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Thermo Fisher anti-wave1 pa5-78273

siRNA-mediated knockdown of <t>WAVE1</t> and WAVE2 inhibits HPV16 infection in HeLa cells. On day 0, HeLa cells were seeded and transfected with siRNA in a 6-well microplate. On day 2, cells were collected and seeded onto a 24-well microplate to establish technical replicates. On day 3, cells were infected with HPV16 PsVs (TCID 30 ) containing a GFP reporter plasmid for 48 h. Protein expression for ( A ) WASP, ( C ) WAVE1, ( E ) WAVE2 or ( G ) WAVE1 and WAVE2 was measured via Western blotting on day 5. NC was the negative control siRNA used in this study, while S1, S2, and S3 refer to each of three separate siRNAs used to target the indicated proteins. For panels ( G , H ), S2 targeting WAVE1 and S3 targeting WAVE2 were employed to achieve knockdown of both proteins. Half volumes of each siRNA were combined for transfection so that the final concentration of siRNA in each experiment remained consistent. The percentage of HPV16 infected cells was also determined on day 5 (48 h post-infection) via flow cytometry for the knockdowns of ( B ) WASP, ( D ) WAVE1, ( F ) WAVE2, or ( H ) both WAVE1 and WAVE2. Each bar represents three biological replicates comprising technical triplicates and shows the mean %GFP+ cells ± standard deviation ( n = 3, normalized to WT). One-way ANOVA with Dunnett’s multiple comparison test was used to statistically determine significance (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

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1) Product Images from "Human Papillomavirus Type 16 Stimulates WAVE1- and WAVE2-Dependent Actin Protrusions for Endocytic Entry"

Article Title: Human Papillomavirus Type 16 Stimulates WAVE1- and WAVE2-Dependent Actin Protrusions for Endocytic Entry

Journal: Viruses

doi: 10.3390/v17040542

siRNA-mediated knockdown of WAVE1 and WAVE2 inhibits HPV16 infection in HeLa cells. On day 0, HeLa cells were seeded and transfected with siRNA in a 6-well microplate. On day 2, cells were collected and seeded onto a 24-well microplate to establish technical replicates. On day 3, cells were infected with HPV16 PsVs (TCID 30 ) containing a GFP reporter plasmid for 48 h. Protein expression for ( A ) WASP, ( C ) WAVE1, ( E ) WAVE2 or ( G ) WAVE1 and WAVE2 was measured via Western blotting on day 5. NC was the negative control siRNA used in this study, while S1, S2, and S3 refer to each of three separate siRNAs used to target the indicated proteins. For panels ( G , H ), S2 targeting WAVE1 and S3 targeting WAVE2 were employed to achieve knockdown of both proteins. Half volumes of each siRNA were combined for transfection so that the final concentration of siRNA in each experiment remained consistent. The percentage of HPV16 infected cells was also determined on day 5 (48 h post-infection) via flow cytometry for the knockdowns of ( B ) WASP, ( D ) WAVE1, ( F ) WAVE2, or ( H ) both WAVE1 and WAVE2. Each bar represents three biological replicates comprising technical triplicates and shows the mean %GFP+ cells ± standard deviation ( n = 3, normalized to WT). One-way ANOVA with Dunnett’s multiple comparison test was used to statistically determine significance (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure Legend Snippet: siRNA-mediated knockdown of WAVE1 and WAVE2 inhibits HPV16 infection in HeLa cells. On day 0, HeLa cells were seeded and transfected with siRNA in a 6-well microplate. On day 2, cells were collected and seeded onto a 24-well microplate to establish technical replicates. On day 3, cells were infected with HPV16 PsVs (TCID 30 ) containing a GFP reporter plasmid for 48 h. Protein expression for ( A ) WASP, ( C ) WAVE1, ( E ) WAVE2 or ( G ) WAVE1 and WAVE2 was measured via Western blotting on day 5. NC was the negative control siRNA used in this study, while S1, S2, and S3 refer to each of three separate siRNAs used to target the indicated proteins. For panels ( G , H ), S2 targeting WAVE1 and S3 targeting WAVE2 were employed to achieve knockdown of both proteins. Half volumes of each siRNA were combined for transfection so that the final concentration of siRNA in each experiment remained consistent. The percentage of HPV16 infected cells was also determined on day 5 (48 h post-infection) via flow cytometry for the knockdowns of ( B ) WASP, ( D ) WAVE1, ( F ) WAVE2, or ( H ) both WAVE1 and WAVE2. Each bar represents three biological replicates comprising technical triplicates and shows the mean %GFP+ cells ± standard deviation ( n = 3, normalized to WT). One-way ANOVA with Dunnett’s multiple comparison test was used to statistically determine significance (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Techniques Used: Knockdown, Infection, Transfection, Plasmid Preparation, Expressing, Western Blot, Negative Control, Concentration Assay, Flow Cytometry, Standard Deviation, Comparison

