Journal: mSphere

Article Title: A Role for the VPS Retromer in Brucella Intracellular Replication Revealed by Genomewide siRNA Screening

doi: 10.1128/mSphere.00380-19

Figure Lengend Snippet: Overview of the high-content screening and analysis. (A) Summary of RNAi screening workflow. Reverse transfection of HeLa cells was performed in 384-well format for 72 h, followed by 48-h infection with GFP-expressing B. abortus , PFA fixation, and staining of HeLa cells with DAPI and DY-547−phalloidin before automated imaging. GW, genomewide. (B) Image analysis was performed with CellProfiler to segment nuclei and bacteria and to extract measurements. (C) Accurate association of segmented bacteria to nuclei enables quantitative single-cell measurements. The naive association (middle image) of segmented pathogen can be affected by oversplitting in dense cell populations (left image). Our proposed solution (right image) based on a nucleus attraction score. (D) The plate histogram shows the bimodal distribution of integrated GFP intensity corresponding to Brucella replication. Intensity on the x axes is log 2 scaled to account for exponential growth. The normal distribution fitted (red curve) to the kernel density estimation of the histogram allows us to compute a robust binary infection threshold (dashed line) separating HeLa cells with (right) and without (left) replicating Brucella . Associated are samples of single-cell images corresponding to the intervals of the intensity distribution (for more details, see Materials and Methods).

Article Snippet: To identify novel host factors important for Brucella intracellular infection, we performed a genomewide small interfering RNA (siRNA) perturbation screen on the human epithelial cell line HeLa (ATCC CCL-2) combined with bacterial infection at biosafety level 3.

Techniques: High Content Screening, Transfection, Infection, Expressing, Staining, Imaging, Bacteria, Quantitative Single Cell

Journal: mSphere

Article Title: A Role for the VPS Retromer in Brucella Intracellular Replication Revealed by Genomewide siRNA Screening

doi: 10.1128/mSphere.00380-19

Figure Lengend Snippet: Entry assay identifies new components required for postentry processes during Brucella infection. (A) Representative images from the entry assay showing nuclei (DAPI) of HeLa cells and intracellular Brucella abortus (GFP) for control condition (mock) and cells treated with siRNAs against CDC42 or VPS35. HeLa cells were infected with B. abortus expressing GFP under a tetracycline-inducible system for 8 h (see Materials and Methods). Bars = 100 μm. (B) Scatter plot in double logarithmic scale showing infection scores measured for the entry assay (8 hpi) versus endpoint assay (48 hpi), normalized to the respective mock data set ( Table S3 ). For the entry assay, cells containing single bacteria were considered infected, and the final readout is proportional to the median number of bacteria per infected cells. For the endpoint assay, only cells containing replicating bacteria were considered infected ( Fig. 1 and Materials and Methods). Each data point corresponds to the average of all siRNAs or esiRNAs targeted against the gene of interest ( n = 3). The straight fit (oblique line, r 2 = 0.763) indicates a globally high correlation between both assays. The blue region shows all points within 1 standard deviation (SD) of the fitted data. The genes falling out of this range are marked in red. For ease of visualization, only the averaged values over all RNAi products targeting a given gene are displayed.

Article Snippet: To identify novel host factors important for Brucella intracellular infection, we performed a genomewide small interfering RNA (siRNA) perturbation screen on the human epithelial cell line HeLa (ATCC CCL-2) combined with bacterial infection at biosafety level 3.

Techniques: Infection, Control, Expressing, End Point Assay, Bacteria, Standard Deviation

Journal: mSphere

Article Title: A Role for the VPS Retromer in Brucella Intracellular Replication Revealed by Genomewide siRNA Screening

