Journal: Experimental & Molecular Medicine

Article Title: Inhibition of GSK3β is synthetic lethal with FHIT loss in lung cancer by blocking homologous recombination repair

doi: 10.1038/s12276-024-01374-0

Figure Lengend Snippet: a Western blotting was used to verify FHIT knockout and overexpression in lung cancer cell pairs. HCC827 and H1650 are FHIT-expressing lines, and H1299 and H460 are FHIT-deficient lines. GAPDH was used as the internal control. b Schematic illustration of highly selective inhibitor library screening to discover synthetic lethal candidates for FHIT deficiency. c The scatter plot presents the screening results as log10-IC 50 values of the inhibitors. Inhibitors with an SI (parental-IC 50 / FHIT −/− -IC 50 ) greater than two were considered synthetic lethal drugs for FHIT defects and are highlighted with colored dots. d – f Cell confluence was acquired from the Incucyte Zoom and normalized to that of the control group to validate the synthetic lethal effects of GSK3β inhibition. HCC827 (parental and FHIT −/− ), H460 ( FHIT OE and parental) and H1299 ( FHIT OE and parental) cell pairs were treated with different doses of CHIR99021 for three days. Representative images of HCC827 ( g ), H460 ( h ) and H1299 ( i ) cells treated with the indicated concentrations of CHIR99021 for three days; scar bar = 50 μm. j Representative images of a colony formation assay in HCC827-FHIT isogenic cells. The cells were treated with 2.5 μM or 10 μM CHIR99021 (CHIR) for 10 days and stained with crystal violet. k Effects of CHIR99021 on the growth rate of HCC827 FHIT-isogenic cell pairs. The cells were incubated with 10 μM CHIR99021, and an IncuCyte Zoom was used to capture images of the cells at different time points to calculate cell confluence. The data are presented as the mean ± SD of each group. The statistical difference between the two groups was analyzed by two-way ANOVA. All FHIT-deficient cells are marked in red.

Article Snippet: Antibodies against the following proteins were used: FHIT (JP18163, IBL International), HA (3724 s, CST), GSK3α (AG2065, Beyotime), GSK3β (9832 s, CST), pho-GSK3α/β (Tyr279/216) (AF1522, Beyotime), PARP (9532 s, CST), cleaved-caspase3 (9664 s, CST), γ-H2A.

Techniques: Western Blot, Knock-Out, Over Expression, Expressing, Control, Library Screening, Inhibition, Colony Assay, Staining, Incubation

Journal: Experimental & Molecular Medicine

Article Title: Inhibition of GSK3β is synthetic lethal with FHIT loss in lung cancer by blocking homologous recombination repair

doi: 10.1038/s12276-024-01374-0

Figure Lengend Snippet: a Synthetic lethality in FHIT-knockdown HCC827 cells treated with CHIR99021 (CHIR). After HCC827 cells were transfected with or without 200 nM FHIT siRNA for 48 h, they were incubated with CHIR99021 (0, 5, 10, or 20 μM) for an additional three days. Cell viability was assessed via the Alamar blue assay. The data are presented as the mean ± SD of each group. Unpaired two-tailed Student’s t tests were used to determine the significance of the differences between two groups. b Representative cell images of a , scale bar = 50 μm. c Western blot analysis of FHIT protein knockdown in a . d Synthetic lethality in FHIT-knockdown H1650 cells treated with CHIR99021. After H1650 cells were transfected with or without 200 nM FHIT siRNA for 48 h, they were incubated with CHIR99021 (0, 5, 10, or 20 μM) for an additional three days. Cell viability was assessed via the Alamar blue assay. The data are presented as the mean ± SD of each group. Unpaired two-tailed Student’s t tests were used to determine differences between two groups. e Representative cell images of d , scale bar = 50 μm. f Western blot analysis of FHIT protein knockdown in d . g ‒ l Synthetic lethality of GSK3β knockdown in HCC827 FHIT −/− cells. After HCC827 parental and FHIT −/− cells were transfected with or without 100 nM GSK3α ( g ) or GSK3β ( j ) siRNA for 72 h, cell viability was determined via the Alamar blue assay. The data are presented as the mean ± SD of each group. Unpaired two-tailed Student’s t tests were used to determine the significance of the differences between two groups. NS denotes not significant. Representative cell images h , k of g and j , scale bar = 50 μm. Western blotting was used to detect the knockdown of the GSK3α ( i ) or GSK3β ( l ) proteins in g and j . GAPDH was used as the internal control. All FHIT-deficient cells are marked in red.

Article Snippet: Antibodies against the following proteins were used: FHIT (JP18163, IBL International), HA (3724 s, CST), GSK3α (AG2065, Beyotime), GSK3β (9832 s, CST), pho-GSK3α/β (Tyr279/216) (AF1522, Beyotime), PARP (9532 s, CST), cleaved-caspase3 (9664 s, CST), γ-H2A.

