Journal: Journal of Virology

Article Title: HPV16 entry requires dynein for minus-end transport and utilizes kinesin Kif11 for plus-end transport along microtubules during mitosis

doi: 10.1128/jvi.00937-24

Figure Lengend Snippet: Kif11 inhibitor reduces transport of EdU-labeled PSVs to mitotic chromosomes. HaCaT cells were infected with WT EdU-labeled PsVs for 24 h and then treated with 1.5 µM Kif11 inhibitor (Eg5 inh+) or left untreated (Ctrl) for 10 min prior to the cells being stained and subjected to the Click-iT reaction as previously described in Materials and Methods. ( A ) AF555 dye was used to stain EdU-labeled pseudogenomes (red), mouse anti-α-tubulin antibody was used to stain for α-tubulin (white), and rabbit anti-Kif11 antibody was used to stain for Kif11 (green). Left-hand panel of Ctrl row of ( A ) is of an infected HaCaT cell in prophase, and the right-hand panel of Ctrl row of ( A ) is of an infected HaCaT cell in metaphase. Since Eg5 inh+ treatment locks cells into a monoastral phenotype , both the left-hand and right-panels of the Eg5 inh+ row of ( A ) are of pre-monoastral cells. Two smaller close-up images, rotated at a 45° angle on the x-, y-, and z-axes, are included on the right-side of the main image(s) to better show close association, or lack thereof in Eg5 inh+, between EdU-labeled HPV genome and Kif11. ( B ) AF555 dye was again used to stain EdU-labeled pseudogenomes in cells undergoing mitosis at prophase or metaphase (red), and mitotic chromosomes were visualized using Hoechst (blue). Images represent single medial slices. ( C ) Quantification of EdU-labeled HPV16 PsV in whole cells +/−Eg5 inh+ treatment; at least 20 cells per group, control and Eg5 inh+ were counted, with 69 total cells counted (group numbers of 21, 26, and 22). Localization of EdU puncta (red) was analyzed with IMARIS using spots/surface analysis. The number of pseudogenomes in whole cells was established based on alpha tubulin and EdU-labeled HPV genome signals; determining genome and the localization on mitotic chromosomes was based on Hoechst staining. Lines in graph represent mean with SEM, and statistical significance was assessed by Student t -test, N = 3, ns: P > 0.05. ( D ) Quantification of EdU-labeled HPV16 PsV (red) co-localized with mitotic chromatin (blue) upon Kif11 inhibitor treatment using IMARIS. At least 20 cells per group, control and Eg5 inh+ were counted, with 66 total cells counted (group numbers of 21, 25, and 20). Lines in graph represent mean with SEM, and statistical significance was assessed by Mann Whitney test, N = 3, ***: P < 0.001. ( E ) Uninfected HaCaT cells were treated with 1.5 µM Eg5 inhibitor for 10 min and then stained for Kif11 and Kif18a as previously described in Materials and Methods, except cells were also subjected to the Click-iT reaction, but without the AF555 dye to stain for kinesins under denaturing conditions. Either Kif11 or Kif18A was stained using anti-rabbit Kif11 or anti-rabbit Kif18A antibodies, respectively (red). Mouse anti-α-tubulin antibody was used to stain α-tubulin (green), and cell nuclei were visualized using Hoechst (blue). Images represent single medial slices.

Article Snippet: Primary antibodies used for the immunofluorescence studies were as follows: mouse monoclonal antibody (mAb) anti-α-tubulin (Cell Signaling; #3873S), mouse mAb AlexaFluor (AF) 488-conjugated anti-α-tubulin (Cell Signaling; #8058), mouse mAb anti-γ-tubulin for the detection of the MTOC (Sigma-Aldrich; #T6557), rabbit mAb anti-dynein intermediate chain 1 (Abcam; #ab171964); all rabbit mAbs for detection of kinesins such as Kif11 (Cell Signaling; #14404S), Kif18a (Novus Biologicals; #NBP1-85126), and Kif25 (Novus Biologicals; #NBP1-92055).