doi: 10.1128/mSphere.00380-19

Figure Lengend Snippet: The VPS retromer is a key component of Brucella intracellular trafficking. (A) Schematic representation of the retromer components and their prominent interactors. (B) Schematic representation of the shRNA constructs used in panel C. The gray box on the shVPS35/rescue construct indicates the silent mutations that prevent base-pairing with the coexpressed shRNA . (C) Infection index from transfected cells. Displayed are the averaged infection index and associated standard deviation after 48 h of Brucella infection. Data were normalized to the YFP-only condition ( n = 3). Values that are statistically significantly different from the value for the scrambled YPF-only condition as determined by paired t test are indicated by an asterisk (*, P value of ≤0.01; ns, not significant). (D) Dot box representation of the z-scored infection score for components of the retromer and interactors, including the positive-control ARPC3. Asterisks indicate statistically significant differences from the values for the scrambled siRNA-treated bacteria (control [Ctrl]) as determined by one-way analysis of variance (ANOVA) and Dunnett’s multiple-comparison test (**, P value of ≤0.001; ns, not significant). (E) Normalized CFU recovered from siRNA-treated cells at 6, 20, or 44 hpi. The presented data correspond to CFU count normalized to control, siRNA-treated cells ( n = 3). Significance was determined using one-way ANOVA with Dunnett’s multiple-comparison test (*, P ≤ 0.01; **, P ≤ 0.001; ns, not significant). (F) Western blot analysis of the indicated proteins in total lysate of HeLa cells treated with siRNA targeting the designated genes, 72 h posttransfection. Displayed is a representative example of an experiment performed in biological triplicate ( n = 3). See Table S4 for the matching averaged intensity quantification.

Article Snippet: To identify novel host factors important for Brucella intracellular infection, we performed a genomewide small interfering RNA (siRNA) perturbation screen on the human epithelial cell line HeLa (ATCC CCL-2) combined with bacterial infection at biosafety level 3.

Techniques: shRNA, Construct, Infection, Transfection, Standard Deviation, Positive Control, Bacteria, Control, Comparison, Western Blot

Journal: mSphere

Article Title: A Role for the VPS Retromer in Brucella Intracellular Replication Revealed by Genomewide siRNA Screening

doi: 10.1128/mSphere.00380-19

Figure Lengend Snippet: VPS35 is required for Brucella to escape the lysosomal pathway. (A) Immunofluorescence approach used to quantify localization of Brucella within LAMP-1-positive vesicles, illustrated with a representative example of control-treated cells 6 hpi. Individual channels and merged picture are presented. Arrows indicate examples of colocalization of bacteria with LAMP-1-positive compartments. Staining of Brucella LPS (anti-LPS [αLPS]) was used to confirm the presence of LAMP-1 in direct proximity of the bacterial surface. Asterisks indicate examples of LAMP-1-negative Brucella . (B) Representative images from Brucella infected cells either mock transfected (ctrl) or after VPS35 knockdown (siVPS35). Samples were fixed 6 and 18 hpi. For clarity, only the LAMP-1 (cyan) and dsRed (magenta) channels are presented. Bars, 5 μm. (C) Global quantification of LAMP-1-negative Brucella. Displayed are the average and associated standard deviation for more than 500 bacteria and more than 50 HeLa cells per time point and condition ( n = 3). (D) Single-cell data representation of the data presented in panel C. Displayed is the distribution of LAMP-1-positive Brucella per cell as a function of the total number of bacteria counted in that given cell.

Article Snippet: To identify novel host factors important for Brucella intracellular infection, we performed a genomewide small interfering RNA (siRNA) perturbation screen on the human epithelial cell line HeLa (ATCC CCL-2) combined with bacterial infection at biosafety level 3.

Techniques: Immunofluorescence, Control, Bacteria, Staining, Infection, Transfection, Knockdown, Standard Deviation

Journal: Nature Communications

Article Title: GAK and PRKCD are positive regulators of PRKN-independent mitophagy

