Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: WT and Ifitm3 -/- mice were intranasally infected with ( a, b ) 1, 10, or 50 TCID50 of H5N1 avian influenza strain (2 independent experiments for doses 1 and 10 (n=10 mice) and 1 experiment for dose of 50 (n=5 mice)) or with ( c, d ) 1 or 10 TCID50 of H7N3 avian influenza strain (n=5 mice). a , c Viral titers from lung homogenates at day 3 post infection. b, d ELISA quantification of IL-6 levels in lung homogenates at day 3 post infection. a-d Error bars represent SEM. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. Only comparisons between WT and Ifitm3 -/- mice for each dose are shown.

Article Snippet: HeLa IFITM1/2/3 knockout and HAP1 IFITM3 knockout were purchased from ATCC (CRL-3452) and Horizon Discovery Biosciences (HZGHC004186c010), respectively.

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: ( a ) Schematic of in vitro infection with potentially zoonotic influenza viruses and representative example from infected A549 human lung cells. ( b ) The indicated A549 cells or THP-1 differentiated macrophages were treated +/- IFNβ for 18 hours, followed by infection with indicated viruses (MOI 1) for 24 hours. Percent infection was determined by flow cytometry and normalized to respective shControl or WT cells without IFNβ pre-treatment. Error bars represent SEM. P values are for the indicated comparisons and were determined by ANOVA followed by Tukey’s multiple comparisons test. Only statistical comparisons between shControl versus shIFITM3 and WT versus IFITM3 -/- are shown. Data are representative of 3 independent experiments each performed in triplicate (n=9). ( c, d ) Western blots of cell lysates at 18 hours +/- IFNβ treatment. Note that commercial IFITM3 antibodies weakly detect IFITM2 in addition to IFITM3. ( e ) The indicated A549 cells were infected with indicated viruses at a range of 0.001 to 10 MOI for 24 hours. Percent infection was determined by flow cytometry. Data are representative of 3 independent experiments each performed in triplicate (n=9). Error bars represent SEM. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. Only comparisons between control and knockdown cells are shown at each dose.

Article Snippet: HeLa IFITM1/2/3 knockout and HAP1 IFITM3 knockout were purchased from ATCC (CRL-3452) and Horizon Discovery Biosciences (HZGHC004186c010), respectively.

Techniques: In Vitro, Infection, Flow Cytometry, Western Blot, Control, Knockdown

Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: HAP1 cells were treated +/- IFNβ for 18 hours, followed by infection with indicated viruses (MOI 1) for 24 hours. ( a ) Percent infection was determined by flow cytometry and normalized to results for WT cells without IFNβ pre-treatment. Error bars represent SEM. P values are for the indicated comparisons and were determined by ANOVA followed by Tukey’s multiple comparisons test. Only statistical comparisons between WT versus IFITM3 -/- are shown. Data are representative of 3 independent experiments each performed in triplicate (n=9). ( b ) Western blots of cell lysates at 18 hours +/- IFNβ treatment. ( c ) Example raw flow cytometry data for determination of normalized infection of WT and IFITM3 -/- HAP1 cells +/- IFNb treatment with each of the indicated virus strains as in ( a ). SSC, side scatter; Anti-influenza nucleoprotein antibody was detected in the APC channel with secondary antibody labeled with Alexafluor 647.

Article Snippet: HeLa IFITM1/2/3 knockout and HAP1 IFITM3 knockout were purchased from ATCC (CRL-3452) and Horizon Discovery Biosciences (HZGHC004186c010), respectively.