Techniques: Labeling, Infection, Staining, Control, MANN-WHITNEY

Journal: Journal of Virology

Article Title: HPV16 entry requires dynein for minus-end transport and utilizes kinesin Kif11 for plus-end transport along microtubules during mitosis

doi: 10.1128/jvi.00937-24

Figure Lengend Snippet: Kinesin motor proteins are in proximity to HPV16 L2 protein during infection. ( A ) HEK293TT cells were transfected with plasmids encoding HA-tagged HPV16 L2 and FLAG-tagged Kif11 to perform a co-immunoprecipitation (CoIP) assay using anti-HA beads; pCMV6 plasmid was used for the CoIP empty vector controls. CoIPs were performed with (right-hand panel) or without (left-hand panel) transfected cells treated with Kif11 inhibitor (1.5 µM; “Eg5i”) 1 or 2 h prior to cell harvesting for CoIP. Tagged HPV16 L2 and Kif11 proteins were probed for via Western blot using anti-HA and anti-FLAG antibodies, respectively, in both whole-cell lysates (“Lysate”) and anti-HA bead elutions (“elute”); anti α-tubulin Western blots were used as a loading control for the lysates. The enclosed immunoblots are representative of at least two independent experiments ( N = 2). ( B ) Mock-infected (“Mock”) and PsV16-infected (“Infected”) HaCaT cells were processed using the proximity ligation assay (PLA) protocol 21 hpi. PLAs were performed using an antibody cocktail to target the HPV16 L2 protein and an individually selected kinesin, either Kif11, Kif18a, or Kif25. The HPV16L2 antibodies used are the same throughout all PLAs, while the specific kinesin antibody used for the indicated PLA is identified in the left-hand margin of the images. PLA puncta were visualized in the representative images as red dots. Confocal images were acquired as z-stacks and processed as described in Materials and Methods. The white arrows highlight mitotic cells in their representative images for the PLAs. ( C ) The average PLA puncta numbers per interphase cells were counted for at least 200 cells per group in one experiment and are presented as median with 95% CI in the corresponding graphs for the PLAs in the middle panels adjacent to their representative images. The average PLA puncta numbers per mock-infected versus PSV-infected interphase cell were (median) 0.43 vs 6.8 (Kif11), 0.83 vs 5.8 (Kif18A), and 1.8 vs 4.1 (Kif25). Differences between mock and infected cells were analyzed using Mann–Whitney test, ( N = 3); **** P < 0.0001. ( D ) The average PLA puncta numbers were counted per at least 20 mitotic cells for each condition and are presented as median with 95% CI. Mock-infected vs PSV-infected PLA puncta numbers per mitotic cell were (median) 0.0 vs 6.0 (Kif11), 0.0 vs 5.0 (Kif18A), and 1.0 vs 5.5 (Kif25). Differences between mock and infected cells were analyzed using Mann–Whitney test, **** P < 0.0001.

Article Snippet: Primary antibodies used for the immunofluorescence studies were as follows: mouse monoclonal antibody (mAb) anti-α-tubulin (Cell Signaling; #3873S), mouse mAb AlexaFluor (AF) 488-conjugated anti-α-tubulin (Cell Signaling; #8058), mouse mAb anti-γ-tubulin for the detection of the MTOC (Sigma-Aldrich; #T6557), rabbit mAb anti-dynein intermediate chain 1 (Abcam; #ab171964); all rabbit mAbs for detection of kinesins such as Kif11 (Cell Signaling; #14404S), Kif18a (Novus Biologicals; #NBP1-85126), and Kif25 (Novus Biologicals; #NBP1-92055).

Techniques: Infection, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Plasmid Preparation, Cell Harvesting, Western Blot, Control, Proximity Ligation Assay, MANN-WHITNEY

Journal: Journal of Virology

Article Title: HPV16 entry requires dynein for minus-end transport and utilizes kinesin Kif11 for plus-end transport along microtubules during mitosis