Techniques: Knockdown, Transfection, Incubation, Alamar Blue Assay, Two Tailed Test, Western Blot, Control

Journal: Experimental & Molecular Medicine

Article Title: Inhibition of GSK3β is synthetic lethal with FHIT loss in lung cancer by blocking homologous recombination repair

doi: 10.1038/s12276-024-01374-0

Figure Lengend Snippet: a Western blotting was used to compare the protein levels of GSK3β and p-GSK3α/β (Y279/Y216) in four lung cancer cell lines, FHIT-expressing (H1650 and HCC827) and FHIT-defective (H460 and H1299) lines. b Western blot analysis of the protein levels of GSK3β and p-GSK3α/β (Y279/Y216) in the HCC827 FHIT-isogenic cell pair. c Western blot analysis of γ-H2A. X protein levels in the HCC827 and H1650 FHIT-isogenic cell pairs. d – i Effects of GSK3βi on DNA double-strand breaks in HCC827 and H1650 FHIT isogenic cells. The cells were treated with or without 20 μM CHIR99021 for 24 h, and the protein level of γ-H2A. X was examined by Western blotting ( d , g ), and the in situ foci of γ-H2A. X were detected via immunofluorescence staining, as shown in the fluorescence images; scale bar = 100 μm. Inset images (red squares) are enlarged and shown below each panel; scale bar = 10 μm. The nuclei were stained with DAPI ( e , h ), and the number of cells containing more than five γ-H2A. X foci in each group were recorded and quantified via ImageJ with a threshold (setting a fixed value: 74, 129) to distinguish positive γ-H2A. X foci from the background staining ( f , i ). The data are presented as the mean ± SD of each group. The difference between two groups was determined by an unpaired two-tailed Student’s t test. j , k Comet assay to test DNA damage in cells upon GSK3βi treatment. HCC827 FHIT-isogenic cells were treated with or without 20 μM CHIR99021 for 24 h to conduct an alkaline comet assay, and representative images from the comet assay are shown; scale bar = 200 μm. Inset images (red squares) are enlarged and shown below each panel; scale bar = 50 μm. Representative images ( j ) were acquired via an Evos microscope (Life Technology), and the percentage of DNA in the tail ( k ) was analyzed via the ImageJ component Open Comet. The data are presented as the mean ± SD of each group. The significance of the difference between two groups was determined by an unpaired two-tailed Student’s t test. GAPDH was used as the internal control. All FHIT-deficient cells are marked in red.

Article Snippet: Antibodies against the following proteins were used: FHIT (JP18163, IBL International), HA (3724 s, CST), GSK3α (AG2065, Beyotime), GSK3β (9832 s, CST), pho-GSK3α/β (Tyr279/216) (AF1522, Beyotime), PARP (9532 s, CST), cleaved-caspase3 (9664 s, CST), γ-H2A.

Techniques: Western Blot, Expressing, In Situ, Immunofluorescence, Staining, Fluorescence, Two Tailed Test, Single Cell Gel Electrophoresis, Alkaline Single Cell Gel Electrophoresis, Microscopy, Control

Journal: Experimental & Molecular Medicine

Article Title: Inhibition of GSK3β is synthetic lethal with FHIT loss in lung cancer by blocking homologous recombination repair

doi: 10.1038/s12276-024-01374-0

Figure Lengend Snippet: a – d Effect of FHIT deficiency on the protein stability of BRCA1 and RAD51. After HCC827 parental and HCC827 FHIT −/− cells were incubated with 50 μg/mL CHX for the indicated times, Western blotting was performed to determine the protein levels of BRCA1 ( a ) and RAD51 ( c ), and ImageJ software was used to quantify the percentage of BRCA1 ( b ) and RAD51 ( d ) protein remaining. e , f HSP90 interacts with FHIT and BRCA1/RAD51. BioID was used to detect the interaction between FHIT and HSP90 in HCC827 cells ( e ), and HSP90 IP was used to analyze the interactions between HSP90 and FHIT, BRCA1, and RAD51 ( f ). g Western blot analysis of the protein levels of BRCA1 and RAD51 in HCC827 FHIT-isogenic cells treated with 125 nM geldanamycin for 48 h. GAPDH was used as the internal control. h – j Effect of a GSK3β inhibitor on the protein stability of BRCA1 and RAD51. After the cells were treated with or without 20 μM CHIR99021 for 30 min and then incubated with 50 μg/mL CHX for the indicated times, Western blotting was performed to determine the protein levels of BRCA1 and RAD51 in HCC827 FHIT −/− cells ( h ), and ImageJ software was used to quantify the percentage of remaining BRCA1 and RAD51 proteins ( i , j ). k Effect of a GSK3β inhibitor on the mRNA levels of BRCA1 and RAD51. After the HCC827 FHIT −/− cells were treated with or without 20 μM CHIR99021 for 12 h, RT‒qPCR was used to examine the mRNA levels of BRCA1 and RAD51. The data are presented as the means ± SDs of each group. The difference between two groups was determined by an unpaired two-tailed Student’s t test. l The effect of si Snail on CHIR99021-induced BRCA1 and RAD51 downregulation. HCC827 FHIT −/− cells were transfected with 200 nM Snail siRNA for 72 hours and then treated with 20 μM CHIR99021 for 24 h. The cell lysates were analyzed via Western blotting for Snail, BRCA1 and RAD51. GAPDH was used as the internal control. All FHIT-deficient cells are marked in red.

Article Snippet: Antibodies against the following proteins were used: FHIT (JP18163, IBL International), HA (3724 s, CST), GSK3α (AG2065, Beyotime), GSK3β (9832 s, CST), pho-GSK3α/β (Tyr279/216) (AF1522, Beyotime), PARP (9532 s, CST), cleaved-caspase3 (9664 s, CST), γ-H2A.