doi: 10.1038/s41467-021-26331-7

Figure Lengend Snippet: a U2OS cells stably expressing an internal MLS-EGFP-mCherry (IMLS) reporter that is pH-responsive (yellow at neutral, red at acidic pH) were incubated for 24 h ± 1 mM DFP followed by PFA fixation, antibody staining for TIM23 (Alexa Fluor-647) and widefield microscopy. Scale bar = 10 µm, inset = 0.5 µm. b U2OS IMLS cells were transfected with 7.5 nM siRNA non-targeting control (siNT) or siULK1 for 48 h prior to 24 h treatment ± 1 mM DFP in the presence or absence of 50 nM BafA1 for the final 2 h. Western blot from cell lysates shows representative ULK1 knockdown level. The graph represents the mean red-only area per cell from fluorescence images normalised to control DMSO siNT cells ± SEM from n = 4 independent experiments. Significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test. c U2OS IMLS cells treated with 1 mM DFP as in ( a ) and fixed for CLEM analysis. Inset of the cell area in the white box is shown by confocal analysis and EM section along with EM overlay. Scale bar = 5 µm, inset = 1 µm. d Citrate synthase activity from U2OS cells treated for 24 h ± 1 mM DFP with final 16 h in the presence of 50 nM BafA1 or DMSO, values are normalised to DMSO control from n = 6 (DMSO) or n = 3 (+BafA1) independent experiments ± SEM. Significance was determined by two-way ANOVA followed by Sidak’s multiple comparisons test. e U2OS whole-cell protein abundance was determined by mass spectrometry following treatment ±1 mM DFP 24 h. Mitochondrial proteins identified by GO analysis (term = mitochondrion) are highlighted in blue. f Mean T test difference between control and DFP samples for peptides identified in e matching GO terms related to cellular organelles. Bars represent Log2 fold change (control vs DFP) ± SEM from n = 4 independent experiments, number on bars indicate how many protein targets are included in GO analysis. ** P < 0.01, *** P < 0.001, **** P < 0.0001 and n.s. = not significant in all relevant panels. For precise P values, see the source data file.

Article Snippet: The tertiary siRNA screen was carried out utilising an ImageXpress Micro Confocal (Molecular Devices) using a ×20 objective (NA 0.45).

Techniques: Stable Transfection, Expressing, Incubation, Staining, Microscopy, Transfection, Western Blot, Fluorescence, Activity Assay, Mass Spectrometry

Journal: Nature Communications

Article Title: GAK and PRKCD are positive regulators of PRKN-independent mitophagy

doi: 10.1038/s41467-021-26331-7

Figure Lengend Snippet: a – e Primary siRNA screen data. U2OS cells were transfected with a pool of three sequence variable siRNA oligonucleotides per gene target (2.5 nM per oligo) for 48 h before the addition of 1 mM DFP for 24 h. Cells were PFA fixed and imaged using a ×20 objective (35 fields of view per well). The red area per cell was normalised to the average of siNT controls and adjusted so that the DFP siNT control was 0 from n = 14 plates ( a , b ) or n = 6 plates ( c – e ) ±SEM. siULK1 and BafA1 (red bars) are positive controls. siRNA targets containing similar lipid-binding domains were assayed and plotted together as shown for a PX domains, b FYVE domains, c C1 domains, d C2 domains, e GRAM, ENTH, PROPPIN domains. f Summary of the significance of different lipid-binding domains relative to the total tested from ( a – e ) proteins containing more than one type of domain are represented in each category. Significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test to the siNT control where * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 and n.s. = not significant in all relevant panels. For precise P values, see the source data file.

Article Snippet: The tertiary siRNA screen was carried out utilising an ImageXpress Micro Confocal (Molecular Devices) using a ×20 objective (NA 0.45).

Techniques: Transfection, Sequencing, Binding Assay

Journal: Nature Communications

Article Title: GAK and PRKCD are positive regulators of PRKN-independent mitophagy