doi: 10.1128/jvi.00937-24

Figure Lengend Snippet: Knocksideways experimental layout. 1) HeLa(Mito)–Kif11–FKBP–3x Flag cells were first treated with doxycycline (doxy) for 24 h to induce expression of the Kif11–FKBP–3x Flag transgene. 2) An endogenous Kif11-targeting siRNA pool was then reverse-transfected into doxy-treated cells to decrease endogenous Kif11 population over 24 h. 3) The doxy and Kif11 siRNA-treated cells, now containing reduced levels of endogenous Ki11 and increased amounts of exogenous Kif11–FKBP–3x Flag protein, were then infected with EdU-labeled PsV16s for either 16 h or infected with PsV16s containing a luciferase reporter genome for 24 h. Infected cells were then treated 16 hpi with 200 nM rapamycin for 1 h or added along with PsV16s for the longer 24 h infection. 4) Rapamycin-induced relocalization of Kif11–FKBP–3x Flag proteins to MitoTrap via dimerization of their respective FKBP and FRB domains. Not all Kif11–FKBP–3x will be re-localized to MitoTrap, some transgenic Kif11 protein will remain on microtubules to transport HPV16. 5) Optimally, the initial goal of the knocksideways experiment was to detect relocalization of EdU-labeled HPV16 genome to mitochondria in infected HeLa(Mito)–Kif11–FKBP–3x Flag cells treated with doxy and rapamycin. 6) Without rapamycin, Kif11–FKBP–3x Flag should perform the same functions as endogenous Kif11, especially during mitosis. 7) With rapamycin addition, a significant portion of the Kif11–FKBP–3x Flag cellular pool will be relocalized to MitoTrap. As a result of cells containing less endogenous Kif11, due to Kif11 siRNA, rapamycin treatment would increase levels of monoastral cells due to less endogenous and transgenic Kif11 available to assist in spindle microtubule sliding. Diagram was generated using BioRender and its available templates.

Article Snippet: Primary antibodies used for the immunofluorescence studies were as follows: mouse monoclonal antibody (mAb) anti-α-tubulin (Cell Signaling; #3873S), mouse mAb AlexaFluor (AF) 488-conjugated anti-α-tubulin (Cell Signaling; #8058), mouse mAb anti-γ-tubulin for the detection of the MTOC (Sigma-Aldrich; #T6557), rabbit mAb anti-dynein intermediate chain 1 (Abcam; #ab171964); all rabbit mAbs for detection of kinesins such as Kif11 (Cell Signaling; #14404S), Kif18a (Novus Biologicals; #NBP1-85126), and Kif25 (Novus Biologicals; #NBP1-92055).

Techniques: Expressing, Transfection, Infection, Labeling, Luciferase, Transgenic Assay, Generated

Journal: Journal of Virology

Article Title: HPV16 entry requires dynein for minus-end transport and utilizes kinesin Kif11 for plus-end transport along microtubules during mitosis

doi: 10.1128/jvi.00937-24

Figure Lengend Snippet: Relocalization of Kif11 via knocksideways significantly decreases transport of EdU-labeled PsVs to mitotic chromosomes. HeLa(Mito) cells containing the tet-inducible Kif11–FKBP–3x Flag construct, were treated with doxycycline (doxy, 5 µg/mL) for 24 h, reverse-transfected with 2.5 pmol of Kif11-targeting siRNA for 24 h, and then infected with PsV16 for 16 h. Cells were then treated with either DMSO or rapamycin (200 nM) for 1 h prior to processing for confocal microscopy or Western blotting (WB) as described in Materials and Methods. ( A ) Doxy-treated (24 h) HeLa(Mito) cells were harvested approximately 42 h post-siRNA-transfection (1–10 pmol control scrambled (SCR) or Kif11-targeting [Kif11] siRNAs) and probed via WB for presence of endogenous Kif11 (WB:Kif11) or exogenous Kif11–FKBP–3x Flag (WB: Flag). Blot is representative of two separate experiments. ( B ) For panels left to right, for HeLa(Mito): Kif11–FKBP–3x flag cells treated with doxy, than 2.5 pmol Kif11 siRNAs, and finally treated with either DMSO or rapamycin; mouse anti-HA antibody was used to stain for MitoTrap protein (green), rabbit anti-Kif11 was used to stain for Kif11 (red), and nuclei were stained with Hoechst (blue). Images with combined staining are on the right-hand side, with a close-up of a selected cell. Colocalization of Kif11 and MitoTrap are visualized as yellow puncta in the combined image, with representative signal(s) shown in the z-stacks provided with the close-up images. White arrows in the DMSO panels show metaphase cells, and blue arrows in the rapamycin panels indicate monoastral cells. ( C,E ) The percentages of monoastral cells for each condition (DMSO or rapamycin treatment) were determined by Hoechst33342 staining. Confocal images were acquired as z-stacks with Olympus CSU W1 Spinning Disk Confocal System using a 20× objective. Monoastral quantifications in ( C ) and ( E ) are based on Hoechst staining and represent individual experiments. Differences in monoastral cell percentages in each condition (DMSO or rapamycin treatment) were analyzed using the Mann–Whitney test; *, P < 0.05. ( D, F, G ) Quantification of EdU-labeled PsV16(s) on mitotic chromatin of either monoastral or metaphase cells, with or without rapamycin treatment. Approximately 23 cells per condition per experiment were counted, with ( D ) 11, 6.3, 7.4 , and 2.2 (median) EdU puncta counted in monoastral, −/+ rapamycin and metaphase, −/+ rapamycin-treated cells, respectively, in one independent experiment and ( F ) 22, 16, 17, and 8.0 (median) EdU puncta counted in monoastral, −/+ rapamycin and metaphase, −/+ rapamycin-treated cells, respectively, in another independent experiment. ( G ) Confocal images were acquired and processed as described in Materials and Methods. AF555 dye coupled with Click-iT® chemistry was used to stain EdU-labeled pseudogenomes (red), and genome localization was determined by proximity to mitotic chromatin stained with Hoechst (blue). Differences of EdU puncta amounts on mitotic chromosomes in each condition were analyzed using the Mann–Whitney test; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Primary antibodies used for the immunofluorescence studies were as follows: mouse monoclonal antibody (mAb) anti-α-tubulin (Cell Signaling; #3873S), mouse mAb AlexaFluor (AF) 488-conjugated anti-α-tubulin (Cell Signaling; #8058), mouse mAb anti-γ-tubulin for the detection of the MTOC (Sigma-Aldrich; #T6557), rabbit mAb anti-dynein intermediate chain 1 (Abcam; #ab171964); all rabbit mAbs for detection of kinesins such as Kif11 (Cell Signaling; #14404S), Kif18a (Novus Biologicals; #NBP1-85126), and Kif25 (Novus Biologicals; #NBP1-92055).