Techniques: Incubation, Western Blot, Software, Control, Two Tailed Test, Transfection

Journal: Cancer cell

Article Title: MYC drives temporal evolution of small cell lung cancer subtypes by reprogramming neuroendocrine fate

doi: 10.1016/j.ccell.2020.05.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies include: ASCL1 (1:300, BD Pharmingen #BD556604), EPCAM (1:1000, Abcam #ab71916), INSM1 (1:300, Santa Cruz sc-271408), NEUROD1 (1:1000, Abcam ab109224), REST (1:1000, Millipore #17-641), YAP (1:1000, CST #14074), NOTCH2 XP (1:1000, CST #5732), HES1 (1:400, CST #11988), MYC (1:1000, CST #5605), NKX2-1 (1:2000, Abcam ab76013), ZEB1 (1:500, Bethyl Labs A301-922A), and HSP90 (1:1000, CST #4877) as loading control.

Techniques: Western Blot, Virus, Plasmid Preparation, Recombinant, Staining, Library Quantification, RNA Sequencing, Software

Journal: Cancer cell

Article Title: MYC drives temporal evolution of small cell lung cancer subtypes by reprogramming neuroendocrine fate

doi: 10.1016/j.ccell.2020.05.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies include: ASCL1 (1:300, BD Pharmingen #BD556604), EPCAM (1:1000, Abcam #ab71916), INSM1 (1:300, Santa Cruz sc-271408), NEUROD1 (1:1000, Abcam ab109224), REST (1:1000, Millipore #17-641), YAP (1:1000, CST #14074), NOTCH2 XP (1:1000, CST #5732), HES1 (1:400, CST #11988), MYC (1:1000, CST #5605), NKX2-1 (1:2000, Abcam ab76013), ZEB1 (1:500, Bethyl Labs A301-922A), and HSP90 (1:1000, CST #4877) as loading control.

Techniques: Western Blot, Virus, Plasmid Preparation, Recombinant, Staining, Library Quantification, RNA Sequencing, Software

Journal: Cancer cell

Article Title: MYC drives temporal evolution of small cell lung cancer subtypes by reprogramming neuroendocrine fate

doi: 10.1016/j.ccell.2020.05.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The primary antibodies include: ASCL1 (BD cat# BD556604) 1:200; UCHL1 (Sigma cat# HPA005993) 1:300; SYP (Thermo-fisher cat# RB1461P1) 1:200; CGRP (Sigma cat# C8198) 1:250; NEUROD1 (Abcam cat# 109224) 1:150; YAP1 (CST cat# 17074S) 1:400; ZEB1 (Abcam cat# ab133357) 1:2000; CD44 (CST cat# 37259) 1:250; POU2F3 (Sigma cat# HPA019652) 1:300; DLL3 (Abcam cat# 198505) 1:200; HES1 (CST cat# 11988) 1:500; MYC (mouse) (Santa Cruz cat#sc-764) and MYC (human) (Abcam cat# ab32072).

Techniques: Western Blot, Virus, Plasmid Preparation, Recombinant, Staining, Library Quantification, RNA Sequencing, Software

Journal: Cancer cell

Article Title: MYC drives temporal evolution of small cell lung cancer subtypes by reprogramming neuroendocrine fate

doi: 10.1016/j.ccell.2020.05.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The primary antibodies include: ASCL1 (BD cat# BD556604) 1:200; UCHL1 (Sigma cat# HPA005993) 1:300; SYP (Thermo-fisher cat# RB1461P1) 1:200; CGRP (Sigma cat# C8198) 1:250; NEUROD1 (Abcam cat# 109224) 1:150; YAP1 (CST cat# 17074S) 1:400; ZEB1 (Abcam cat# ab133357) 1:2000; CD44 (CST cat# 37259) 1:250; POU2F3 (Sigma cat# HPA019652) 1:300; DLL3 (Abcam cat# 198505) 1:200; HES1 (CST cat# 11988) 1:500; MYC (mouse) (Santa Cruz cat#sc-764) and MYC (human) (Abcam cat# ab32072).

Techniques: Western Blot, Virus, Plasmid Preparation, Recombinant, Staining, Library Quantification, RNA Sequencing, Software

Journal: Cancer cell

Article Title: MYC drives temporal evolution of small cell lung cancer subtypes by reprogramming neuroendocrine fate

doi: 10.1016/j.ccell.2020.05.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Alternatively, CST primary antibodies were detected using 150 μL of SignalStain Boost IHC Detection Reagent (CST cat# 8114).

Techniques: Western Blot, Virus, Plasmid Preparation, Recombinant, Staining, Library Quantification, RNA Sequencing, Software

Journal: eLife

Article Title: Genetic interactions of G-quadruplexes in humans

doi: 10.7554/eLife.46793

Figure Lengend Snippet: ( A ) Number of depleted hairpins and median log 2 FC values for DEAH/DEAD-box helicase genes within the 758 genes identified in the genome-wide screen. Those highlighted in blue caused sensitivity to both PDS and PhenDC3. ( B ) Representative immunoblots showing cytoplasmic (C) and nuclear ( N ) lysates for HT1080, human embryonic kidney (HEK) and HeLa cells probed for DDX42, laminB1 and GAPDH2. ( C, D ) GAPDH and laminB1 protein levels for ( C ) cytoplasmic and ( D ) nuclear lysates (mean for two biological replicates ± standard deviation). ( E ) DDX42 nuclear protein levels (normalised to cytoplasmic levels, mean for two biological replicates ± standard deviation). ( F, G ) DDX42 binding curves G4s by ELISA. ( F ) NRAS 5’ UTR RNA G4 (rG4), mutated G4 sequence (rG4 mut) and RNA hairpin. ( G ) MYC DNA G4 (dG4) and mutated control (dG4 mut). Apparent K d is calculated from five replicates (values are indicative as the model assumes saturation kinetics). 10.7554/eLife.46793.023 Figure 10—source data 1. Source files for western blots. ( A ) Full-length western blots and ( B ) capillary traces obtained from Compass Software (Simple Western) for results shown in .