Techniques: Infection, Flow Cytometry, Western Blot, Virus, Labeling

Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: HeLa cells were treated +/- IFNβ for 18 hours, followed by infection with indicated viruses (MOI 1) for 24 hours. ( a ) Percent infection was determined by flow cytometry and normalized to results for WT cells without IFNβ pre-treatment. Error bars represent SEM. P values are for the indicated comparisons and were determined by ANOVA followed by Tukey’s multiple comparisons test. Only statistical comparisons between WT versus IFITM3 -/- are shown. Data are representative of 3 independent experiments each performed in triplicate (n=9). ( b ) Western blots of cell lysates at 18 hours +/- IFNβ treatment. ( c ) Example raw flow cytometry data for determination of normalized infection of WT and IFITM1/2/3 -/- HeLa cells +/- IFNb treatment with each of the indicated virus strains as in a . SSC, side scatter; Anti-influenza nucleoprotein antibody was detected in the APC channel with secondary antibody labeled with Alexafluor 647.

Article Snippet: HeLa IFITM1/2/3 knockout and HAP1 IFITM3 knockout were purchased from ATCC (CRL-3452) and Horizon Discovery Biosciences (HZGHC004186c010), respectively.

Techniques: Infection, Flow Cytometry, Western Blot, Virus, Labeling

Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: ( a ) Schematic of mouse passaging experiments. Initial intranasal infections were performed with 1,000 TCID50 of parental viruses. ( b ) Schematic of WT mouse challenge with parental or passaged viruses. ( c-h and j-l ) Groups of WT mice were challenged with equal doses of virus passaged 1, 5, or 10 times through WT or Ifitm3 -/- mice and compared to the parent virus (passage 0). ( c, e ) Viral titers from lung homogenates taken at day 7 ( c represents 2 independent experiments). Error bars represent SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s post hoc test. ( d, f ) ELISA quantification of IL-6 levels in lung homogenates of WT and IFITM3 KO mice at day 7 post infection ( d represents 2 independent experiments). Error bars represent SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. ( g, j ) Viral titers from lung homogenates taken at day 7 ( g ) or day 6 ( j ) post infection. Error bars represent SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s post hoc test. ( h, k ) ELISA quantification of IL-6 levels in lung homogenates of WT and IFITM3 KO mice at day 7 ( h ) or day 6 ( k ) post infection. Error bars represent SD of the mean. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. ( i, m ) Mutations found in the segments of A/California/04/2009 (H1N1) after serial passage through WT or Ifitm3 -/- mice. ( l ) Weight loss. Skull and crossbones indicate humane euthanasia of all animals infected with KO passage 10. Error bars represent SD of the mean, comparisons were made using the Mann-Whitney test (* P = 0.0022, ** P < 0.0001).

Article Snippet: HeLa IFITM1/2/3 knockout and HAP1 IFITM3 knockout were purchased from ATCC (CRL-3452) and Horizon Discovery Biosciences (HZGHC004186c010), respectively.

Techniques: Passaging, Virus, Enzyme-linked Immunosorbent Assay, Infection, MANN-WHITNEY

Journal: bioRxiv

Article Title: Innate immune control of influenza virus interspecies adaptation

doi: 10.1101/2023.08.23.554491

Figure Lengend Snippet: ( a ) Schematic of mouse passaging experiments. Initial intranasal infections were performed with 1,000 TCID50 of parental viruses. ( b ) Schematic of WT mouse challenge with parental or passaged viruses. ( c,d ) Groups of WT mice were challenged with equal doses of virus passaged 1, 5, or 10 times through WT or Stat1 -/- mice and compared to the parent virus (passage 0). ( c ) Viral titers from lung homogenates taken at day 7 post infection. Error bars represent SEM. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test. ( d ) ELISA quantification of IL-6 levels in lung homogenates of WT and IFITM3 KO mice at day 7 post infection. Error bars represent SEM. Comparisons were analyzed by ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: HeLa IFITM1/2/3 knockout and HAP1 IFITM3 knockout were purchased from ATCC (CRL-3452) and Horizon Discovery Biosciences (HZGHC004186c010), respectively.