doi: 10.1038/s41467-021-26331-7

Figure Lengend Snippet: a Summary of significant targets identified across primary, secondary (7.5 nM individual siRNA oligos) and tertiary screens (15 nM each oligo) siRNA screens. Cells were transfected for 48 h prior to 24 h of 1 mM DFP treatment. Bars represent mean fold change in mitophagy relative to the siNT controls ± SEM from n = 12 (siNT, primary + secondary), n = 13 (SNX10, primary), n = 14 (SYTL5 + ULK1, primary), n = 17 (siNT, secondary) or n = 6 (all others) independent plates. Significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test to the siNT control. b Protein–protein interaction networks for candidate proteins (see “Methods”) were plotted by % of interacting proteins belonging to each highlighted compartment, value in brackets represents total number of interacting proteins. Dashed lines indicate average values from all screened proteins for mitochondria (red) or autophagosome (orange). c siRNA treatment with indicated oligos for 48 h prior to 24 h treatment ± 1 mM DFP and subsequent analysis of citrate synthase activity levels. Values were normalised to the siNT control and plotted ± SEM for n = 4 independent experiments. Significance was determined by two-way ANOVA followed by Sidak’s multiple comparison test. d U2OS cells treated ± 1 mM DFP for 24 h were enriched from a post-nuclear supernatant (PNS) for mitochondria followed by western blotting for the indicated proteins. e U2OS IMLS cells treated ± 1 mM DFP for 24 h followed by PFA fixation and staining for endogenous PRKCD (Alexa Fluor-647). Scale bar = 20 µm. f U2OS cells treated ± 1 mM DFP for 24 h ± 50 nM BafA1 for the final 16 h and blotted for the indicated proteins. g Quantitation of PRKCD and TIM23 levels to β-actin in ( f ) from n = 3 independent experiments ± SEM. Significance was determined by two-way ANOVA followed by Dunnett’s multiple comparison test to the control. h U2OS cells (−) or those stably expressing PRKCD wild type (WT) or PRKCD ∆C1 ∆C2 (∆lipid-binding domains—LBD) were enriched from a post-nuclear supernatant (PNS) for cytosol and mitochondrial fractions followed by western blotting for the indicated proteins. Representative from n = 2 experiments * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 and n.s. = not significant in all relevant panels. For precise P values, see the source data file.

Article Snippet: The tertiary siRNA screen was carried out utilising an ImageXpress Micro Confocal (Molecular Devices) using a ×20 objective (NA 0.45).

Techniques: Transfection, Activity Assay, Western Blot, Staining, Quantitation Assay, Stable Transfection, Expressing, Binding Assay

Journal: Cell Reports Medicine

Article Title: Deletion of pancreas-specific miR-216a reduces beta-cell mass and inhibits pancreatic cancer progression in mice

doi: 10.1016/j.xcrm.2021.100434

Figure Lengend Snippet: miR-216a regulates cell migration and apoptosis (A) Percentage of calcein-stained PANC-1 cells migrating through a transwell following transfection with miR-216a or control (Ctrl) mimetics or untransfected (UTR). n = 3 technical replicates. (B) Percentage of islets isolated from 10-week-old male WT or KO mice that appeared as having defined, transitionary (transit.), or spread boundaries 3 days after plating on collagen wells. n = 3 mice. (C) Distance to duct was quantified using the H&E stains of WT and miR-216a KO adult pancreata. (D and E) Human EndoC-βH1 cells were treated with varying concentrations of TGF-β or TGF-β inhibitor SB431542, and miR-216a levels were quantified using qRT-PCR. n = 6 technical replicates. Ct levels for miR-216a in untreated human EndoC-βH1 cells = ∼21. (F) Islets from 10-week-old male WT and KO mice were isolated and expression of Smad7 , Pten , Becn1 , and Ybx1 was quantified with qRT-PCR. n = 3 or 4 mice. (G) MIN6 cells were transfected with miR-216a and control mimetics and expression of 84 ECM-related genes was quantified using qRT-PCR. Genes displaying >2-fold difference are shown. Each value is the mean of three independent transfections. (H and I) INS1-E cells were transfected with the indicated miRNA inhibitors (inh.) or with a scrambled control miRNA inhibitor (Ctrl inh.), and cell viability was assessed using XTT (2,3-bis-[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) assay (H) or live cell imaging using Hoechst and Alexa 647 annexin V (I). TNF-α, IFN-γ, and IL-1β were added to media prior to imaging cells at 37°C and 5% CO 2 in an ImageXpress Micro. n = 4 technical replicates. (J) Mouse islets were transfected with a control miRNA (miR-negi) and miR-216a inhibitor. Expression of Bad and Bcl-xl was quantified using qRT-PCR. n = 5 mice. A two-tailed Student’s t test (A–F, H, and J) or two-way ANOVA with Bonferroni’s multiple-comparison post-test (I) was performed to assess significance. ∗p < 0.05 and ∗∗p < 0.01. Individual data points are shown in (B)–(F), (H), and (J). Data represent mean ± SEM.

Article Snippet: Images were captured using a ImageXpress MicroTM Imaging System and analyzed using MetaXpress 5.3.0.5 Software (Molecular Devices Corporation, Sunnyvale, CA, USA). β-cell mass was analyzed in three pancreas sections per mouse, at least 200 μm apart, on insulin-labeled sections.