WAVE1 knockout (W1KO), W2KO, and W1/W2KO alters cellular morphology, but not proliferation, and inhibits HPV16 infection in multiple cell lines. ( A ) WAVE1 (W1) WAVE2 (W2) or both (W1-W2KO) proteins were knocked out in wild-type (WT) HeLa cells via CRISPR/Cas9 and confirmed by Western blotting. ( B ) Representative phase-contrast images of WT, W1KO, W2KO, and W1–W2KO HeLa cells were taken on the FloID Cell Imaging Station (20× magnification, scale bar = 50 µm). Black arrows indicate narrow protrusions, while white arrows indicate lamellipodial protrusions. ( C ) W1KO, W2KO, and W1/W2KO HeLa cells were seeded in equal amounts, grown for 48 h, and then analyzed for differences in DNA quantity via CyQUANT cell proliferation assay (Thermo Fisher) compared to WT. WT, W1KO, W2KO, and W1/W2KO ( D ) HeLa or ( E ) B16-F1 cells were treated with HPV16 PsVs (TCID 30 ) containing a GFP reporter plasmid. The percentage of infected cells (based on GFP reporter gene expression) was measured at 48 h post-infection via flow cytometry. Background from mock-infected cells was subtracted. For HeLa cells, at least 2 independent clones of each knockout were screened for consistent inhibition of HPV16 infection. Each bar represents three biological repeats comprising technical triplicates and shows DNA quantification over 48 h for panel ( C ) or the mean %GFP+ cells ± standard deviation ( n = 3, normalized to WT) for panels ( D , E ). One-way ANOVA with Dunnett’s multiple comparison test was used to statistically determine significance (ns = not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure Legend Snippet: WAVE1 knockout (W1KO), W2KO, and W1/W2KO alters cellular morphology, but not proliferation, and inhibits HPV16 infection in multiple cell lines. ( A ) WAVE1 (W1) WAVE2 (W2) or both (W1-W2KO) proteins were knocked out in wild-type (WT) HeLa cells via CRISPR/Cas9 and confirmed by Western blotting. ( B ) Representative phase-contrast images of WT, W1KO, W2KO, and W1–W2KO HeLa cells were taken on the FloID Cell Imaging Station (20× magnification, scale bar = 50 µm). Black arrows indicate narrow protrusions, while white arrows indicate lamellipodial protrusions. ( C ) W1KO, W2KO, and W1/W2KO HeLa cells were seeded in equal amounts, grown for 48 h, and then analyzed for differences in DNA quantity via CyQUANT cell proliferation assay (Thermo Fisher) compared to WT. WT, W1KO, W2KO, and W1/W2KO ( D ) HeLa or ( E ) B16-F1 cells were treated with HPV16 PsVs (TCID 30 ) containing a GFP reporter plasmid. The percentage of infected cells (based on GFP reporter gene expression) was measured at 48 h post-infection via flow cytometry. Background from mock-infected cells was subtracted. For HeLa cells, at least 2 independent clones of each knockout were screened for consistent inhibition of HPV16 infection. Each bar represents three biological repeats comprising technical triplicates and shows DNA quantification over 48 h for panel ( C ) or the mean %GFP+ cells ± standard deviation ( n = 3, normalized to WT) for panels ( D , E ). One-way ANOVA with Dunnett’s multiple comparison test was used to statistically determine significance (ns = not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Techniques Used: Knock-Out, Infection, CRISPR, Western Blot, Imaging, CyQUANT Assay, Proliferation Assay, Plasmid Preparation, Gene Expression, Flow Cytometry, Clone Assay, Inhibition, Standard Deviation, Comparison

HPV infectivity is functionally recovered by WAVE1 or WAVE2 expression in HeLa cells. WT and ( A ) W1KO or ( C ) W2KO cells were transduced with a mammalian gene expression lentiviral control vector or a vector containing either GFP-WAVE1 or GFP-WAVE2, respectively (Vector Builder). Transduced cells received an antibiotic resistance gene and underwent selection. WT, ( B ) W1KO, ( D ) W2KO and cells with WAVE protein expression restored were treated with HPV16 PsVs (TCID 30 ) containing an RFP reporter plasmid. The percentage of infected cells (RFP reporter gene transduction) was measured at 48 h post-infection via flow cytometry. Background from mock infected cells was subtracted. Each bar represents three biological repeats comprising technical triplicates and show the mean %RFP+ cells ± standard deviation ( n = 3, normalized to WT). One-way ANOVA with Dunnett’s multiple comparison test was used to statistically determine significance (ns = not significant, **** p < 0.0001).

Figure Legend Snippet: HPV infectivity is functionally recovered by WAVE1 or WAVE2 expression in HeLa cells. WT and ( A ) W1KO or ( C ) W2KO cells were transduced with a mammalian gene expression lentiviral control vector or a vector containing either GFP-WAVE1 or GFP-WAVE2, respectively (Vector Builder). Transduced cells received an antibiotic resistance gene and underwent selection. WT, ( B ) W1KO, ( D ) W2KO and cells with WAVE protein expression restored were treated with HPV16 PsVs (TCID 30 ) containing an RFP reporter plasmid. The percentage of infected cells (RFP reporter gene transduction) was measured at 48 h post-infection via flow cytometry. Background from mock infected cells was subtracted. Each bar represents three biological repeats comprising technical triplicates and show the mean %RFP+ cells ± standard deviation ( n = 3, normalized to WT). One-way ANOVA with Dunnett’s multiple comparison test was used to statistically determine significance (ns = not significant, **** p < 0.0001).

Techniques Used: Infection, Expressing, Transduction, Gene Expression, Control, Plasmid Preparation, Selection, Flow Cytometry, Standard Deviation, Comparison

HPV16 colocalizes with actin and WAVE proteins at the cellular dorsal surface. WT HeLa cells expressing LifeAct-GFP seeded in chambered microscope slides were first cooled from 37 °C to 4 °C for 0.5 h to inhibit endocytosis prior to the addition of HPV16 L1L2 VLPs (10 ng/1 × 10 6 cells) in ice-cold media for 1 h. Cells were then returned to 37 °C for 10 min prior to fixation with 4% paraformaldehyde for 10 min at room temperature, which was the temperature for subsequent steps. Samples were then permeabilized with 0.1% Triton X-100, blocked with 1% BSA, and immunostained against HPV16 L1 and ( A ) WAVE1 or ( C ) WAVE2. Hoechst 33342 was added during secondary antibody addition as a counterstain. Z-stacked images were generated via laser scanning confocal microscopy. ( A , C ) depict maximum-intensity projections of Z-stacks of images depicting representative cells. The color channels are labeled at the upper left of each image. ( B ) and ( D ) depict the analysis of the spatial relationship between signals. To generate these images, we utilized Imaris 10.1.1 Microscopy Image Analysis Software (Oxford Instruments). Briefly, a “surface” was created for each signal, which is an Imaris segmentation algorithm. Surfaces were generated to provide object–object statistics. Parameters included the smoothing of surface details to 0.2 um with the method of absolute intensity thresholding. Background signal was subtracted through voxel size filtration (voxels smaller than 10 were excluded). Next, colocalization between channels was determined by the colocalization tool. Colocalized voxels (as determined by a Manders coefficient of 1) between surfaces were determined by first thresholding images to include true signals and restrict noise. New channels were then created of colocalization voxels. For both conditions, 3 fields containing 5–15 cells across 3 biological replicates were imaged. Scale = 10 µm.