Techniques: Passaging, Virus, Infection, Enzyme-linked Immunosorbent Assay

Journal: Nature

Article Title: Targeting PIKfyve-driven lipid metabolism in pancreatic cancer.

doi: 10.1038/s41586-025-08917-z

Figure Lengend Snippet: Fig. 3 | PIKfyve inhibition obligates PDAC cells to stimulate a lipogenic transcriptional and metabolic program. a, Schematic of the metabolism- focused CRISPR screen in MIA PaCa-2 cells. Created in BioRender. Cheng, C. (2025) https://BioRender.com/d149928. b,c, Gene enrichment rank plot-based differential sgRNA representation (b) and scatter plot of gene fitness scores (c) of high dose (2,000 nM) and low dose (100 nM) apilimod-treated versus DMSO- treated end-point populations of the CRISPR screen experiment. Graphs show the top 30 synthetically lethal genes involved in fatty acid and sphingolipid synthesis (red), the top 30 synthetically lethal genes involved in cholesterol synthesis (purple) and genes that confer sensitivity to apilimod (blue). d, Metabolic map of sphingolipid and cholesterol synthesis. The figure shows the top 90 synthetically lethal genes involved in fatty acid and sphingolipid

Article Snippet: The human CRISPR metabolic gene knockout library was a gift from David Sabatini (Addgene, 110066)58.

Techniques: Inhibition, CRISPR

Journal: bioRxiv

Article Title: Genome-Wide CRISPR Screen Identifies Non-Canonical NF-κB Signaling as a Potent Regulator of Density-dependent Proliferation

doi: 10.1101/2020.10.02.324301

Figure Lengend Snippet: Whole-genome screening for genes that inhibit proliferation at homeostatic cell density. (A) A Schematic of FUCCI color transitions through cell cycle. (B) EpH4-FUCCI stable cell line grown at 1 and 4 DPC. (C) Whole-genome CRISPR KO screening strategy. (D) Read count distribution for samples before sorting and after different rounds of sorting. Data are logit transformed (f(p) = log 2 (p/1 - p) where p is the proportion of a given sgRNA in the total number of sgRNAs in a sample). Color coding shows depleted sgRNAs in blue, enriched sgRNAs in red and sgRNA with no enrichment in white. Gray shows lost sgRNAs. (E) Genes plotted based on their RRA enrichment score (3 rd sorting). (F) List of genes with FDR below 0.25 and >= 3 sgRNAs enriched compared to control after 3 rd sort.

Article Snippet: Mouse CRISPR GeCKO v2 Knockout Pooled Library ( , ) was purchased from Addgene.

Techniques: Stable Transfection, CRISPR, Transformation Assay, Control

Journal: bioRxiv

Article Title: Parallel CRISPR screens reveal pathways controlling the cell surface levels of the attractant receptor FPR1

doi: 10.1101/2025.04.21.649864

Figure Lengend Snippet: Clonally selected β-arrestin double CRISPR knockout cells are compared to pooled double β-arrestin knockout cell lines using the FPR1 endocytosis assay. Cells were untreated or stimulated with 1 μM fMLF. (A) A representative plot showing fMLF-induced decrease in surface FPR1 in the control sgRNA expressing cells. (B) Pooled double β-arrestin knockout cells have a smaller shift after fMLF stimulation. (C) Representative plots obtained from twelve clonally selected β-arrestin double knockout cell lines. Phenotypes observed in clonally selected and pooled knockout cell lines resemble each other.

Article Snippet: Bassik Lab Human CRISPR Deletion Library , Addgene catalog #101926- 101934, was packaged into lentiviral particles using TransIT-2020 transfection reagent (VWR, catalog #10767-014).