Techniques: Labeling, Construct, Transfection, Infection, Confocal Microscopy, Western Blot, Control, Staining, MANN-WHITNEY

Journal: Journal of Virology

Article Title: HPV16 entry requires dynein for minus-end transport and utilizes kinesin Kif11 for plus-end transport along microtubules during mitosis

doi: 10.1128/jvi.00937-24

Figure Lengend Snippet: Knocksideways relocalization of Kif11 over 24 h also significantly decreases PsV infectivity of cells. HeLa(Mito): Kif11–FKBP–3x Flag cells were subjected to the same general knocksideways protocol as in , with the main differences being the infection time, and rapamycin time treatments were extended, 24 h each; the resultant infection readout is based on luciferase activity instead of EdU signal on mitotic chromosomes. ( A ) HeLa(Mito) cells containing the tet-inducible Kif11–FKBP–3x Flag construct were treated with or without doxycycline (doxy, 5 µg/mL) and then transfected with control (SCR) or Kif11-targeting siRNAs as before. Cells were then infected with luciferase-containing PsV16s at a VGE of 500 with or without rapamycin (200 nM). At 24 hpi, cells were harvested and lysed using OneGlo substrate, with subsequent luciferase activity ascertained with a Tecan Spark plate reader. “100%” infectivity was set for the luciferase activity of infected cells transfected with control (SCR) siRNAs, with or without doxycycline; the luciferase activity (infectivity) of infected cells for subsequent conditions, with or without doxycycline, was charted relative to the respective “100%” infectivity set points of SCR siRNAs. The % infectivity and standard deviation values for SCR siRNA transfection, −/+ rapamycin, and Kif11siRNA transfection, −/+ rapamycin, under “no doxycycline” conditions were 100.0% ± 14.8, 82.9% ± 14.5, 52.7% ± 46.2, and 31.5% ± 31.3, respectively. The % infectivity and standard deviation values for SCR siRNA transfection, −/+ rapamycin, and Kif11siRNA transfection, −/+ rapamycin, under “with doxycycline” conditions were 100.7% ± 9.3, 94.8% ± 15.5, 166.2% ± 37.9, and 64.1% ± 70.5, respectively. Differences of “% Infectivity” amounts in each condition were analyzed using Student’s t -test ( N = 4, with technical triplicates per condition); **, P < 0.01; ****, P < 0.0001. (B and C) Representative images of cells (10× magnification) in the stated conditions (−/+ rapamycin, with SCR or Kif11-siRNA transfected cells) in the ( B ) “no doxycycline” or ( C ) “with doxycycline” conditions were taken immediately before cell harvesting and luciferase activity determination. The white arrows show representative cells and clusters of cells that are in the monoastral phenotype. Brightfield images were acquired with a Leica DMI6000 B microsope using a 10x objective.

