # 59452S), IKKα (Cell Signaling Technology, cat. # 2682s, RRID: AB_331626), IKKγ (Cell Signaling Technology, cat. # 2685s, RRID: AB_2124829), NF-κB p65 (Cell Signaling Technology, cat. # 8242s, RRID: AB_10859369), IκBα (Cell Signaling Technology, cat. # 4812T, RRID: AB_10694416), Anti-Nuclear Matrix Protein p84 (Abcam, cat. # ab487, RRID:AB_304696), NUP214 (Abcam, cat. # ab70497, RRID:AB_1269607), NUP98 (Cell Signaling Technology, cat. # 2598T, RRID: AB_2267700), NUP96 (Thermo Fisher Scientific, cat. # A301784A, RRID:AB_1211487), NUP35 (ABclonal, cat. # A12762, RRID: AB_2759608), MCL1 (Cell Signaling Technology, cat. # 94296S, RRID: AB_2722740), GLE1 (Proteintech, cat. # 26466-1-AP, RRID: AB_2880525), BCLXL (Cell Signaling Technology, cat. # 2764T, RRID: AB_2228008), BCL2 (Cell Signaling Technology, cat. # 4223T, RRID: AB_1903909), Cleaved Caspase-3 (Cell Signaling Technology, cat. # 9661s, RRID: AB_2341188), Caspase-3 (Cell Signaling Technology, cat. # 9668s, RRID: AB_2069870), Cleaved Caspase-7 (Cell Signaling Technology, cat. # 8438s, RRID:AB_11178377), Caspase-7 (Cell Signaling Technology, cat. # 9494s, RRID: AB_2068141), PARP (Cell Signaling Technology, cat. # 9532s, RRID:AB_659884), MDM2 (Cell Signaling Technology, cat. # 86934s, RRID:AB_2784534), c-MYC Antibody (Cell Signaling Technology, cat. # 9402s, RRID: AB_2151827), β-actin (Cell Signaling Technology, cat. # 4970s, RRID: AB_2223172), β-actin (Cell Signaling Technology, cat. # 3700S, RRID: AB_2242334), α-tubulin (Cell Signaling Technology, cat. # 2125, RRID: AB_2619646), and GAPDH (Cell Signaling Technology, cat. # 97166, RRID: AB_2756824).

Techniques: In Vivo, Expressing, Western Blot, Clinical Proteomics, Injection