Techniques: Migration, Staining, Transfection, Control, Isolation, Quantitative RT-PCR, Expressing, Live Cell Imaging, Imaging, Two Tailed Test, Comparison

Journal: Cell Reports Medicine

Article Title: Deletion of pancreas-specific miR-216a reduces beta-cell mass and inhibits pancreatic cancer progression in mice

doi: 10.1016/j.xcrm.2021.100434

Figure Lengend Snippet: RNA-seq analysis of pancreata from miR-216a-KO mice (A) RNA from the pancreata of 1-day old male WT and miR-216a KO mice was isolated and subjected to RNA sequencing and top 50 most abundant genes are shown by heatmap. (B) A treemap plot, combining statistically significant GO terms in the biological processes category into similar terms. The size of the square increases with a decreasing p value, and the color of the square indicates the grouping of like terms (labeled in larger text with a gray background). Statistically significant terms were identified using hypergeometric tests with a false discovery rate of 0.1. (C) All statistically significant KEGG terms are shown in a network map, with nodes representing KEGG terms and edges connecting nodes representing differentially expressed genes in common between KEGG terms. Statistically significant terms were identified using hypergeometric tests with a false discovery rate of 0.1. (D) Normalized gene expression data for key genes of interest. All genes shown are differentially expressed, with adjusted p values < 0.05 (adjusted by the Benjamini-Hochberg correction). (E and F) PANC-1 cells were transfected with the indicated miRNAs, and 48 h later TGF-β was added to cell culture media and cell number was counted (E). Individual data points are shown. n = 3 technical replicates. (F) Cell death was assessed with live cell imaging using Hoechst and Alexa 647 annexin V. Cells were imaged at 37°C and 5% CO 2 in an ImageXpress Micro. n = 5 technical replicates. A two-tailed Student’s t test (E) or two-way ANOVA with Bonferroni’s multiple-comparison post-test (F) was performed to assess significance. ∗p < 0.05. Data represent mean ± SEM.

Article Snippet: Images were captured using a ImageXpress MicroTM Imaging System and analyzed using MetaXpress 5.3.0.5 Software (Molecular Devices Corporation, Sunnyvale, CA, USA). β-cell mass was analyzed in three pancreas sections per mouse, at least 200 μm apart, on insulin-labeled sections.

Techniques: RNA Sequencing, Isolation, Labeling, Gene Expression, Transfection, Cell Culture, Live Cell Imaging, Two Tailed Test, Comparison

Journal: The EMBO Journal

Article Title: Maintenance of p-eIF2α levels by the eIF2B complex is vital for colorectal cancer

doi: 10.1038/s44318-025-00381-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: IRDye® 680RD Goat-Anti-Rabbit (IgG) , Li-Cor , 926-68071.

Techniques: Recombinant, Sequencing, Protease Inhibitor, Viability Assay, Western Blot, SYBR Green Assay, Membrane, RNA Sequencing, Software, Imaging, Control, Real-time Polymerase Chain Reaction, High Content Screening

Journal: Cell Chemical Biology

Article Title: Target identification for small-molecule discovery in the FOXO3a tumor-suppressor pathway using a biodiverse peptide library

doi: 10.1016/j.chembiol.2021.05.009

Figure Lengend Snippet: High-content screening of a peptide library to identify FOXO3a nuclear translocation in a U2OS reporter cell line (A) Representative images of U2OS-GFP-FOXO3a cells showing GFP-tagged FOXO3a localization after treatment with small-molecule inhibitors or (B) transfection with siRNAs targeting the AKT and nuclear export pathways. PI3Ki, 1 μM PI-103; AKTi, 1 μM GSK690693; XPO1i, 2 nM leptomycin. The nuclear area is marked by an orange dotted line. Scale bars, 50 μm. See Figure S1 for quantitation. (C) Schematic of screening workflow. (D) Scatterplot of primary screen results showing a ranked distribution of peptides according to the robust Z score. Red dots indicate a Z score greater than 3. (E) Bar graph showing validation of 59 selected primary hits, ranked by fold change in percentage of cells with nuclear FOXO3a relative to vector. Those over 2-fold were selected for further work.