Article Snippet: Primary antibodies used for the immunofluorescence studies were as follows: mouse monoclonal antibody (mAb) anti-α-tubulin (Cell Signaling; #3873S), mouse mAb AlexaFluor (AF) 488-conjugated anti-α-tubulin (Cell Signaling; #8058), mouse mAb anti-γ-tubulin for the detection of the MTOC (Sigma-Aldrich; #T6557), rabbit mAb anti-dynein intermediate chain 1 (Abcam; #ab171964); all rabbit mAbs for detection of kinesins such as Kif11 (Cell Signaling; #14404S), Kif18a (Novus Biologicals; #NBP1-85126), and Kif25 (Novus Biologicals; #NBP1-92055).

Techniques: Infection, Luciferase, Activity Assay, Construct, Transfection, Control, Standard Deviation, Cell Harvesting

Journal: Nature Genetics

Article Title: Base editing screens define the genetic landscape of cancer drug resistance mechanisms

doi: 10.1038/s41588-024-01948-8

Figure Lengend Snippet: a , Overview of base editing screens to identify drug resistance variants in cancer cell models. b , Base editor screens in HT-29 cells across 11 cancer genes show depletion of gRNAs targeting essential genes demonstrating base editing activity. Unpaired, two-tailed Student’s t -test comparing NT gRNAs ( n = 114) with gRNAs targeting essential gene splice sites ( n = 632) in CBE and ABE screens. Boxplots represent the median, interquartile range (IQR) and whiskers are the lowest and highest values within 1.5 × IQR. c , Comparison of gRNA z -scores from base editor screens in PC9 ( EGFR -mutant, MYC -dependent) and HT-29 ( BRAF -mutant, MYC -dependent) reveals shared and disparate oncogene dependencies. d , Base editing mutagenesis screens of the driving oncogene, BRAF , in HT-29 cells reveal functional protein domains, sites of post-translational modification and driver variants. Data are the average of two independent experiments. See also Extended Data Fig. . Schematic in a created with BioRender.com .

Article Snippet: Proteins were transferred to a polyvinylidenedifluoride membrane before blotting with the following primary antibodies: EGFR total (1068 epitope, cat. no. 2232, 1:1,000 dilution), p-EGFR (1148 region, cat. no. 4404, 1:1,000 dilution), β-actin (cat. no. 4970, 1:1,000 dilution), p-ERK (cat. no. 9101, 1:1,000 dilution), ERK total (cat. no. 9102, 1:1,000 dilution) (Cell Signaling Technology), EGFR epitope 1020-1046 (cat. no. 610017 BD Biosciences, 1:1,000 dilution).

Techniques: Activity Assay, Two Tailed Test, Comparison, Mutagenesis, Functional Assay, Modification

Journal: Nature Genetics

Article Title: Base editing screens define the genetic landscape of cancer drug resistance mechanisms

doi: 10.1038/s41588-024-01948-8

Figure Lengend Snippet: Replicate correlation for CBE and ABE screens across four cancer cell models; HT-29, H23, PC9 and MHH-ES-1. Pearson correlation coefficient values (r) between independent replicate screens are shown. Low correlation was observed for replicates of PC9 screens with gefitinib, which may relate to a high degree of enrichment of resistant, EGFR T790M base edit harbouring cells.

Article Snippet: Proteins were transferred to a polyvinylidenedifluoride membrane before blotting with the following primary antibodies: EGFR total (1068 epitope, cat. no. 2232, 1:1,000 dilution), p-EGFR (1148 region, cat. no. 4404, 1:1,000 dilution), β-actin (cat. no. 4970, 1:1,000 dilution), p-ERK (cat. no. 9101, 1:1,000 dilution), ERK total (cat. no. 9102, 1:1,000 dilution) (Cell Signaling Technology), EGFR epitope 1020-1046 (cat. no. 610017 BD Biosciences, 1:1,000 dilution).

Techniques:

Journal: Nature Genetics

Article Title: Base editing screens define the genetic landscape of cancer drug resistance mechanisms