Figure Legend Snippet: HPV16 colocalizes with actin and WAVE proteins at the cellular dorsal surface. WT HeLa cells expressing LifeAct-GFP seeded in chambered microscope slides were first cooled from 37 °C to 4 °C for 0.5 h to inhibit endocytosis prior to the addition of HPV16 L1L2 VLPs (10 ng/1 × 10 6 cells) in ice-cold media for 1 h. Cells were then returned to 37 °C for 10 min prior to fixation with 4% paraformaldehyde for 10 min at room temperature, which was the temperature for subsequent steps. Samples were then permeabilized with 0.1% Triton X-100, blocked with 1% BSA, and immunostained against HPV16 L1 and ( A ) WAVE1 or ( C ) WAVE2. Hoechst 33342 was added during secondary antibody addition as a counterstain. Z-stacked images were generated via laser scanning confocal microscopy. ( A , C ) depict maximum-intensity projections of Z-stacks of images depicting representative cells. The color channels are labeled at the upper left of each image. ( B ) and ( D ) depict the analysis of the spatial relationship between signals. To generate these images, we utilized Imaris 10.1.1 Microscopy Image Analysis Software (Oxford Instruments). Briefly, a “surface” was created for each signal, which is an Imaris segmentation algorithm. Surfaces were generated to provide object–object statistics. Parameters included the smoothing of surface details to 0.2 um with the method of absolute intensity thresholding. Background signal was subtracted through voxel size filtration (voxels smaller than 10 were excluded). Next, colocalization between channels was determined by the colocalization tool. Colocalized voxels (as determined by a Manders coefficient of 1) between surfaces were determined by first thresholding images to include true signals and restrict noise. New channels were then created of colocalization voxels. For both conditions, 3 fields containing 5–15 cells across 3 biological replicates were imaged. Scale = 10 µm.

Techniques Used: Expressing, Microscopy, Generated, Confocal Microscopy, Labeling, Software, Filtration

Knockout of WAVE1, WAVE2, or both, prevents HPV16 stimulated HeLa cells from expressing dorsal surface actin protrusions. Cells were prepared as described in ; however, cells were not permeabilized during immunostaining. Untreated (top row, − symbol) or HPV16-infected WT, W1KO, or W1/W2KO HeLa cells (10 ng/1 × 10 6 cells) (middle row, + symbol) treated with CellLight Actin-GFP were imaged via laser scanning confocal microscopy to obtain Z-stacks. Z-stacks were then stitched together and rotated to view the XZ-oriented volume. Scale: images 1, 2, 4–7 = 10 µm; image 3 = 8 µm; image 8 = 14 µm. 22 cells were analyzed per condition.

Figure Legend Snippet: Knockout of WAVE1, WAVE2, or both, prevents HPV16 stimulated HeLa cells from expressing dorsal surface actin protrusions. Cells were prepared as described in ; however, cells were not permeabilized during immunostaining. Untreated (top row, − symbol) or HPV16-infected WT, W1KO, or W1/W2KO HeLa cells (10 ng/1 × 10 6 cells) (middle row, + symbol) treated with CellLight Actin-GFP were imaged via laser scanning confocal microscopy to obtain Z-stacks. Z-stacks were then stitched together and rotated to view the XZ-oriented volume. Scale: images 1, 2, 4–7 = 10 µm; image 3 = 8 µm; image 8 = 14 µm. 22 cells were analyzed per condition.

Techniques Used: Knock-Out, Expressing, Immunostaining, Infection, Confocal Microscopy

Knockout of WAVE1, WAVE2, or both results in a significant reduction of dorsal surface actin protrusions. Dorsal surface actin protrusions were quantified using the same method as described in . The graph depicts the average number of protrusions per cell ± standard deviation. Statistics: 2-way ANOVA with comparison of means was used to statistically determine significance, corrected for multiple comparisons using Tukey’s test (ns = not significant, **** p < 0.0001).

Figure Legend Snippet: Knockout of WAVE1, WAVE2, or both results in a significant reduction of dorsal surface actin protrusions. Dorsal surface actin protrusions were quantified using the same method as described in . The graph depicts the average number of protrusions per cell ± standard deviation. Statistics: 2-way ANOVA with comparison of means was used to statistically determine significance, corrected for multiple comparisons using Tukey’s test (ns = not significant, **** p < 0.0001).

Techniques Used: Knock-Out, Standard Deviation, Comparison

Image Search Results

Journal: Viruses

Article Title: Human Papillomavirus Type 16 Stimulates WAVE1- and WAVE2-Dependent Actin Protrusions for Endocytic Entry

doi: 10.3390/v17040542

Figure Lengend Snippet: siRNA-mediated knockdown of WAVE1 and WAVE2 inhibits HPV16 infection in HeLa cells. On day 0, HeLa cells were seeded and transfected with siRNA in a 6-well microplate. On day 2, cells were collected and seeded onto a 24-well microplate to establish technical replicates. On day 3, cells were infected with HPV16 PsVs (TCID 30 ) containing a GFP reporter plasmid for 48 h. Protein expression for ( A ) WASP, ( C ) WAVE1, ( E ) WAVE2 or ( G ) WAVE1 and WAVE2 was measured via Western blotting on day 5. NC was the negative control siRNA used in this study, while S1, S2, and S3 refer to each of three separate siRNAs used to target the indicated proteins. For panels ( G , H ), S2 targeting WAVE1 and S3 targeting WAVE2 were employed to achieve knockdown of both proteins. Half volumes of each siRNA were combined for transfection so that the final concentration of siRNA in each experiment remained consistent. The percentage of HPV16 infected cells was also determined on day 5 (48 h post-infection) via flow cytometry for the knockdowns of ( B ) WASP, ( D ) WAVE1, ( F ) WAVE2, or ( H ) both WAVE1 and WAVE2. Each bar represents three biological replicates comprising technical triplicates and shows the mean %GFP+ cells ± standard deviation ( n = 3, normalized to WT). One-way ANOVA with Dunnett’s multiple comparison test was used to statistically determine significance (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Anti-WAVE1 (PA5-78273), anti-WAVE2 (PA5-60975), anti-ITGβ4 (MA5-17104), anti-SERCA2 (MA3-919), anti-VPS25 (PA5-99005) anti-GAPDH (1D4), Texas Red-X goat anti-rabbit (T6391), Texas Red-X goat anti-mouse (T862), Alexa Fluor 488 goat anti-mouse (A11029), Alexa Fluor 488 goat anti-rabbit (A11034), and Alexa Fluor 680 goat anti-mouse (A21058) antibodies were purchased from ThermoFisher Scientific.

