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Santa Cruz Biotechnology dock7 hdr h

Dock7 Hdr H, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/dock7+hdr+h/DOCK+7+HDR+Plasmid/bio_rxiv__2023__01__03__522657-236-12-15
Average 94 stars, based on 1 article reviews

dock7 hdr h - by Bioz Stars, 2026-09

94/100 stars

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1) Product Images from "Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells"

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

Journal: bioRxiv

doi: 10.1101/2023.01.03.522657

Figure Legend Snippet: ( A ) The Cancer Genome Atlas (TGCA) expression profile for Dock-C family members, Dock6, Dock7, and Dock8, in triple-negative breast cancer and normal tissue. ( B ) Protein expression of Dock7 in a breast cancer cell-line cohort. ( C ) Dock7 mRNA levels across patients with either receptor-positive (BC) or triple-negative breast cancers (TNBC). ( D and E ) Quantification of colonies formed in soft agar suspension for SKRB3 and MCF7, respectively. (Below) Western blot showing Dock7 protein expression after either control or Dock7 KD for each cell line. ( F ) Dock7 expression profile for MDA-MB-231 cells that were used for either (G) soft agar assay or ( H ) survival in serum-free media for 4 days. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in ( C ), ( D - E ), and ( G - H ) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01

Techniques Used: Expressing, Suspension, Western Blot, Control, Soft Agar Assay

( A and B ) Soft agar colony formation assay and survival in serum-free media assays for HeLa cells and A549 cells, respectively, where Dock7 has been knocked down using shRNA. Western blot analysis shows Dock7 protein expression for each cell line. ( C ) Kaplan– Meier survival plot from The Protein Atlas showing a correlation between Dock7 mRNA expression and survival in liver cancer patients. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in ( A - B ) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01, and not significant (n.s.)

Figure Legend Snippet: ( A and B ) Soft agar colony formation assay and survival in serum-free media assays for HeLa cells and A549 cells, respectively, where Dock7 has been knocked down using shRNA. Western blot analysis shows Dock7 protein expression for each cell line. ( C ) Kaplan– Meier survival plot from The Protein Atlas showing a correlation between Dock7 mRNA expression and survival in liver cancer patients. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in ( A - B ) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01, and not significant (n.s.)

Techniques Used: Soft Agar Assay, shRNA, Western Blot, Expressing

( A ) Either Myc-tagged TSC1, Flag-tagged TSC2, or ( B ) Myc-mTOR were transiently expressed in HEK293T cells and isolated with anti-Myc, or anti-Flag beads. Immunoprecipitated endogenous Dock7 protein was separated and analyzed by Western blot. ( C ) HeLa cells were transiently transfected with either WT Dock7 or Rheb for 48 h after which, the cells were starved overnight and collected for analysis. Heregulin treated (1 nmol/L, 30 mins) and mock-transfected cells were used as positive and negative controls, respectively. Western blots are representative of three separate biological replicates.

Figure Legend Snippet: ( A ) Either Myc-tagged TSC1, Flag-tagged TSC2, or ( B ) Myc-mTOR were transiently expressed in HEK293T cells and isolated with anti-Myc, or anti-Flag beads. Immunoprecipitated endogenous Dock7 protein was separated and analyzed by Western blot. ( C ) HeLa cells were transiently transfected with either WT Dock7 or Rheb for 48 h after which, the cells were starved overnight and collected for analysis. Heregulin treated (1 nmol/L, 30 mins) and mock-transfected cells were used as positive and negative controls, respectively. Western blots are representative of three separate biological replicates.

Techniques Used: Isolation, Immunoprecipitation, Western Blot, Transfection

( A ) Cells were either mock-transfected or transiently transfected with plasmids to overexpress HA-tagged WT Rheb or the active forms of Cdc42 and Rac. Heregulin treatment (1 nmol/L for 1 h) was used as a control to confirm mTORC1 signaling stimulation. ( B ) HA-tagged Cdc42 was overexpressed, and different Rac1 siRNAs were used to knock-down Rac protein. ( C ) HA-tagged Rac1 was overexpressed, and different Cdc42 siRNAs were used to knock-down Cdc42 protein. ( D ) Cells were either mock-transfected or transiently transfected with plasmids to overexpress either V5-tagged Dock7 WT or its GEF-defective mutant (GDM). ( E ) V5-tagged Dock7 was transiently overexpressed, while either Cdc42 or Rac was knocked down using siRNA. All experiments were performed in HeLa cells. After the genetic manipulation specified, cells were serum starved overnight (16-20 h) and collected for Western blot analysis. Western blots are representative of three separate biological replicates, and numbers under western blots represent fold difference between phosphorylation states when normalized to endogenous protein levels.