Techniques: CRISPR, Knock-Out, Endocytosis Assay, Control, Expressing, Double Knockout

Journal: bioRxiv

Article Title: Parallel CRISPR screens reveal pathways controlling the cell surface levels of the attractant receptor FPR1

doi: 10.1101/2025.04.21.649864

Figure Lengend Snippet: (A) Schematic representation of a selection of hits found in the parallel whole-genome CRISPR screens. Genes shown are significant hits in at least one screen, and they were organized based on prior literature. The effect scores for each gene are represented as colored rectangles. The scores are only shown if the gene was a hit in the particular screen (pFDR< 0.05). Negative scores indicate a decrease in surface FPR1, while positive scores indicate an increase. For hits regulating FPR1 surface expression post-stimulation (shown in pink and green), the effect scores obtained from the integrated analysis of the two screens were presented. (B) Top hits from the surface FPR1 expression screen. FPR2 and FPR3 were shown for comparison and are not identified as hits in either of the screens. (C) Significant hits from the integrated analysis of FPR1 internalization, recycling, or exocytosis. Scatter plots in B and C represent the same dataset (log Pscore, with the basal surface expression score on the y-axis and post-stimulation score on the x-axis) with different genes highlighted.

Article Snippet: Bassik Lab Human CRISPR Deletion Library , Addgene catalog #101926- 101934, was packaged into lentiviral particles using TransIT-2020 transfection reagent (VWR, catalog #10767-014).

Techniques: Selection, CRISPR, Expressing, Comparison

Journal: bioRxiv

Article Title: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC reprogramming

doi: 10.1101/2023.07.31.551297

Figure Lengend Snippet: ( A ) Experimental design. X-GFP iCas9 ESCs were infected with a genome-wide lentiviral gRNA library and differentiated into NPCs. Next, NPCs were treated with doxycycline to activate the expression of the reprogramming cassette and the iCas9 . The knockouts took place during reprogramming. At day 10 of reprogramming, three populations were FACS-separated: non-pluripotent (SSEA1-X-GFP-), early pluripotent (SSEA1+ X-GFP-) and late pluripotent, X-reactivated (SSEA1+ X-GFP+). For these three populations and the NPCs, genomic DNA extraction, PCR-amplification of the gRNA sequences, sequencing and analysis of gRNA abundance were performed. n=2 biological replicates (independent reprogramming rounds). ( B ) Pathways related to overrepresented genes in the three reprogramming populations (non-pluripotent, early pluripotent and late pluripotent) compared to NPCs (WikiPathways Mouse 2019). For pathway enrichment analysis, the top 250 genes from each individual comparison to NPCs ranked by RRA score with MAGeCK software were merged and selected. ( C ) gRNA abundance comparison (early pluripotent vs non-pluripotent) and representation of genes with negative Log2FC (underrepresented) vs −log10 RRA (RRA cutoff = 0.05, Log2FC cutoff = −0.75) (activators of early pluripotency). ( D ) gRNA abundance comparison (late pluripotent vs early pluripotent) and representation of genes with negative Log2FC (underrepresented) vs −log10 RRA (RRA cutoff = 0.05, Log2FC cutoff = −0.75) (activators of late pluripotency, X-reactivation). Genes highlighted in yellow are related to pluripotency, genes highlighted in red are involved in Notch or interferon γ signaling. ( E ) Pathways (WikiPathways Mouse 2019) related to underrepresented genes in “late pluripotent vs early pluripotent” comparison (activators of late pluripotency, X-reactivation, n=1313 genes). Pathways related to proliferation, differentiation and metabolism are shown in gray. The rest of the pathways are highlighted in green. RRA score < 0.05 and Log2FC < −0.8 filtering was applied. ( F ) Experimental design for (G). Treatment with molecules targeting pathways identified in the CRISPR screen was done at the beginning of reprogramming (day 0 - day 5), later in reprogramming (day 5 - day 7) or during the whole process (day 0 - day 7). At day 7, flow cytometry analysis of SSEA1 and X-GFP percentages was performed. ( G ) Pathway validation by molecule treatment at the beginning of reprogramming (day 0 - day 5), later in reprogramming (day 5 - day 7) or during the whole process (day 0 - day 7) in three independent reprogramming rounds, n=3 (except for TGFβ, n=2). Flow cytometry analysis at day 7 of SSEA1 and X-GFP percentages is shown. Data represented as mean +/− SD. Statistics (paired t-tests): where not specified = non-significant; * = p<0.05; ** = p<0.01; *** = p<0.001.