Techniques: Knockdown, Infection, Transfection, Plasmid Preparation, Expressing, Western Blot, Negative Control, Concentration Assay, Flow Cytometry, Standard Deviation, Comparison

Journal: Viruses

Article Title: Human Papillomavirus Type 16 Stimulates WAVE1- and WAVE2-Dependent Actin Protrusions for Endocytic Entry

doi: 10.3390/v17040542

Figure Lengend Snippet: WAVE1 knockout (W1KO), W2KO, and W1/W2KO alters cellular morphology, but not proliferation, and inhibits HPV16 infection in multiple cell lines. ( A ) WAVE1 (W1) WAVE2 (W2) or both (W1-W2KO) proteins were knocked out in wild-type (WT) HeLa cells via CRISPR/Cas9 and confirmed by Western blotting. ( B ) Representative phase-contrast images of WT, W1KO, W2KO, and W1–W2KO HeLa cells were taken on the FloID Cell Imaging Station (20× magnification, scale bar = 50 µm). Black arrows indicate narrow protrusions, while white arrows indicate lamellipodial protrusions. ( C ) W1KO, W2KO, and W1/W2KO HeLa cells were seeded in equal amounts, grown for 48 h, and then analyzed for differences in DNA quantity via CyQUANT cell proliferation assay (Thermo Fisher) compared to WT. WT, W1KO, W2KO, and W1/W2KO ( D ) HeLa or ( E ) B16-F1 cells were treated with HPV16 PsVs (TCID 30 ) containing a GFP reporter plasmid. The percentage of infected cells (based on GFP reporter gene expression) was measured at 48 h post-infection via flow cytometry. Background from mock-infected cells was subtracted. For HeLa cells, at least 2 independent clones of each knockout were screened for consistent inhibition of HPV16 infection. Each bar represents three biological repeats comprising technical triplicates and shows DNA quantification over 48 h for panel ( C ) or the mean %GFP+ cells ± standard deviation ( n = 3, normalized to WT) for panels ( D , E ). One-way ANOVA with Dunnett’s multiple comparison test was used to statistically determine significance (ns = not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Techniques: Knock-Out, Infection, CRISPR, Western Blot, Imaging, CyQUANT Assay, Proliferation Assay, Plasmid Preparation, Gene Expression, Flow Cytometry, Clone Assay, Inhibition, Standard Deviation, Comparison

Journal: Viruses

Article Title: Human Papillomavirus Type 16 Stimulates WAVE1- and WAVE2-Dependent Actin Protrusions for Endocytic Entry

doi: 10.3390/v17040542

Figure Lengend Snippet: HPV infectivity is functionally recovered by WAVE1 or WAVE2 expression in HeLa cells. WT and ( A ) W1KO or ( C ) W2KO cells were transduced with a mammalian gene expression lentiviral control vector or a vector containing either GFP-WAVE1 or GFP-WAVE2, respectively (Vector Builder). Transduced cells received an antibiotic resistance gene and underwent selection. WT, ( B ) W1KO, ( D ) W2KO and cells with WAVE protein expression restored were treated with HPV16 PsVs (TCID 30 ) containing an RFP reporter plasmid. The percentage of infected cells (RFP reporter gene transduction) was measured at 48 h post-infection via flow cytometry. Background from mock infected cells was subtracted. Each bar represents three biological repeats comprising technical triplicates and show the mean %RFP+ cells ± standard deviation ( n = 3, normalized to WT). One-way ANOVA with Dunnett’s multiple comparison test was used to statistically determine significance (ns = not significant, **** p < 0.0001).

Techniques: Infection, Expressing, Transduction, Gene Expression, Control, Plasmid Preparation, Selection, Flow Cytometry, Standard Deviation, Comparison

Journal: Viruses

Article Title: Human Papillomavirus Type 16 Stimulates WAVE1- and WAVE2-Dependent Actin Protrusions for Endocytic Entry

doi: 10.3390/v17040542

Figure Lengend Snippet: HPV16 colocalizes with actin and WAVE proteins at the cellular dorsal surface. WT HeLa cells expressing LifeAct-GFP seeded in chambered microscope slides were first cooled from 37 °C to 4 °C for 0.5 h to inhibit endocytosis prior to the addition of HPV16 L1L2 VLPs (10 ng/1 × 10 6 cells) in ice-cold media for 1 h. Cells were then returned to 37 °C for 10 min prior to fixation with 4% paraformaldehyde for 10 min at room temperature, which was the temperature for subsequent steps. Samples were then permeabilized with 0.1% Triton X-100, blocked with 1% BSA, and immunostained against HPV16 L1 and ( A ) WAVE1 or ( C ) WAVE2. Hoechst 33342 was added during secondary antibody addition as a counterstain. Z-stacked images were generated via laser scanning confocal microscopy. ( A , C ) depict maximum-intensity projections of Z-stacks of images depicting representative cells. The color channels are labeled at the upper left of each image. ( B ) and ( D ) depict the analysis of the spatial relationship between signals. To generate these images, we utilized Imaris 10.1.1 Microscopy Image Analysis Software (Oxford Instruments). Briefly, a “surface” was created for each signal, which is an Imaris segmentation algorithm. Surfaces were generated to provide object–object statistics. Parameters included the smoothing of surface details to 0.2 um with the method of absolute intensity thresholding. Background signal was subtracted through voxel size filtration (voxels smaller than 10 were excluded). Next, colocalization between channels was determined by the colocalization tool. Colocalized voxels (as determined by a Manders coefficient of 1) between surfaces were determined by first thresholding images to include true signals and restrict noise. New channels were then created of colocalization voxels. For both conditions, 3 fields containing 5–15 cells across 3 biological replicates were imaged. Scale = 10 µm.