Article Snippet: Rabbit monoclonal anti FOXO3a , Cell Signaling Technology , Cat#12829; RRID: AB_2636990.

Techniques: High Content Screening, Translocation Assay, Transfection, Quantitation Assay, Biomarker Discovery, Plasmid Preparation

Journal: Cell Chemical Biology

Article Title: Target identification for small-molecule discovery in the FOXO3a tumor-suppressor pathway using a biodiverse peptide library

doi: 10.1016/j.chembiol.2021.05.009

Figure Lengend Snippet: Peptide 9J10 interacts with 14-3-3 (A) Silver-stained gels of eluted proteins following immunoprecipitation with V5 antibody from U2OS-GFP-FOXO3a cells transfected with the indicated peptide for 48 h. Red arrowheads indicate differential bands (relative to vector), which were excised for protein identification by mass spectrometry. (B) Graph showing proteins identified by mass spectrometry for the band from 9J10, ranked by emPAI score. Members of the 14-3-3 family are highlighted in red. Proteins with an emPAI >1 are displayed; see for complete list. (C) Western blotting for 14-3-3 following immunoprecipitation (IP) with V5 antibody from U2OS-GFP-FOXO3a cells transfected with 9J10 or vector for 48 h. A representative blot from three independent replicates is shown. (D) Graphical representation of yeast two-hybrid experiment using 9J10 as bait, showing prey fragments for interacting proteins. Members of the 14-3-3 family are highlighted in red.

Article Snippet: Rabbit monoclonal anti FOXO3a , Cell Signaling Technology , Cat#12829; RRID: AB_2636990.

Techniques: Staining, Immunoprecipitation, Transfection, Plasmid Preparation, Mass Spectrometry, Western Blot

Journal: Cell Chemical Biology

Article Title: Target identification for small-molecule discovery in the FOXO3a tumor-suppressor pathway using a biodiverse peptide library

doi: 10.1016/j.chembiol.2021.05.009

Figure Lengend Snippet: The interaction between 9J10 and 14-3-3 is phospho-dependent (A) Western blotting for 14-3-3 following immunoprecipitation with V5 antibody from HEK293T cells transfected with 9J10 or vector for 24 h. Lysates were incubated in the presence or absence of lambda phosphatase. (B) Western blotting for 14-3-3 following immunoprecipitation (I.P.) with V5 antibody from HEK293T cells transfected for 24 h with vector, wild-type 9J10 (9J10 WT ), or 9J10 Ser94>Ala mutant (9J10 AMut ). The asterisk marks the antibody light chain. All western blots are representative of at least two independent experiments. (C) Representative images of HEK293T cells co-transfected with GFP-FOXO3a and vector, 9J10 WT or 9J10 AMut , fixed at 24 h post transfection. GFP-FOXO3a localization is shown. Inset shows an enlarged region with the nucleus marked by an orange dotted line. Scale bar, 25 μm. (D) Quantitation of FOXO3a localization from cells treated as in (C). Data represent the mean of three independent experiments ± SD.

Article Snippet: Rabbit monoclonal anti FOXO3a , Cell Signaling Technology , Cat#12829; RRID: AB_2636990.

Techniques: Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation, Incubation, Mutagenesis, Quantitation Assay

Journal: Cell Chemical Biology

Article Title: Target identification for small-molecule discovery in the FOXO3a tumor-suppressor pathway using a biodiverse peptide library