Article Snippet: Samples were also immunoblotted with antibodies against nuclear laminB1 (CST 12586; 1:250) and cytoplasmic GAPDH to confirm subcellular fractionation efficiency (CST 5174, 1:50).

Techniques: Genome Wide, Western Blot, Standard Deviation, Binding Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Control, Software, Simple Western

Journal: eLife

Article Title: Genetic interactions of G-quadruplexes in humans

doi: 10.7554/eLife.46793

Figure Lengend Snippet:

Article Snippet: Samples were also immunoblotted with antibodies against nuclear laminB1 (CST 12586; 1:250) and cytoplasmic GAPDH to confirm subcellular fractionation efficiency (CST 5174, 1:50).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Plasmid Preparation, shRNA, Genome Wide, Sequencing, Adapter Ligation, Biomarker Discovery, Transfection, Western Blot, Marker, Library Quantification, Purification, Lysis, Protease Inhibitor, Software

Journal: Cancer cell

Article Title: MYC drives temporal evolution of small cell lung cancer subtypes by reprogramming neuroendocrine fate

doi: 10.1016/j.ccell.2020.05.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies include: ASCL1 (1:300, BD Pharmingen #BD556604), EPCAM (1:1000, Abcam #ab71916), INSM1 (1:300, Santa Cruz sc-271408), NEUROD1 (1:1000, Abcam ab109224), REST (1:1000, Millipore #17-641), YAP (1:1000, CST #14074), NOTCH2 XP (1:1000, CST #5732), HES1 (1:400, CST #11988), MYC (1:1000, CST #5605), NKX2-1 (1:2000, Abcam ab76013), ZEB1 (1:500, Bethyl Labs A301-922A), and HSP90 (1:1000, CST #4877) as loading control.

Techniques: Western Blot, Virus, Plasmid Preparation, Recombinant, Staining, Library Quantification, RNA Sequencing, Software

Journal: eLife

Article Title: Genetic interactions of G-quadruplexes in humans

doi: 10.7554/eLife.46793

Figure Lengend Snippet: ( A–C ) Venn diagrams for: ( A ) significantly differentially expressed individual shRNAs (FDR ≤ 0.05); ( B ) significantly depleted genes (50% or three hairpins, FDR ≤ 0.05, median log 2 FC < 0) following DMSO, PDS and PhenDC3 treatment and ( C ) Significant PDS and PhenDC3 sensitiser genes not in DMSO and after applying a median log 2 FC ≤ −1 cut off. ( D–F ) Tables showing the number of depleted hairpins and median log 2 FC values for: ( D ) known G4 ligand sensitisers, ATRX, HERC2 , BRCA1 and BRCA2, that are independently validated in our screen; ( E ) sensitisers annotated with a G4-associated term in GO, UniprotKB or G4IPBD databases and ( F ) sensitisers identified as G4-related by text-mining showing the associated PolySearch2 algorithm score and summary of the G4 association. Sensitisers are defined as a gene where 50% or three hairpins were significantly differentially expressed (FDR ≤ 0.05) with median log 2 FC ≤ −1. See also .

Article Snippet: Lysates from non-transfected and siRNA-treated (targeting and non-targeting) samples were probed with antibodies against BRCA1 (Cell Signalling Technology, cat # 4970-CST), TOP1 (Abcam, cat # AB109374), GAR1 (NovusBio cat #NBP2-31742) or DDX42 (Abcam cat #AB80975), plus anti-beta actin antibody (mouse Merck cat # A5441; rabbit cat # 4970-CST) by multiplexing.

Techniques:

Journal: eLife

Article Title: Genetic interactions of G-quadruplexes in humans

doi: 10.7554/eLife.46793

Figure Lengend Snippet: ( A ) HT1080 cells were treated with non-targeting (NT) or targeting (T) siRNAs for BRCA1 , TOP1, DDX42 and GAR1 . 48 hr and 144 hr after transfection, cell lysates and a non-transfected cell lysate (U) were probed with appropriate antibodies and actin control by western blotting. ( B ) Protein levels for targeting (T) and non-targeting (NT) 48 hr lysates were normalised to the internal actin control and then normalised to NT levels for three biological replicates (mean ± standard deviation). ( C–F ) HT1080 cells were transfected with targeting siRNAs for 24 hr before PDS, PhenDC3 or DMSO treatment. Comparative box plots of confluency differences and significance (unpaired parametric t-test) at selected timepoints for ( C ) BRCA1 , ( D ) TOP1 , ( E ) DDX42 , ( F ) GAR (ns = not significant) for three separate siRNA transfections. See also , and . 10.7554/eLife.46793.017 Figure 8—source data 1. Source files for western blots. ( A ) Full length western blots and ( B ) capillary traces obtained from Compass Software (Simple Western) for results shown in .

Article Snippet: Lysates from non-transfected and siRNA-treated (targeting and non-targeting) samples were probed with antibodies against BRCA1 (Cell Signalling Technology, cat # 4970-CST), TOP1 (Abcam, cat # AB109374), GAR1 (NovusBio cat #NBP2-31742) or DDX42 (Abcam cat #AB80975), plus anti-beta actin antibody (mouse Merck cat # A5441; rabbit cat # 4970-CST) by multiplexing.