Techniques: Expressing, Microscopy, Generated, Confocal Microscopy, Labeling, Software, Filtration

Journal: Viruses

Article Title: Human Papillomavirus Type 16 Stimulates WAVE1- and WAVE2-Dependent Actin Protrusions for Endocytic Entry

doi: 10.3390/v17040542

Figure Lengend Snippet: Knockout of WAVE1, WAVE2, or both, prevents HPV16 stimulated HeLa cells from expressing dorsal surface actin protrusions. Cells were prepared as described in ; however, cells were not permeabilized during immunostaining. Untreated (top row, − symbol) or HPV16-infected WT, W1KO, or W1/W2KO HeLa cells (10 ng/1 × 10 6 cells) (middle row, + symbol) treated with CellLight Actin-GFP were imaged via laser scanning confocal microscopy to obtain Z-stacks. Z-stacks were then stitched together and rotated to view the XZ-oriented volume. Scale: images 1, 2, 4–7 = 10 µm; image 3 = 8 µm; image 8 = 14 µm. 22 cells were analyzed per condition.

Techniques: Knock-Out, Expressing, Immunostaining, Infection, Confocal Microscopy

Journal: Viruses

Article Title: Human Papillomavirus Type 16 Stimulates WAVE1- and WAVE2-Dependent Actin Protrusions for Endocytic Entry

doi: 10.3390/v17040542

Figure Lengend Snippet: Knockout of WAVE1, WAVE2, or both results in a significant reduction of dorsal surface actin protrusions. Dorsal surface actin protrusions were quantified using the same method as described in . The graph depicts the average number of protrusions per cell ± standard deviation. Statistics: 2-way ANOVA with comparison of means was used to statistically determine significance, corrected for multiple comparisons using Tukey’s test (ns = not significant, **** p < 0.0001).

Techniques: Knock-Out, Standard Deviation, Comparison

Boc interacts with the WRC (A and D) Boc and tagged WRC constructs were expressed in HEK293T cells as indicated. The lysates were immunoprecipitated with an anti-Boc antibody, and interacting proteins were analyzed through SDS-PAGE and western blot. (B, C, E, F) The relative amount (mean ± SEM) of CYFIP2, NCKAP1, WAVE1, or ABI1 interacting with Boc was calculated by normalizing the amount of corresponding protein in the immunoprecipitate to its amount in the cell lysate and expressed relative to the “Boc + WRC” condition in each experiment. “WRC” refers to the condition in which NCKAP1, CYFIP2, WAVE1, and ABI1 were co-expressed. (B) One-way ANOVA ( p = 0.0001), Tukey’s post hoc test, n = 4 independent experiments. (C, E, F) Unpaired t tests, n = 3 independent experiments for (C) and (F) and n = 4 independent experiments for (E). ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (G) Boc and tagged WRC constructs were expressed in HEK293 cells as indicated. The lysates were immunoprecipitated with an anti-Flag antibody, and interacting proteins were analyzed through SDS-PAGE and western blot. Boc and ABI1-EGFP co-immunoprecipitated with CYFIP2-Flag/NCKAP1-Flag/WAVE1-Flag. See also <xref ref-type=

Figures S1–S3 . " width="100%" height="100%">

Journal: iScience

Article Title: The WAVE regulatory complex interacts with Boc and is required for Shh-mediated axon guidance

doi: 10.1016/j.isci.2024.111333

Figure Lengend Snippet: Boc interacts with the WRC (A and D) Boc and tagged WRC constructs were expressed in HEK293T cells as indicated. The lysates were immunoprecipitated with an anti-Boc antibody, and interacting proteins were analyzed through SDS-PAGE and western blot. (B, C, E, F) The relative amount (mean ± SEM) of CYFIP2, NCKAP1, WAVE1, or ABI1 interacting with Boc was calculated by normalizing the amount of corresponding protein in the immunoprecipitate to its amount in the cell lysate and expressed relative to the “Boc + WRC” condition in each experiment. “WRC” refers to the condition in which NCKAP1, CYFIP2, WAVE1, and ABI1 were co-expressed. (B) One-way ANOVA ( p = 0.0001), Tukey’s post hoc test, n = 4 independent experiments. (C, E, F) Unpaired t tests, n = 3 independent experiments for (C) and (F) and n = 4 independent experiments for (E). ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (G) Boc and tagged WRC constructs were expressed in HEK293 cells as indicated. The lysates were immunoprecipitated with an anti-Flag antibody, and interacting proteins were analyzed through SDS-PAGE and western blot. Boc and ABI1-EGFP co-immunoprecipitated with CYFIP2-Flag/NCKAP1-Flag/WAVE1-Flag. See also Figures S1–S3 .

Article Snippet: NAP1-Flag-WT was subcloned from pFLAG-CMV-2-NAP1 to pCAGGS (in Kpn1 at the multiple cloning site (MCS)) using In-Fusion to generate pCAGGS-NCKAP1-Flag. pcDNA3.1(+)Myc-Cyfip2 (mouse) and pCMV-Tag2B-Nap1 (Flag tagged) (mouse) were gifts from Dr. Jia-Jia Liu. pEGFP-C2-Cyfip1 (mouse) was generated by Steffen et al. pcDNA3-Human WAVE1-Flag was a gift from Dr. Greg Bashaw (generated by Westphal et al. ). pCMV6-Entry-BRK1(HSPC300)-Myc-DDK(Flag) was obtained from Origene (cat# RC200804). pCAGGS-NCKAP1 sm -Flag, the shRNA resistant form of human NCKAP1, was made by subcloning NAP1-Flag-WT from pFLAG-CMV-2-NAP1 to pCAGGS (into the Kpn1 site) to have better expression in commissural neurons, along with making silent mutations in NCKAP1, using In-Fusion Cloning Technology.