Figure Legend Snippet: ( A ) Cells were either mock-transfected or transiently transfected with plasmids to overexpress HA-tagged WT Rheb or the active forms of Cdc42 and Rac. Heregulin treatment (1 nmol/L for 1 h) was used as a control to confirm mTORC1 signaling stimulation. ( B ) HA-tagged Cdc42 was overexpressed, and different Rac1 siRNAs were used to knock-down Rac protein. ( C ) HA-tagged Rac1 was overexpressed, and different Cdc42 siRNAs were used to knock-down Cdc42 protein. ( D ) Cells were either mock-transfected or transiently transfected with plasmids to overexpress either V5-tagged Dock7 WT or its GEF-defective mutant (GDM). ( E ) V5-tagged Dock7 was transiently overexpressed, while either Cdc42 or Rac was knocked down using siRNA. All experiments were performed in HeLa cells. After the genetic manipulation specified, cells were serum starved overnight (16-20 h) and collected for Western blot analysis. Western blots are representative of three separate biological replicates, and numbers under western blots represent fold difference between phosphorylation states when normalized to endogenous protein levels.

Techniques Used: Transfection, Control, Knockdown, Mutagenesis, Western Blot, Phospho-proteomics

CRISPR-Cas9 with Dock7 gene guiding sequence was expressed in cells and antibiotic resistance was applied. Once cells were selected, Dock7 protein expression was determined using western blotting ( A ) and then these cells were used as follows: ( B and C ) Cells were seeded and allowed to grow in full media or ( B ) in soft agar suspension for two weeks ( C ). Colonies formed in soft agar suspension were counted two weeks after seeding. ( D ) Cells were seeded and allowed to recover for a day before changing media to serum-free media for 24 h. Cells were then either collected (Lane 1 and 4), treated with 100 nM insulin for 1 h (Lane 3 and 6), or media was changed to HBBS to remove amino acids for 1 h (Lane 2 and 5) before being collected and used for Western blot analysis. ( E ) Cells were grown in either serum-free or glutamine-free media for two days before they were trypsinized and counted. ( F ) Cells were grown in either complete media or serum-free conditions for 20-24 h and then treated with EdU (10 μm) for 4 h. Cells were then fixed and EdU incorporation was determined. ( G ) Dock7 KO and WT HeLa cells were seeded, starved for 24 h, then stained with TUNEL and cleaved caspase 3 antibody to determine apoptotic index. DAPI stain was used to normalize for cell number. Western blots are representative of three separate biological replicates, and numbers under western blots represent fold difference between phosphorylation states when normalized to endogenous protein levels. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in (C and E-G) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01, ∗p < 0.1, and not significant (n.s.)

Figure Legend Snippet: CRISPR-Cas9 with Dock7 gene guiding sequence was expressed in cells and antibiotic resistance was applied. Once cells were selected, Dock7 protein expression was determined using western blotting ( A ) and then these cells were used as follows: ( B and C ) Cells were seeded and allowed to grow in full media or ( B ) in soft agar suspension for two weeks ( C ). Colonies formed in soft agar suspension were counted two weeks after seeding. ( D ) Cells were seeded and allowed to recover for a day before changing media to serum-free media for 24 h. Cells were then either collected (Lane 1 and 4), treated with 100 nM insulin for 1 h (Lane 3 and 6), or media was changed to HBBS to remove amino acids for 1 h (Lane 2 and 5) before being collected and used for Western blot analysis. ( E ) Cells were grown in either serum-free or glutamine-free media for two days before they were trypsinized and counted. ( F ) Cells were grown in either complete media or serum-free conditions for 20-24 h and then treated with EdU (10 μm) for 4 h. Cells were then fixed and EdU incorporation was determined. ( G ) Dock7 KO and WT HeLa cells were seeded, starved for 24 h, then stained with TUNEL and cleaved caspase 3 antibody to determine apoptotic index. DAPI stain was used to normalize for cell number. Western blots are representative of three separate biological replicates, and numbers under western blots represent fold difference between phosphorylation states when normalized to endogenous protein levels. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in (C and E-G) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01, ∗p < 0.1, and not significant (n.s.)