Techniques: Transfection, Control, Western Blot, Standard Deviation, Software, Simple Western

Journal: eLife

Article Title: Genetic interactions of G-quadruplexes in humans

doi: 10.7554/eLife.46793

Figure Lengend Snippet: ( A ) HT1080 cells were transfected with targeting siRNAs (orange) against BRCA1, TOP1, DDX42 or GAR1 for 24 hr before treatment with PDS (0.25 μM and 0.5 μM), PhenDC3 (20 μM and 40 μM) or vehicle control (DMSO). For each knockdown, confluency over 144 hr was monitored and plotted against confluency of non-transfected cells (blue) and cells transfected with a non-targeting control siRNA (green). Experiments were performed in triplicate and average confluency accumulation shown (mean ± standard deviation). ( B–D ) Confluency differences (mean ± standard deviation) compared to NT siRNA were plotted across three replicates for ( B ) BRCA1 , ( C ) TOP1 , ( D ) DDX42 , ( E ) GAR1 .

Article Snippet: Lysates from non-transfected and siRNA-treated (targeting and non-targeting) samples were probed with antibodies against BRCA1 (Cell Signalling Technology, cat # 4970-CST), TOP1 (Abcam, cat # AB109374), GAR1 (NovusBio cat #NBP2-31742) or DDX42 (Abcam cat #AB80975), plus anti-beta actin antibody (mouse Merck cat # A5441; rabbit cat # 4970-CST) by multiplexing.

Techniques: Transfection, Control, Knockdown, Standard Deviation

Journal: eLife

Article Title: Genetic interactions of G-quadruplexes in humans

doi: 10.7554/eLife.46793

Figure Lengend Snippet: A375 cells were transfected with targeting siRNAs (orange) against BRCA1, TOP1, DDX42 or GAR1 for 24 hr before treatment with PDS (5 μM and 10 μM), PhenDC3 (20 μM and 40 μM) or vehicle control (DMSO). For each knockdown, confluency over 144 hr was monitored and plotted against confluency of non-transfected cells (blue) and cells transfected with a non-targeting control siRNA (green). Experiments were performed in triplicate and average confluency accumulation shown (mean ± standard deviation).

Article Snippet: Lysates from non-transfected and siRNA-treated (targeting and non-targeting) samples were probed with antibodies against BRCA1 (Cell Signalling Technology, cat # 4970-CST), TOP1 (Abcam, cat # AB109374), GAR1 (NovusBio cat #NBP2-31742) or DDX42 (Abcam cat #AB80975), plus anti-beta actin antibody (mouse Merck cat # A5441; rabbit cat # 4970-CST) by multiplexing.

Techniques: Transfection, Control, Knockdown, Standard Deviation

Journal: eLife

Article Title: Genetic interactions of G-quadruplexes in humans

doi: 10.7554/eLife.46793

Figure Lengend Snippet: ( A ) For BRCA1 , TOP1 , DDX42 and GAR1 , A375 cells were transfected with non-targeting (NT) or targeting (T) siRNAs. 48 hr and 144 hr after transfection, lysates were probed appropriate antibodies alongside a non-transfected lysate (U). ( B ) BRCA1, TOP1, DDX42 and GAR1 protein levels for targeting (T) and non-targeting (NT) lysates were normalised to the internal actin control and then normalised to NT levels for three independent blots 48 hr (top) and two independent blots 144 hr (bottom) after transfection (mean ± standard deviation). ( C–F ) A375 cells were transfected with the targeting siRNAs for 24 hr before PDS, PhenDC3 or DMSO treatment. Average confluency difference (mean ± standard deviation) compared to NT siRNA plotted across three replicates for ( C ) BRCA1, ( D ) TOP1, ( E ) DDX42, ( F ) GAR1. Confluency differences at 72, 96 and 120 hr plotted for comparison for ( G ) BRCA1, ( H ) TOP1, ( I ) DDX42, ( J ) GAR1. 10.7554/eLife.46793.016 Figure 8—figure supplement 3—source data 1. Source files for western blots. ( A ) Full length western blots and ( B ) capillary traces obtained from Compass Software (Simple Western) for results shown in .

Article Snippet: Lysates from non-transfected and siRNA-treated (targeting and non-targeting) samples were probed with antibodies against BRCA1 (Cell Signalling Technology, cat # 4970-CST), TOP1 (Abcam, cat # AB109374), GAR1 (NovusBio cat #NBP2-31742) or DDX42 (Abcam cat #AB80975), plus anti-beta actin antibody (mouse Merck cat # A5441; rabbit cat # 4970-CST) by multiplexing.

Techniques: Transfection, Control, Standard Deviation, Comparison, Western Blot, Software, Simple Western

Journal: eLife

Article Title: Genetic interactions of G-quadruplexes in humans

doi: 10.7554/eLife.46793

Figure Lengend Snippet: Possible chemotherapeutic combinations for G4-stabilising ligands with clinically relevant pharmacological drugs

Article Snippet: Lysates from non-transfected and siRNA-treated (targeting and non-targeting) samples were probed with antibodies against BRCA1 (Cell Signalling Technology, cat # 4970-CST), TOP1 (Abcam, cat # AB109374), GAR1 (NovusBio cat #NBP2-31742) or DDX42 (Abcam cat #AB80975), plus anti-beta actin antibody (mouse Merck cat # A5441; rabbit cat # 4970-CST) by multiplexing.