Techniques: Construct, Immunoprecipitation, SDS Page, Western Blot

The interaction between Boc and the WRC occurs in live cells and is direct (A–D) The NanoBiT structural complementation reporter system was used to detect interaction between Boc and CYFIP2 in live cells. Boc was fused to the Large BiT (LgBiT) subunit (Boc-LgBiT), and CYFIP2 was fused to the Small BiT (SmBiT) subunit either N-terminally (CYFIP2-SmBiT) or C-terminally (SmBiT-CYFIP2) and expressed in HEK293 or COS7 cells. When the two proteins interact, a luminescent signal is generated in the presence of substrate. (A) Expression of Boc-LgBiT and CYFIP2-SmBit alone or (B) expression of Boc-LgBiT and SmBit-CYFIP2 alone does not generate a luminescent signal. (Left) When Boc-LgBiT is expressed with CYFIP2-SmBiT (A) or SmBit-CYFIP2 (B), they interact to generate a luminescent signal. (Right) Boc-LgBiT also interacts with CYFIP2-SmBiT (A) or SmBit-CYFIP2 (B) in the presence of co-expressed WRC components NCKAP1-Flag, WAVE1-Emerald, ABI1-EGFP, and BRK1-Myc-Flag (“+WRC”). Data are represented as mean ± SEM; error bars representing the SEM are too small to be visible. (Left) Repeated measures one-way ANOVA with Dunnett’s multiple comparison test, (right) paired t test. n = 2–6 independent experiments. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. (C) Boc lacking an intracellular domain (ICD), BocΔICD-LgBiT, has a significantly lower interaction with CYFIP2-SmBiT and (D) SmBiT-CYFIP2 compared to full-length Boc-LgBiT in the presence of the WRC components NCKAP1-Flag, WAVE1-Emerald, ABI1-EGFP, and BRK1-Myc-Flag. Data are represented as mean ± SEM. Paired t test, n = 3–5 independent experiments. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. (E) Schematic of GST-Boc ICD constructs. (F) Coomassie-blue-stained SDS-PAGE gel showing MBP pull-down between purified (MBP) 2 -tagged HSPC300 or WRC and the indicated purified GST-Boc ICD proteins. Only GST-Boc ICD FL and GST-Boc ICD NT are pulled down by (MBP) 2 -WRC.

Journal: iScience

Article Title: The WAVE regulatory complex interacts with Boc and is required for Shh-mediated axon guidance

doi: 10.1016/j.isci.2024.111333

Figure Lengend Snippet: The interaction between Boc and the WRC occurs in live cells and is direct (A–D) The NanoBiT structural complementation reporter system was used to detect interaction between Boc and CYFIP2 in live cells. Boc was fused to the Large BiT (LgBiT) subunit (Boc-LgBiT), and CYFIP2 was fused to the Small BiT (SmBiT) subunit either N-terminally (CYFIP2-SmBiT) or C-terminally (SmBiT-CYFIP2) and expressed in HEK293 or COS7 cells. When the two proteins interact, a luminescent signal is generated in the presence of substrate. (A) Expression of Boc-LgBiT and CYFIP2-SmBit alone or (B) expression of Boc-LgBiT and SmBit-CYFIP2 alone does not generate a luminescent signal. (Left) When Boc-LgBiT is expressed with CYFIP2-SmBiT (A) or SmBit-CYFIP2 (B), they interact to generate a luminescent signal. (Right) Boc-LgBiT also interacts with CYFIP2-SmBiT (A) or SmBit-CYFIP2 (B) in the presence of co-expressed WRC components NCKAP1-Flag, WAVE1-Emerald, ABI1-EGFP, and BRK1-Myc-Flag (“+WRC”). Data are represented as mean ± SEM; error bars representing the SEM are too small to be visible. (Left) Repeated measures one-way ANOVA with Dunnett’s multiple comparison test, (right) paired t test. n = 2–6 independent experiments. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. (C) Boc lacking an intracellular domain (ICD), BocΔICD-LgBiT, has a significantly lower interaction with CYFIP2-SmBiT and (D) SmBiT-CYFIP2 compared to full-length Boc-LgBiT in the presence of the WRC components NCKAP1-Flag, WAVE1-Emerald, ABI1-EGFP, and BRK1-Myc-Flag. Data are represented as mean ± SEM. Paired t test, n = 3–5 independent experiments. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. (E) Schematic of GST-Boc ICD constructs. (F) Coomassie-blue-stained SDS-PAGE gel showing MBP pull-down between purified (MBP) 2 -tagged HSPC300 or WRC and the indicated purified GST-Boc ICD proteins. Only GST-Boc ICD FL and GST-Boc ICD NT are pulled down by (MBP) 2 -WRC.

Techniques: Generated, Expressing, Comparison, Construct, Staining, SDS Page, Purification

Journal: iScience

Article Title: The WAVE regulatory complex interacts with Boc and is required for Shh-mediated axon guidance

doi: 10.1016/j.isci.2024.111333

Figure Lengend Snippet:

Techniques: Virus, Recombinant, Activity Assay, Protease Inhibitor, Molecular Weight, Concentration Assay, Sequencing, Modification, Affinity Purification, Expressing, shRNA, Generated, Plasmid Preparation, Software, Blocking Assay

On day 0 HeLa cells were seeded and transfected with siRNA in a 6-well microplate. On day 2, cells were collected and seeded onto a 24-well microplate to establish technical replicates. On day 3, cells were infected with HPV16 PsVs (TCID 30 ) containing a GFP reporter plasmid for 48 hours. Protein expression of relevant proteins was measured via Western blotting on day 5 (A, C, E, G). NC is the negative control siRNA used in this study, while S1, S2, and S3 refer to each of three separate siRNAs used to target the indicated proteins. For panels G and H, S2 targeting WAVE1 and S3 targeting WAVE2 were employed to achieve knockdown of both proteins. Half volumes of each siRNA were combined for transfection so that the final concentration of siRNA in each experiment remained consistent. The percentage of HPV16 infected cells was also determined on day 5 (48 hours post infection) via flow cytometry (B, D, F, H). Each bar represents three biological replicates comprised of technical triplicates and show the mean %GFP+ cells ± standard deviation (n=3, normalized to WT). 1-way ANOVA with Dunnett’s multiple comparisons test was used to statistically determine significance (ns=not significant, **p<0.001, ***p<0.0001, ****p<0.0001).