Article Snippet: The gRNA library used for the screening was the Mouse Improved Genome-wide Knockout CRISPR Library v2 (a gift from Kosuke Yusa, Addgene, #67988) , with 90,230 gRNAs targeting 18,424 genes (average of 5 gRNAs per gene).

Techniques: Infection, Genome Wide, Expressing, DNA Extraction, Amplification, Sequencing, Comparison, Software, CRISPR, Flow Cytometry, Biomarker Discovery

Journal: bioRxiv

Article Title: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC reprogramming

doi: 10.1101/2023.07.31.551297

Figure Lengend Snippet: A genome-wide CRISPR knockout screen reveals molecular networks involved in reprogramming and X-chromosome reactivation. Related to . (A) Validation of knockout efficiency by flow cytometry. Flow cytometry analysis during 6 days of doxycycline treatment in the X-GFP iCas9 ESC line was done to measure the X-GFP percentage decay in cells containing a gRNA targeting the GFP gene. Gating shows the X-GFP+ population. (B) Percentage of gRNA representation in the plasmid library, infected ESCs and the 4 populations analyzed in two independent screening rounds: NPCs and day 10 reprogramming populations (non-pluripotent, early pluripotent, late pluripotent). Error bars represent SD. ( C ) gRNA abundance comparisons (related to D-G): NPCs to non-pluripotent, early pluripotent and late pluripotent populations. ( D ) Pathways related to common underrepresented genes (n=927 genes) in the three reprogramming populations compared to NPCs (WikiPathways Mouse 2019). For all comparisons, a RRA score < 0.05 and Log2FC < −0.75 (underrepresented) filtering was applied. ( E-G ) Representation of genes with negative Log2FC (underrepresented) vs −log10 RRA in the non-pluripotent (E), early pluripotent (F) and late pluripotent (G) populations compared to NPCs (RRA cutoff = 0.05, Log2FC cutoff = −0.75). ( H ) Pathways (WikiPathways Mouse 2019) related to underrepresented genes in the “early pluripotent vs non-pluripotent” comparison (activators of early pluripotency, n=1361 genes) (RRA score < 0.05 and Log2FC < −0.8 filtering was applied). ( I ) Pathways (WikiPathways Mouse 2019) related to overrepresented genes in the “early pluripotent vs non-pluripotent” comparison (repressors of early pluripotency, n=693 genes) (RRA score < 0.05 and Log2FC < −0.8 filtering was applied). ( J ) Representation of genes with positive Log2FC (overrepresented) vs −log10 RRA (RRA cutoff = 0.05, Log2FC cutoff = 0.75) in the “early pluripotent vs non-pluripotent” comparison (repressors of early pluripotency). ( K ) Representation of genes with positive Log2FC (overrepresented) vs −log10 RRA (RRA cutoff = 0.05, Log2FC cutoff = 0.75) in the “late pluripotent vs early pluripotent” comparison (repressors of late pluripotency, X-reactivation). ( L ) Pathways (WikiPathways Mouse 2019) related to overrepresented genes in the “late pluripotent vs early pluripotent” comparison (repressors of late pluripotency, X-reactivation, n=839 genes) (RRA score < 0.05 and Log2FC < −0.8 filtering was applied).

Article Snippet: The gRNA library used for the screening was the Mouse Improved Genome-wide Knockout CRISPR Library v2 (a gift from Kosuke Yusa, Addgene, #67988) , with 90,230 gRNAs targeting 18,424 genes (average of 5 gRNAs per gene).

Techniques: Genome Wide, CRISPR, Knock-Out, Biomarker Discovery, Flow Cytometry, Plasmid Preparation, Infection, Comparison