doi: 10.1038/s41588-024-01948-8

Figure Lengend Snippet: a) Base editor screens in H23, PC9 and MHH-ES-1 cancer cells targeting 11 cancer genes show depletion of gRNAs targeting essential genes demonstrating base editing activity. Unpaired, two-tailed Student’s t-test comparing non-targeting gRNAs ( n = 114) to gRNAs targeting essential gene splice sites ( n = 632) in CBE and ABE screens. For MHH-ES-1, ABE screens are shown (NT; n = 57; essential-targeting, n = 306). Boxplots represent the median and interquartile range (IQR), and whiskers represent the lowest and highest values within 1.5 x the IQR. b) Number of off-target sites plotted against the z-score for base editing gRNAs. A high number of off-targets for a small number of KRAS UTR -targeting gRNAs is associated with severe gRNA depletion. These were filtered out of downstream analysis. c) Our previously reported whole-genome CRISPR-Cas9 KO screen in HT-29 cells in the presence of dabrafenib (0.1 µM) across three time-points. Volcano plot showing EGFR KO as the top sensitising hit. Data are the average of two independent screens and significance was determined with MAGeCK, with a threshold of p-value < 0.05 and FDR < 0.05. d) TCGA oncoprint (pan-cancer cohort, n = 526) of colorectal adenocarcinomas with alterations in KRAS and BRAF . Mutual exclusivity p-value < 0.001 derived from two-sided Fisher exact test, q-value < 0.001 derived from Benjamini-Hochberg FDR correction procedure for multiple hypothesis testing.

Article Snippet: Proteins were transferred to a polyvinylidenedifluoride membrane before blotting with the following primary antibodies: EGFR total (1068 epitope, cat. no. 2232, 1:1,000 dilution), p-EGFR (1148 region, cat. no. 4404, 1:1,000 dilution), β-actin (cat. no. 4970, 1:1,000 dilution), p-ERK (cat. no. 9101, 1:1,000 dilution), ERK total (cat. no. 9102, 1:1,000 dilution) (Cell Signaling Technology), EGFR epitope 1020-1046 (cat. no. 610017 BD Biosciences, 1:1,000 dilution).

Techniques: Activity Assay, Two Tailed Test, CRISPR, Derivative Assay

Journal: Nature Genetics

Article Title: Base editing screens define the genetic landscape of cancer drug resistance mechanisms

doi: 10.1038/s41588-024-01948-8

Figure Lengend Snippet: a , Variants conferring resistance or sensitivity to the MEK inhibitor, trametinib, in HT-29 cells. Comparison of gRNA z -scores for the control treated arm versus plasmid library, and the drug-treated arm versus plasmid library is shown. b , Variants conferring resistance to the combination of BRAF and EGFR inhibitors, dabrafenib and cetuximab, in HT-29 cells. Comparison of gRNA z -scores for the control treated arm versus plasmid library, and the drug-treated arm versus plasmid library is shown. c , Crystal structure of the complex of EGFR and cetuximab (PDB 1yy9 ) , and MEK1 and trametinib (PDB 7jur ) , highlights canonical drug resistance variants discovered in base editor screens predicted to disrupt drug binding. d , Cell growth of base-edited HT-29 cells harboring canonical and drug-addiction drug-resistance variants. Cells were left untreated or treated with trametinib (3 nM) or the combination of dabrafenib (80 nM) and cetuximab (1 µg ml −1 ), and cell proliferation was monitored using an incucyte. Data represent the mean ± s.d. of biological triplicates and are representative of two independent experiments. e , Western blotting of WT HT-29 ABE cells and cells harboring drug-resistance mutations activating the MAPK signaling pathway. Cells were treated with the combination of dabrafenib (80 nM) and cetuximab (1 µg ml −1 ) or DMSO as a control for 24 h before analysis. f , β-galactosidase staining for senescent cells; β-galactosidase positive senescent foci (blue) are indicated with arrows. HT-29 cells were treated with the combination of dabrafenib (80 nM) and cetuximab (1 µg ml −1 ) or DMSO as a control for 48 h before analysis. Representative images are shown for the drug addiction variant MAP2K1 Y130C. Scale bar, 500 µm. Predicted amino acid editing consequences are labeled for drug resistance screens and genotyped edits are shown in d , e and f . Data are the average of two independent experiments performed on separate days, or representative of two independent experiments ( e and f ). See also Extended Data Figs. and .

Article Snippet: Proteins were transferred to a polyvinylidenedifluoride membrane before blotting with the following primary antibodies: EGFR total (1068 epitope, cat. no. 2232, 1:1,000 dilution), p-EGFR (1148 region, cat. no. 4404, 1:1,000 dilution), β-actin (cat. no. 4970, 1:1,000 dilution), p-ERK (cat. no. 9101, 1:1,000 dilution), ERK total (cat. no. 9102, 1:1,000 dilution) (Cell Signaling Technology), EGFR epitope 1020-1046 (cat. no. 610017 BD Biosciences, 1:1,000 dilution).