Techniques: Ubiquitin Proteomics, Clinical Proteomics

Journal: eLife

Article Title: Genetic interactions of G-quadruplexes in humans

doi: 10.7554/eLife.46793

Figure Lengend Snippet: Examples of cancer-associated genetic vulnerabilities to G4 ligands.

Article Snippet: Lysates from non-transfected and siRNA-treated (targeting and non-targeting) samples were probed with antibodies against BRCA1 (Cell Signalling Technology, cat # 4970-CST), TOP1 (Abcam, cat # AB109374), GAR1 (NovusBio cat #NBP2-31742) or DDX42 (Abcam cat #AB80975), plus anti-beta actin antibody (mouse Merck cat # A5441; rabbit cat # 4970-CST) by multiplexing.

Techniques: Homologous Recombination, Translesion Synthesis, Inhibition, Activity Assay, Ubiquitin Proteomics, Expressing

Journal: eLife

Article Title: Genetic interactions of G-quadruplexes in humans

doi: 10.7554/eLife.46793

Figure Lengend Snippet:

Article Snippet: Lysates from non-transfected and siRNA-treated (targeting and non-targeting) samples were probed with antibodies against BRCA1 (Cell Signalling Technology, cat # 4970-CST), TOP1 (Abcam, cat # AB109374), GAR1 (NovusBio cat #NBP2-31742) or DDX42 (Abcam cat #AB80975), plus anti-beta actin antibody (mouse Merck cat # A5441; rabbit cat # 4970-CST) by multiplexing.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Plasmid Preparation, shRNA, Genome Wide, Sequencing, Adapter Ligation, Biomarker Discovery, Transfection, Western Blot, Marker, Library Quantification, Purification, Lysis, Protease Inhibitor, Software

Journal: eLife

Article Title: Genetic interactions of G-quadruplexes in humans

doi: 10.7554/eLife.46793

Figure Lengend Snippet:

Article Snippet: Lysates from non-transfected and siRNA-treated (targeting and non-targeting) samples were probed with antibodies against BRCA1 (Cell Signalling Technology, cat # 4970-CST), TOP1 (Abcam, cat # AB109374), GAR1 (NovusBio cat #NBP2-31742) or DDX42 (Abcam cat #AB80975), plus anti-beta actin antibody (mouse Merck cat # A5441; rabbit cat # 4970-CST) by multiplexing.

Techniques: Sequencing

Journal: eLife

Article Title: A conserved major facilitator superfamily member orchestrates a subset of O-glycosylation to aid macrophage tissue invasion

doi: 10.7554/eLife.41801

Figure Lengend Snippet:

Article Snippet: Anti-Rab7 (rabbit monoclonal) , CST, RRID: SCR_004431 , Clone D95F2 , CST Cat# 9367, RRID: AB_1904103 , (1:200) for immunochemistry.

Techniques: Amplification, Clone Assay, cDNA Library Assay, Expressing, Plasmid Preparation, Staining, Software

Journal: eLife

Article Title: The origins and consequences of UPF1 variants in pancreatic adenosquamous carcinoma

doi: 10.7554/eLife.62209

Figure Lengend Snippet: ( A ) Schematic of UPF1 gene structure and corresponding encoded protein domains. Intron 10 (I10) contains the bulk of the mutations reported by Liu et al. Scissors indicate the sites targeted by the paired guide RNAs used to excise exons 10 and 11 (E10 and E11). Red nucleotides represent positions subject to point mutations reported in Liu et al. Arrows indicate specific mutations that we modeled in 293 T cells. The horizontal black line indicates the nucleotide within the protospacer adjacent motif (PAM) site that we mutated to prevent repeated cutting by Cas9 in 293 T cells. ( B ) Top, experimental strategy for testing whether mimicking UPF1 mis-splicing by deleting exons 10 and 11 promoted pancreatic cancer growth. Mice were orthotopically injected with mouse pancreatic cancer cells (KPC cells: Kras G12D ; Trp53 R172H/null ; Pdx1-Cre) lacking Upf1 exons 10 and 11. Bottom, hematoxylin and eosin (H and E) stain of pancreatic tumor tissue harvested from the mice. ( C ) Line graph comparing tumor volume between mice injected with control (AdCas9; Cas9 only) or treatment (AdUpf1; Cas9 with Upf1 -targeting guide RNAs) KPC cells. Tumor volume measured by ultrasound imaging. Error bars, standard deviation computed over surviving animals (n = 10 at first time point). n.s., not significant (p>0.05). p-values at each timepoint were calculated relative to the control group with an unpaired, two-tailed t -test. ( D ) Survival curves for the control (AdCas9) or treatment (AdUpf1) cohorts. Error bars, standard deviation computed over biological replicates (n = 10, each group). p-value was calculated relative to the control group by a logrank test. ( E ) Representative hematoxylin and eosin (H and E) staining of a pancreatic tumor resulting from orthotopic injection of control KPC cells displaying features of a moderately to poorly differentiated pancreatic ductal adenocarcinoma. Tumors were composed of medium-size duct-like structures and small tubular glands with lower mucin production. ( F ) Representative H and E image illustrating a moderately to poorly differentiated pancreatic ductal adenocarcinoma resulting from orthotopic injection of Upf1 -targeted KPC cells. Depicted here is a section of the poorly differentiated component (arrow), which was characterized by solid sheets of tumor cells with large eosinophilic cytoplasms and marked nuclear polymorphism. ( G ) Representative H and E image of a pancreatic tumor resulting from orthotopic injection of Upf1 -targeted KPC cells. The dashed circle marks a moderately differentiated component; the remainder is poorly differentiated. ( H ) Representative IHC image of a pancreatic tumor resulting from orthotopic injection of Upf1 -targeted KPC cells for the squamous marker p40 (ΔNp63). No expression of the marker was observed in tumor cells. Figure 1—source data 1. Source data for mouse tumor volume . Figure 1—source data 2. Source data for mouse survival .