doi: 10.1016/j.chembiol.2021.05.009

Figure Lengend Snippet: Expression of 9J10 reduces the interaction between 14-3-3 and FOXO3a, modulates transcription, and impairs cell growth in HEK293T cells (A) Western blotting for FOXO3a and 14-3-3γ following immunoprecipitation (I.P.) with FLAG antibody from HEK293T cells co-transfected with FLAG-14-3-3ε and vector, 9J10 WT , or 9J10 Amut for 24 h. All western blots are representative of at least two independent experiments. (B) Normalized FHRE (Forkhead Response Element)-luciferase reporter activity in HEK293T transfected with vector, 9J10 WT , or 9J10 AMut for 48 h. Data represent the mean of three independent experiments ± SD. (C) Venn diagrams showing numbers of differentially expressed genes in response to 9J10 or FOXO3a-AAA relative to respective controls. (D) Cell confluency measurements from live-cell imaging of HEK293T cells transfected with Vector, 9J10 WT or 9J10 AMut for 48 h. A representative experiment from three independent experiments is shown, data represent the average of four fields ± SD. (E) Western blotting for expression of FOXO3a in cells transfected with vector in the presence of siNT or siFOXO3a. Samples were harvested at 48 h after plasmid transfection (72 h after siRNA transfection). β-Actin is included as a control for total protein levels. A representative example from three independent experiments is shown. (F) Cell confluency measurements from live-cell imaging of HEK293T cells transfected with vector, 9J10 WT , or 9J10 AMut in the presence of a non-targeting (siNT) or FOXO3a (siFOXO3a) siRNA at 48 h after plasmid transfection (72 h after siRNA transfection). Bars represent the mean of three independent experiments ± SD.

Article Snippet: Rabbit monoclonal anti FOXO3a , Cell Signaling Technology , Cat#12829; RRID: AB_2636990.

Techniques: Expressing, Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Live Cell Imaging, Control

Journal: Cell Chemical Biology

Article Title: Target identification for small-molecule discovery in the FOXO3a tumor-suppressor pathway using a biodiverse peptide library

doi: 10.1016/j.chembiol.2021.05.009

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti FOXO3a , Cell Signaling Technology , Cat#12829; RRID: AB_2636990.

Techniques: Virus, Recombinant, Silver Staining, Plasmid Preparation, DNA Purification, Transfection, Western Blot, Reverse Transcription, Labeling, Luciferase, RNA Sequencing, Expressing, Software, Translocation Assay

Journal: iScience

Article Title: CRISPR-Cas9 screening identifies INTS3 as an anti-apoptotic RNA-binding protein and therapeutic target for colorectal cancer

doi: 10.1016/j.isci.2024.109676

Figure Lengend Snippet: INTS3 is overexpressed in CRC and correlated with clinical severity (A) INTS3 expression in various cancer types from TCGA were validated by UALCAN. (B–D) ENCORI, GEPIA2, and TNMplot were analyzed for INTS3 expression levels in CRC tumor tissues and normal tissues. (E) Comparison of INTS3 protein expression in tumor and normal tissues of CRC in Clinical Proteomic Tumor Analysis Consortium (CPTAC) database. (F) Effect of INTS3 expression level on CRC patient survival. The cut-off value is the median expression of INTS3. (G) RT-qPCR (top) and western blot (bottom) analysis of INTS3 expression in CRC cell lines and the human normal colonic epithelial cell NCM460. (H) IHC analysis of INTS3 expression in 11 paired CRC patient samples. Representative images (left) and associated statistics (right). (I) Western blot analysis of INTS3 expression in 11 paired CRC patient samples. Scale bars: 500 μm. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: INTS3 antibody , Proteintech , Cat#16620-1-AP; RRID: AB_2127274.

Techniques: Expressing, Comparison, Quantitative RT-PCR, Western Blot

Journal: iScience

Article Title: CRISPR-Cas9 screening identifies INTS3 as an anti-apoptotic RNA-binding protein and therapeutic target for colorectal cancer

doi: 10.1016/j.isci.2024.109676

Figure Lengend Snippet: INTS3-LOF significantly reduces CRC cell survival (A) Propagation/survival assay in INTS3-LOF CRC cell lines ( n = 3). (B) Colony formation assays showed the INTS3-LOF effect on clone forming ability ( n = 3). Representative images (left) and associated statistics (right). (C) Sphere formation assays of INTS3-LOF and control cells ( n = 3, scale bars: 400 μm). Representative images (left) and associated statistics (right). (D) EdU staining assays of INTS3-LOF and control cells ( n = 4). Representative images (left, scale bars: 50 μm) and associated statistics (right). (E) Flow cytometry analysis of Annexin V/7-AAD staining of INTS3-LOF and control cells ( n = 3). Representative images (left) and associated statistics (right). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: INTS3 antibody , Proteintech , Cat#16620-1-AP; RRID: AB_2127274.