Techniques: Comparison, Control, Plasmid Preparation, Binding Assay, Western Blot, Staining, Variant Assay, Labeling

Journal: Nature Genetics

Article Title: Base editing screens define the genetic landscape of cancer drug resistance mechanisms

doi: 10.1038/s41588-024-01948-8

Figure Lengend Snippet: a , Drug resistance variants to the EGFR inhibitor gefitinib, profiled with CBE and ABE base editors in PC9 lung cancer cells. Comparison of gRNA z -scores for the control treated arm versus plasmid library, and the drug-treated arm versus plasmid library is shown. b , Drug resistance variants to the EGFR inhibitor, osimertinib, profiled with CBE and ABE base editors in PC9 lung cancer cells. Comparison of gRNA z -scores for the control treated arm versus plasmid library, and the drug-treated arm versus plasmid library is shown. Data represent the average of two independent screens performed on separate days. c , Prime editing mutagenesis screens of EGFR in the presence and absence of osimertinib. PC9 ∆ MLH1 cells were prime edited for 7 days with doxycycline (1 µg ml −1 ) before growth for 10 days in DMSO (control) or osimertinib (75 nM). Data represent the z -score for each pegRNA derived from the average of two independent screens performed on separate days. Samples were compared with the plasmid library. d , Competition flow cytometry assays in PC9 ∆ MLH1 cells comparing the growth of NT gRNA GFP cells with epegRNA BFP cells harboring different EGFR variants in the presence and absence of osimertinib (75 nM) for 5 days. Data are normalized to day 0 ratios and represent the mean ± s.d. of biological triplicates. Unpaired, two-tailed Student’s t -test comparing with the EGFR C797C synonymous variant control; * P = 0.0003, ** P = 0.0002, *** p < 0.0001. Predicted amino acid editing consequences are labeled for drug resistance variant screens. See also Extended Data Fig. .

Article Snippet: Proteins were transferred to a polyvinylidenedifluoride membrane before blotting with the following primary antibodies: EGFR total (1068 epitope, cat. no. 2232, 1:1,000 dilution), p-EGFR (1148 region, cat. no. 4404, 1:1,000 dilution), β-actin (cat. no. 4970, 1:1,000 dilution), p-ERK (cat. no. 9101, 1:1,000 dilution), ERK total (cat. no. 9102, 1:1,000 dilution) (Cell Signaling Technology), EGFR epitope 1020-1046 (cat. no. 610017 BD Biosciences, 1:1,000 dilution).

Techniques: Comparison, Control, Plasmid Preparation, Mutagenesis, Derivative Assay, Flow Cytometry, Two Tailed Test, Variant Assay, Labeling

Journal: Nature Genetics

Article Title: Base editing screens define the genetic landscape of cancer drug resistance mechanisms

doi: 10.1038/s41588-024-01948-8

Figure Lengend Snippet: a) Western blot for MLH1 verifies KO of MLH1 in PC9 cells. PC9 cells were transfected with a Cas9-GFP plasmid encoding an MLH1 targeting gRNA. FACS of GFP positive single cells gave clonal populations, or a pooled population (“pool”). Cells were expanded before analysis by Western blotting. Actin serves as a loading control. Data are representative of two independent experiments. b) Sanger sequencing of prime editing of EGFR C797S in PC9 cells. PC9-PE MLH1 KO (clone 1 from above), or MLH1 WT PC9-PE cells were infected with a pegRNA encoding the C797S edit, puromycin selected and prime editing was initiated with the addition of doxycycline for 5 days. Control (untreated) or osimertinib selected cells (5 nM) are shown. The EGFR C797 locus was PCR amplified and then analysed with Sanger sequencing. c) Replicate correlation between pegRNA z-scores from EGFR prime editing mutagenesis screens performed in PC9 MLH1 KO PE2 cells. Data are from two independent screens performed on different days. Labelled are predicted mutations in EGFR installed by the pegRNAs. Pearson correlation coefficient values (r) between independent replicate screens are shown. pegRNA, prime editing gRNA.

Article Snippet: Proteins were transferred to a polyvinylidenedifluoride membrane before blotting with the following primary antibodies: EGFR total (1068 epitope, cat. no. 2232, 1:1,000 dilution), p-EGFR (1148 region, cat. no. 4404, 1:1,000 dilution), β-actin (cat. no. 4970, 1:1,000 dilution), p-ERK (cat. no. 9101, 1:1,000 dilution), ERK total (cat. no. 9102, 1:1,000 dilution) (Cell Signaling Technology), EGFR epitope 1020-1046 (cat. no. 610017 BD Biosciences, 1:1,000 dilution).