Journal: bioRxiv

Article Title: WAVE1 and WAVE2 facilitate human papillomavirus-driven actin polymerization during cellular entry

doi: 10.1101/2024.10.28.620484

Figure Lengend Snippet: On day 0 HeLa cells were seeded and transfected with siRNA in a 6-well microplate. On day 2, cells were collected and seeded onto a 24-well microplate to establish technical replicates. On day 3, cells were infected with HPV16 PsVs (TCID 30 ) containing a GFP reporter plasmid for 48 hours. Protein expression of relevant proteins was measured via Western blotting on day 5 (A, C, E, G). NC is the negative control siRNA used in this study, while S1, S2, and S3 refer to each of three separate siRNAs used to target the indicated proteins. For panels G and H, S2 targeting WAVE1 and S3 targeting WAVE2 were employed to achieve knockdown of both proteins. Half volumes of each siRNA were combined for transfection so that the final concentration of siRNA in each experiment remained consistent. The percentage of HPV16 infected cells was also determined on day 5 (48 hours post infection) via flow cytometry (B, D, F, H). Each bar represents three biological replicates comprised of technical triplicates and show the mean %GFP+ cells ± standard deviation (n=3, normalized to WT). 1-way ANOVA with Dunnett’s multiple comparisons test was used to statistically determine significance (ns=not significant, **p<0.001, ***p<0.0001, ****p<0.0001).

Article Snippet: Anti-WAVE1 (PA5- 78273), anti-WAVE2 (PA5-60975), anti-ITGβ4 (MA5-17104), anti-GAPDH (1D4), Texas Red-X goat anti-rabbit (T6391), Texas Red-X goat anti-mouse (T862), Alexa Fluor 488 goat anti-mouse (A11029), Alexa Fluor 488 goat anti-rabbit (A11034), and Alexa Fluor 680 goat anti-mouse (A21058) antibodies were purchased from ThermoFisher Scientific.

Techniques: Transfection, Infection, Plasmid Preparation, Expressing, Western Blot, Negative Control, Knockdown, Concentration Assay, Flow Cytometry, Standard Deviation

(A) WAVE1 (W1) WAVE2 (W2) or both (DKO) proteins were knocked out in wild type (WT) HeLa cells via CRISPR/Cas9 and confirmed by Western blotting. (B) Representative phase-contrast images of WT, W1KO, W2KO, and W1/W2KO HeLa cells were taken on the FloID Cell Imaging Station (20x magnification, scale bar = 50μm). (C) W1KO, W2KO, and W1/W2KO HeLa cells were seeded in equal amounts, grown for 48 hours, and then analyzed for differences in DNA quantity via CyQUANT Cell Proliferation Assay (Thermo Fisher) compared to WT. (D and E) WT, W1KO, W2KO, and W1/W2KO HeLa or B16- F1 cells were treated with HPV16 PsVs (TCID 30 ) containing a GFP reporter plasmid. The percentage of infected cells (based on GFP reporter gene expression) was measured at 48 hours post infection via flow cytometry. Background from mock infected cells was subtracted. For HeLa cells, at least 2 independent clones of each knockout were screened for consistent inhibition of HPV16 infection. Each bar represents three biological repeats comprised of technical triplicates and show DNA quantification over 48 hours (Panel C) or the mean %GFP+ cells ± standard deviation (n=3, normalized to WT) (Panels D and E). 1-way ANOVA with Dunnett’s multiple comparisons test was used to statistically determine significance (ns=not significant, **p<0.001, ***p<0.0001, ****p<0.0001).

Journal: bioRxiv

Article Title: WAVE1 and WAVE2 facilitate human papillomavirus-driven actin polymerization during cellular entry

doi: 10.1101/2024.10.28.620484

Figure Lengend Snippet: (A) WAVE1 (W1) WAVE2 (W2) or both (DKO) proteins were knocked out in wild type (WT) HeLa cells via CRISPR/Cas9 and confirmed by Western blotting. (B) Representative phase-contrast images of WT, W1KO, W2KO, and W1/W2KO HeLa cells were taken on the FloID Cell Imaging Station (20x magnification, scale bar = 50μm). (C) W1KO, W2KO, and W1/W2KO HeLa cells were seeded in equal amounts, grown for 48 hours, and then analyzed for differences in DNA quantity via CyQUANT Cell Proliferation Assay (Thermo Fisher) compared to WT. (D and E) WT, W1KO, W2KO, and W1/W2KO HeLa or B16- F1 cells were treated with HPV16 PsVs (TCID 30 ) containing a GFP reporter plasmid. The percentage of infected cells (based on GFP reporter gene expression) was measured at 48 hours post infection via flow cytometry. Background from mock infected cells was subtracted. For HeLa cells, at least 2 independent clones of each knockout were screened for consistent inhibition of HPV16 infection. Each bar represents three biological repeats comprised of technical triplicates and show DNA quantification over 48 hours (Panel C) or the mean %GFP+ cells ± standard deviation (n=3, normalized to WT) (Panels D and E). 1-way ANOVA with Dunnett’s multiple comparisons test was used to statistically determine significance (ns=not significant, **p<0.001, ***p<0.0001, ****p<0.0001).