Techniques: Clonogenic Cell Survival Assay, Control, Staining, Flow Cytometry

Journal: iScience

Article Title: CRISPR-Cas9 screening identifies INTS3 as an anti-apoptotic RNA-binding protein and therapeutic target for colorectal cancer

doi: 10.1016/j.isci.2024.109676

Figure Lengend Snippet: INTS3 reduces the expression of pro-apoptotic genes by regulating mRNA stability (A) Volcano plot of RNA-seq data showing the distribution of mapped different expressed genes (DEGs) between sgINTS3 #3 and sgControl. (B) Overlap between sgINTS3 #3 and sgINTS3 #4 upregulated genes (top). Biological process analysis of overlap genes (bottom). (C) Heatmap of upregulated genes in apoptotic process from the RNA-seq experiment. (D) Gene Set Enrichment Analysis (GSEA) was used to analyze the distribution of differentially expressed genes between sgINTS3 #3 and sgControl in apoptotic process. (E) Nascent RNA labeling assay. (F) Schematic of RNA stability assay. (G) Overlap between ten pro-apoptotic genes and genes with extended mRNA half-life (lifetime sgINTS3 #4 /lifetime control > 1.5). (H) TXNIP, CLU, and NR4A1 expression levels in CRC tumor tissues and normal tissues. (I) After treatment of SW620 cells with 5 μg/mL ActD for 0, 1, 2, 4, and 8 h, the effects of INTS3 knockout on TXNIP, CLU, and NR4A1 mRNA stability ( n = 3). (J) Western blot analysis of TXNIP, CLU, and NR4A1 expression.

Article Snippet: INTS3 antibody , Proteintech , Cat#16620-1-AP; RRID: AB_2127274.

Techniques: Expressing, RNA Sequencing, Labeling, Stability Assay, Control, Knock-Out, Western Blot

Journal: iScience

Article Title: CRISPR-Cas9 screening identifies INTS3 as an anti-apoptotic RNA-binding protein and therapeutic target for colorectal cancer

doi: 10.1016/j.isci.2024.109676

Figure Lengend Snippet: INTS3 depletion inhibits CRC propagation in vivo (A) Images of SW620-transduced cell tumor-bearing mice treated with DOX drinking water for 12 days (top). sgINTS3 #4 and control tumor tissues (bottom). (B–D) The growth curves (B), tumor weight (C), and tumor inhibition rate (D) of sgINTS3 #4 and control SW620 cell tumors ( n = 10). (E) Images of HCT116-transduced cell tumor-bearing mice (top). shINTS3 #2 and control tumor tissues (bottom). (F–H) The growth curves (F), tumor weight (G), and tumor inhibition rate (H) of shINTS3 #2 and control HCT116 cell tumors ( n = 6). (I) Images of RKO-transduced cell tumor-bearing mice (top). INTS3-LOF and control tumor tissues (bottom). (J–L) The growth curves (J), tumor weight (K), and tumor inhibition rate (L) of shINTS3 #2 and control RKO cell tumors ( n = 6). (M and N) TUNEL staining showed the SW620 (M) and HCT116 (N) tumor cell apoptosis in each group ( n = 5, scale bars: 50 μm). (O) Western blot analysis of TXNIP, CLU, and NR4A1 protein expression. SW620 tumor tissues (top, n = 7) and HCT116 tumor tissues (bottom, n = 6). (P) RT-qPCR analysis of TXNIP, CLU, and NR4A1 mRNA expression. SW620 tumor tissues (left, n = 7) and HCT116 tumor tissues (right, n = 6). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: INTS3 antibody , Proteintech , Cat#16620-1-AP; RRID: AB_2127274.

Techniques: In Vivo, Control, Inhibition, TUNEL Assay, Staining, Western Blot, Expressing, Quantitative RT-PCR

Journal: iScience

Article Title: CRISPR-Cas9 screening identifies INTS3 as an anti-apoptotic RNA-binding protein and therapeutic target for colorectal cancer

doi: 10.1016/j.isci.2024.109676

Figure Lengend Snippet:

Article Snippet: INTS3 antibody , Proteintech , Cat#16620-1-AP; RRID: AB_2127274.

Techniques: Virus, Recombinant, DNA Extraction, Plasmid Preparation, Imaging, Transfection, CRISPR, Knock-Out, Software, High Content Screening