Article Snippet: The membranes were incubated with rabbit UPF1 antibody (CST #9435) used at 1:1000 dilution in 5% BSA 1× TBST overnight at 4°C or mouse Tubulin (Sigma T9206) used at 1:2000 dilution in 5% milk 1× TBST for 1 hr at room temperature.

Techniques: Injection, Staining, Control, Imaging, Standard Deviation, Two Tailed Test, Marker, Expressing

Journal: eLife

Article Title: The origins and consequences of UPF1 variants in pancreatic adenosquamous carcinoma

doi: 10.7554/eLife.62209

Figure Lengend Snippet: ( A ) Titration of adenovirus expressing Cas9 only (AdCas9) or Cas9 and guide RNAs targeting intron 9 and intron 11 of mouse Upf1 (AdUpf1) in KPC cells. Gel corresponds to PCR using genomic DNA with primers in exons 9 and 12 (E9, E12). The lower band, corresponding to excision of exons 10 and 11, was only observed in AdUpf1 conditions. This band was excised and sequence-verified by Sanger sequencing (sequence trace displayed below). ( B ) As ( A ), but assaying a cDNA library instead of genomic DNA using RT-PCR. The lower band, corresponding to spliced mRNA lacking exons 10 and 11, was only observed in AdUpf1 conditions. ( C ) Immunoblot with a probe against UPF1. Levels of full-length UPF1 protein are lower in AdUpf1 conditions, as expected. 1 and 2 indicate biological replicates. Tubulin serves as a loading control. ( D ) Raw gel image for the genomic DNA PCR shown in ( A ). The cropped region is boxed in white. ( E ) Raw gel image for the RT-PCR shown in ( B ). The cropped region is boxed in white. ( F ) Raw image of the western blot shown in ( C ). The cropped region is boxed in white. ( G ) Raw image of the western blot for the mouse tubulin loading control shown in ( C ). The cropped region is boxed in white.

Article Snippet: The membranes were incubated with rabbit UPF1 antibody (CST #9435) used at 1:1000 dilution in 5% BSA 1× TBST overnight at 4°C or mouse Tubulin (Sigma T9206) used at 1:2000 dilution in 5% milk 1× TBST for 1 hr at room temperature.

Techniques: Titration, Expressing, Sequencing, cDNA Library Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: eLife

Article Title: The origins and consequences of UPF1 variants in pancreatic adenosquamous carcinoma

doi: 10.7554/eLife.62209

Figure Lengend Snippet: ( A ) Sanger sequencing of genomic DNA from engineered 293 T cells verifying introduction of the mutation IVS10+31G>A in a homozygous state, as reported by Liu et al. for patient P1, as well as a single protospacer adjacent motif (PAM) site mutation. ( B ) Sanger sequencing of genomic DNA from engineered 293 T cells verifying introduction of the IVS10-17G>A mutation in a heterozygous state, as reported by Liu et al. for patient P9, as well as a single PAM site mutation. ( C ) Sanger sequencing of genomic DNA from engineered 293 T cells verifying introduction of a single PAM site mutation as a wild-type control. ( D ) Raw image of the western blot from . The cropped regions for UPF1 and GAPDH are boxed in red. ( E ) Raw image of the RT-PCR gel from . The cropped region for full-length UPF1 is boxed in red.

Article Snippet: The membranes were incubated with rabbit UPF1 antibody (CST #9435) used at 1:1000 dilution in 5% BSA 1× TBST overnight at 4°C or mouse Tubulin (Sigma T9206) used at 1:2000 dilution in 5% milk 1× TBST for 1 hr at room temperature.

Techniques: Sequencing, Mutagenesis, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: eLife

Article Title: The origins and consequences of UPF1 variants in pancreatic adenosquamous carcinoma