Techniques: CRISPR, Western Blot, Imaging, CyQUANT Assay, Proliferation Assay, Plasmid Preparation, Infection, Expressing, Flow Cytometry, Clone Assay, Knock-Out, Inhibition, Standard Deviation

(A and C) WT and W1KO or W2KO cells were transduced with a mammalian gene expression lentiviral control vector or a vector containing either GFP-WAVE1 or GFP-WAVE2, respectively (Vector Builder). Transduced cells received an antibiotic resistance gene and underwent selection. (B and D) WT, KO, and cells with WAVE protein expression restored were treated with HPV16 PsVs (TCID 30 ) containing an RFP reporter plasmid. The percentage of infected cells (RFP reporter gene transduction) was measured at 48 hours post infection via flow cytometry. Background from mock infected cells was subtracted. Each bar represents three biological repeats comprised of technical triplicates and show the mean %RFP+ cells ± standard deviation (n=3, normalized to WT). 1-way ANOVA with Dunnett’s multiple comparisons test was used to statistically determine significance (ns=not significant, **p<0.001, ***p<0.0001, ****p<0.0001).

Journal: bioRxiv

Article Title: WAVE1 and WAVE2 facilitate human papillomavirus-driven actin polymerization during cellular entry

doi: 10.1101/2024.10.28.620484

Figure Lengend Snippet: (A and C) WT and W1KO or W2KO cells were transduced with a mammalian gene expression lentiviral control vector or a vector containing either GFP-WAVE1 or GFP-WAVE2, respectively (Vector Builder). Transduced cells received an antibiotic resistance gene and underwent selection. (B and D) WT, KO, and cells with WAVE protein expression restored were treated with HPV16 PsVs (TCID 30 ) containing an RFP reporter plasmid. The percentage of infected cells (RFP reporter gene transduction) was measured at 48 hours post infection via flow cytometry. Background from mock infected cells was subtracted. Each bar represents three biological repeats comprised of technical triplicates and show the mean %RFP+ cells ± standard deviation (n=3, normalized to WT). 1-way ANOVA with Dunnett’s multiple comparisons test was used to statistically determine significance (ns=not significant, **p<0.001, ***p<0.0001, ****p<0.0001).

Techniques: Transduction, Expressing, Control, Plasmid Preparation, Selection, Infection, Flow Cytometry, Standard Deviation

WT HeLa cells expressing LifeAct-GFP seeded in chambered microscope slides were first cooled from 37°C to 4°C for 0.5 h to inhibit endocytosis prior to the addition of HPV16 VLPs (10 ng/1E6 cells) in ice cold media for 1 hour. Cells were then returned to 37°C for 10 minutes prior to fixation with 4% paraformaldehyde for 10 minutes at room temperature, which was the temperature for subsequent steps. Samples were then permeabilized with 0.1% Triton X-100, blocked with 1% BSA, and immunostained against HPV16 L1 and (A) WAVE1 or (C) WAVE2. Hoescht 33342 was added during secondary antibody addition as a counterstain. Z-stacked images were generated via laser scanning confocal microscopy. (A and C) maximum intensity projections of Z-stacks of images depicting candidate cells. The color channels are labeled at the upper left of each image. (B and D) to analyze the spatial relationship between signals, we utilized Imaris 10.1.1 Microscopy Image Analysis Software (Oxford Instruments). Briefly, a “surface” was created for each signal, which is an Imaris segmentation algorithm. Surfaces were generated to provide object-object statistics. Parameters included the smoothing of surface details to 0.2 um with the method of absolute intensity thresholding. Background signal was subtracted through voxel size filtration (voxels smaller than 10 were excluded). Next, colocalization between channels was determined by the colocalization tool. Colocalized voxels (as determined by a Manders’ coefficient of 1) between surfaces were determined by first thresholding images to include true signals and restrict noise. New channels were then created of colocalization voxels. For both conditions, 3 fields containing 5-15 cells across 3 biological replicates were imaged. Scale = 10 µm.

Journal: bioRxiv

Article Title: WAVE1 and WAVE2 facilitate human papillomavirus-driven actin polymerization during cellular entry

doi: 10.1101/2024.10.28.620484

Figure Lengend Snippet: WT HeLa cells expressing LifeAct-GFP seeded in chambered microscope slides were first cooled from 37°C to 4°C for 0.5 h to inhibit endocytosis prior to the addition of HPV16 VLPs (10 ng/1E6 cells) in ice cold media for 1 hour. Cells were then returned to 37°C for 10 minutes prior to fixation with 4% paraformaldehyde for 10 minutes at room temperature, which was the temperature for subsequent steps. Samples were then permeabilized with 0.1% Triton X-100, blocked with 1% BSA, and immunostained against HPV16 L1 and (A) WAVE1 or (C) WAVE2. Hoescht 33342 was added during secondary antibody addition as a counterstain. Z-stacked images were generated via laser scanning confocal microscopy. (A and C) maximum intensity projections of Z-stacks of images depicting candidate cells. The color channels are labeled at the upper left of each image. (B and D) to analyze the spatial relationship between signals, we utilized Imaris 10.1.1 Microscopy Image Analysis Software (Oxford Instruments). Briefly, a “surface” was created for each signal, which is an Imaris segmentation algorithm. Surfaces were generated to provide object-object statistics. Parameters included the smoothing of surface details to 0.2 um with the method of absolute intensity thresholding. Background signal was subtracted through voxel size filtration (voxels smaller than 10 were excluded). Next, colocalization between channels was determined by the colocalization tool. Colocalized voxels (as determined by a Manders’ coefficient of 1) between surfaces were determined by first thresholding images to include true signals and restrict noise. New channels were then created of colocalization voxels. For both conditions, 3 fields containing 5-15 cells across 3 biological replicates were imaged. Scale = 10 µm.

Techniques: Expressing, Microscopy, Generated, Confocal Microscopy, Labeling, Software, Filtration

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1) Product Images from "Human Papillomavirus Type 16 Stimulates WAVE1- and WAVE2-Dependent Actin Protrusions for Endocytic Entry"

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