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Sequencing, Infection, Amplification, Mutagenesis

Journal: Nature Genetics

Article Title: Base editing screens define the genetic landscape of cancer drug resistance mechanisms

doi: 10.1038/s41588-024-01948-8

Figure Lengend Snippet: a) Drug titration experiments in PC9 CBE and ABE cells using Cell-titre Glo to measure cell proliferation in the presence of EGFR inhibitors (cetuximab, erlotinib, lapatinib), or chemotherapy agents (cisplatin, paclitaxel). Data represent the mean ± SD of two independent experiments performed on separate days, each in biological triplicate. b) Sanger sequencing of DNA from WT or base edited PC9 cells harbouring the EGFR-inhibitor sensitising splice variant. CBE editing and ABE editing of a known (GT) splice donor is shown. The position of each gRNA is indicated. c) Sanger sequencing cDNA from WT or base edited PC9 cells harbouring the EGFR-inhibitor sensitising splice variant. WT cells display exon-exon splicing as expected, whereas mutant cells display intron retention by utilising an alternative splice donor in the downstream intron. d) Gating strategy for flow cytometry analysis of EGFR expression on PC9 cells (FITC). Gating was performed on cells, singlets, viable cells, BFP+ cells (gRNA expression).

Article Snippet: Proteins were transferred to a polyvinylidenedifluoride membrane before blotting with the following primary antibodies: EGFR total (1068 epitope, cat. no. 2232, 1:1,000 dilution), p-EGFR (1148 region, cat. no. 4404, 1:1,000 dilution), β-actin (cat. no. 4970, 1:1,000 dilution), p-ERK (cat. no. 9101, 1:1,000 dilution), ERK total (cat. no. 9102, 1:1,000 dilution) (Cell Signaling Technology), EGFR epitope 1020-1046 (cat. no. 610017 BD Biosciences, 1:1,000 dilution).

Techniques: Titration, Sequencing, Variant Assay, Mutagenesis, Flow Cytometry, Expressing

Journal: Nature Genetics

Article Title: Base editing screens define the genetic landscape of cancer drug resistance mechanisms

doi: 10.1038/s41588-024-01948-8

Figure Lengend Snippet: a , Increased gefitinib and osimertinib sensitivity in PC9 cells with EGFR C-terminal truncating mutations. Data represent the mean ± s.e.m. of two independent experiments, each performed in biological triplicate. Two-way ANOVA (analysis of variance) comparing with parental (Par.) response; *** P < 0.0001. CTG; CellTiter-Glo. b , A drug-sensitizing base edit in EGFR causes loss of a splice donor site. The EGFR RNA splice variants are shown by migration of PCR products from cDNA. The larger PCR product in the mutant samples is due to retention of a short region of a downstream intronic sequence after exon 27, where an alternative splice donor is used. EGFR protein after residue 1,091 is not translated due to a frameshift leading to a stop codon. c , Western blotting of drug-sensitizing mutants reveals a C-terminal truncation in EGFR and confirms drug sensitization. PC9 CBE or ABE control cells (NT gRNA) or cells mutant for EGFR were treated with gefitinib (gefit.), osimertinib (osim.) or DMSO vehicle control (ø) for 24 h before analysis. Data are representative of two independent experiments. d , Flow cytometry analysis of EGFR protein surface expression in PC9 cells with WT EGFR or base-edited EGFR. Data are represented as a histogram or quantified as EGFR-FITC mean fluorescence intensity (MFI), and represent the mean of three independent experiments ± s.d. Unpaired, two-tailed Student’s t -test; *** P = 0.0004, ** P = 0.0096, * P = 0.0217. See also Extended Data Fig. .

Article Snippet: Proteins were transferred to a polyvinylidenedifluoride membrane before blotting with the following primary antibodies: EGFR total (1068 epitope, cat. no. 2232, 1:1,000 dilution), p-EGFR (1148 region, cat. no. 4404, 1:1,000 dilution), β-actin (cat. no. 4970, 1:1,000 dilution), p-ERK (cat. no. 9101, 1:1,000 dilution), ERK total (cat. no. 9102, 1:1,000 dilution) (Cell Signaling Technology), EGFR epitope 1020-1046 (cat. no. 610017 BD Biosciences, 1:1,000 dilution).

Techniques: Migration, Mutagenesis, Sequencing, Residue, Western Blot, Control, Flow Cytometry, Expressing, Fluorescence, Two Tailed Test