doi: 10.7554/eLife.62209

Figure Lengend Snippet: ( A ) Box plot of NMD efficiency in 293 T cells engineered to contain wild-type (WT) or mutant (P1, P9) UPF1 . P1 and P9 correspond to the IVS10+31G>A and IVS10-17G>A mutations reported by Liu et al. All cells have the protospacer adjacent motif (PAM) site mutation illustrated in . NMD efficiency estimated via the beta-globin reporter assay 11 . Middle line, notches, and whiskers indicate median, first and third quartiles, and range of data. Each point corresponds to a single biological replicate. n.s., not significant (p>0.05). p-values were calculated for each variant relative to the control by a two-sided Mann–Whitney U test (p=0.40 for P1, 0.30 for P9). ( B ) Scatter plot showing transcriptome-wide quantification of transcripts containing NMD-promoting features in 293 T cells carrying the UPF1 mutation that was reportedly observed in patient 1 relative to control, wild-type cells. Each point corresponds to a single isoform that is a predicted NMD substrate (NMD(+)). Purple points represent NMD substrates that are significantly increased in UPF1 -mutant cells relative to wild-type cells; black points represent NMD substrates that exhibit the opposite behavior. Plot is restricted to NMD substrates arising from differential inclusion of cassette exons. Significantly increased/decreased NMD substrates were defined as transcripts that displayed either an absolute increase/decrease in isoform ratio of ≥10% or an absolute log fold-change in expression of ≥2 with associated p≤0.05 (two-sided t -test). ( C ) As ( B ), but for 293 T cells carrying the UPF1 mutation that was reportedly observed in patient 9. Gold points represent NMD substrates that are significantly increased in UPF1 -mutant cells relative to wild-type cells. ( D ) Summary of the numbers of NMD substrates arising from differential alternative splicing that exhibit significantly higher or lower levels in UPF1 -mutant cells relative to wild-type cells. Analysis is identical to ( B ) and ( C ), but extended to the illustrated different types of alternative splicing. ( E ) Left, immunoblot of full-length UPF1 protein for the 293 T cell lines. Each lane represents a single biological replicate with the indicated genotype. GAPDH serves as a loading control. Equal amounts of protein were loaded in each lane (measured by fluorescence). Right, box plot illustrating UPF1 protein levels relative to GAPDH for each genotype. Middle line, notches, and whiskers indicate median, first and third quartiles, and range of data. Each point corresponds to a single biological replicate. Data was quantified with Fiji (v2.0.0). A.U., arbitrary units. n.s., not significant (p>0.05). p-values were calculated for each variant relative to the control by a two-sided Mann–Whitney U test (p=0.10 for P1, 1.0 for P9). ( F ) PCR using primers that amplify both full-length UPF1 mRNA (FL) and mRNA lacking exons 10 and 11 (ΔE10-11). UPF1 mRNA lacking exons 10 and 11 was only detected in the positive control lanes (ΔE10-11 spike in), in which DNA corresponding to UPF1 cDNA lacking exons 10 and 11 was synthesized and added to cDNA libraries created from WT cells prior to PCR. Numbers above each lane indicate biological replicates. Numbers below each lane represent the abundance of the lower band as a percentage of total intensity (see Materials and methods). Data was quantified with Fiji (v2.0.0). ( G ) RNA-seq read coverage across the genomic locus containing UPF1 exons 9–12 in the indicated 293 T cell lines. Each sample corresponds to a distinct biological replicate. Numbers represent read counts that supported each indicated splice junction . Figure 2—source data 1. Source data for qPCR in HEK 293 T cell lines . Figure 2—source data 2. Source data for western blot in HEK 293 T cell lines . Figure 2—source data 3. Source data for RT-PCR in HEK 293 T cell lines .

Article Snippet: The membranes were incubated with rabbit UPF1 antibody (CST #9435) used at 1:1000 dilution in 5% BSA 1× TBST overnight at 4°C or mouse Tubulin (Sigma T9206) used at 1:2000 dilution in 5% milk 1× TBST for 1 hr at room temperature.

Techniques: Mutagenesis, Reporter Assay, Variant Assay, Control, MANN-WHITNEY, Expressing, Alternative Splicing, Western Blot, Fluorescence, Positive Control, Synthesized, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction

Journal: eLife

Article Title: The origins and consequences of UPF1 variants in pancreatic adenosquamous carcinoma

doi: 10.7554/eLife.62209

Figure Lengend Snippet: ( A ) Illustration of the mutations in UPF1 intron 10 (I10) reported by Liu et al. Each row indicates the wild-type (WT) sequence from the reference human genome or mutations reported by Liu et al. (P1, patient 1). Purple and gold arrows indicate the mutations that we modeled with genome engineering in 293 T cells for patient 1 and patient 9, respectively. Red nucleotides represent positions subject to point mutations reported in Liu et al. The horizontal black line indicates the nucleotide within the protospacer adjacent motif (PAM) site that we mutated to prevent repeated cutting by Cas9. Parentheses indicate where we found genetic variation at a reported mutation position that differed from the specific mutated nucleotide reported by Liu et al. ( B ) As ( A ), but for UPF1 exon 10 (E10). ( C ) As ( A ), but for UPF1 exon 11 (E11). ( D ) As ( A ), but for UPF1 exon 21 (E21). ( E ) As ( A ), but for UPF1 intron 22 (I22). ( F ) As ( A ), but for UPF1 exon 23 (E23).

Article Snippet: The membranes were incubated with rabbit UPF1 antibody (CST #9435) used at 1:1000 dilution in 5% BSA 1× TBST overnight at 4°C or mouse Tubulin (Sigma T9206) used at 1:2000 dilution in 5% milk 1× TBST for 1 hr at room temperature.

Techniques: Sequencing, Mutagenesis

Journal: eLife

Article Title: The origins and consequences of UPF1 variants in pancreatic adenosquamous carcinoma

doi: 10.7554/eLife.62209

Figure Lengend Snippet:

Article Snippet: The membranes were incubated with rabbit UPF1 antibody (CST #9435) used at 1:1000 dilution in 5% BSA 1× TBST overnight at 4°C or mouse Tubulin (Sigma T9206) used at 1:2000 dilution in 5% milk 1× TBST for 1 hr at room temperature.

Techniques: Expressing, CRISPR, Generated, Recombinant, Plasmid Preparation, Control, Transfection, Construct, Sequencing, Positive Control, Variant Assay, PCR Cloning, Protease Inhibitor, Software, Reverse Transcription