Techniques Used: CRISPR, Sequencing, Expressing, Western Blot, Suspension, Staining, TUNEL Assay, Phospho-proteomics

( A ) WT HeLa cells were seeded and culturedfor one day before media was changed to serum-free media containing either vehicle DMSO, Rapamycin (1 nM) or Torin (100 nM). Cells were treated for 20 h, then collected, and lysed for Western blot analysis. ( B ) WT HeLa cells were seeded in soft agar suspension and allowed to recover for a day before treatment began. Cells were then treated with 200 μl of complete media containing either vehicle DMSO, Rapamycin (1 nM), Torin (100 nM), or MK2206 (10 μM) on day 2, and every 3 days subsequently. Colonies were quantified 2 weeks after first drug treatment. ( C ) Either Rictor or Raptor was semi-stably knocked down with shRNA, and then full-length V5-tagged Dock7 protein was overexpressed. After 48 h, cells were starved overnight and collected for Western blot. ( D ) Dock7-V5 containing plasmid was transiently transfected in WT HeLa cells and cells were treated with serum-free media either containing vehicle control (DMSO, 1:100) or Rapamycin (1 nM). Cells were then collected and used for Western blot analysis. ( E ) V5-tagged Dock7 was transiently overexpressed in HeLa cells, and Rheb was knocked down using siRNA the next day. After 48 h, cells were starved overnight and collected for Western blot analysis. ( F ) S6 Kinase was knocked down using siRNA, and cells were seeded in soft agar suspension. Colonies formed were counted two weeks after. S6K protein levels were determined using western blotting. Western blots are representative of three separate biological replicates, and numbers under western blots represent fold difference between phosphorylation states normalized to endogenous protein levels. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in ( B and F ) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01.

Figure Legend Snippet: ( A ) WT HeLa cells were seeded and culturedfor one day before media was changed to serum-free media containing either vehicle DMSO, Rapamycin (1 nM) or Torin (100 nM). Cells were treated for 20 h, then collected, and lysed for Western blot analysis. ( B ) WT HeLa cells were seeded in soft agar suspension and allowed to recover for a day before treatment began. Cells were then treated with 200 μl of complete media containing either vehicle DMSO, Rapamycin (1 nM), Torin (100 nM), or MK2206 (10 μM) on day 2, and every 3 days subsequently. Colonies were quantified 2 weeks after first drug treatment. ( C ) Either Rictor or Raptor was semi-stably knocked down with shRNA, and then full-length V5-tagged Dock7 protein was overexpressed. After 48 h, cells were starved overnight and collected for Western blot. ( D ) Dock7-V5 containing plasmid was transiently transfected in WT HeLa cells and cells were treated with serum-free media either containing vehicle control (DMSO, 1:100) or Rapamycin (1 nM). Cells were then collected and used for Western blot analysis. ( E ) V5-tagged Dock7 was transiently overexpressed in HeLa cells, and Rheb was knocked down using siRNA the next day. After 48 h, cells were starved overnight and collected for Western blot analysis. ( F ) S6 Kinase was knocked down using siRNA, and cells were seeded in soft agar suspension. Colonies formed were counted two weeks after. S6K protein levels were determined using western blotting. Western blots are representative of three separate biological replicates, and numbers under western blots represent fold difference between phosphorylation states normalized to endogenous protein levels. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in ( B and F ) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01.

Techniques Used: Western Blot, Suspension, Stable Transfection, shRNA, Plasmid Preparation, Transfection, Control, Phospho-proteomics

( A ) Flag-tagged AKT was transiently overexpressed in HEK293T cells semi-stably expressing either V5-DHR1L or V5-DHR2. Cells were then either treated with vehicle control (DMSO, 1:1000) or with MK2206 (10 μM) for 16 h before being collected and lysed. Immunoprecipitation was performed using anti-V5 beads and complexes were resolved on an SDS-PAGE gel for Western analysis. ( B ) HEK293T cells semi-stably expressing either V5-DHR1L or V5-DHR2 were grown in full-serum or serum starved for 16 h, collected, lysed then used for immunoprecipitation and western blot analysis as described in ( A ). Proximity Ligation Assays (PLA) were performed to determine the number of direct interactions between endogenous Dock7 and either ( C ) endogenous AKT or ( D ) phospho-AKT in HeLa cells, respectively. ( E ) Cirspr-Cas9 Dock7 KO and WT HeLa cells were seeded and allowed to recover for 24 h. Media was then changed to either full serum media or serum-free media for 24 h. Serum-starved cells were then treated with either vehicle control DMSO (10 µl), Okadaic Acid (10 nM), or Calyculin A (50 nM) for 1 h before being collected for Western blot analysis. ( F ) Proximity ligation assay was performed on Cirspr-Cas9 Dock7 KO and WT HeLa cells to measure the number of complexes formed between AKT and PHLPP in the presence and absence of Dock7.

Figure Legend Snippet: ( A ) Flag-tagged AKT was transiently overexpressed in HEK293T cells semi-stably expressing either V5-DHR1L or V5-DHR2. Cells were then either treated with vehicle control (DMSO, 1:1000) or with MK2206 (10 μM) for 16 h before being collected and lysed. Immunoprecipitation was performed using anti-V5 beads and complexes were resolved on an SDS-PAGE gel for Western analysis. ( B ) HEK293T cells semi-stably expressing either V5-DHR1L or V5-DHR2 were grown in full-serum or serum starved for 16 h, collected, lysed then used for immunoprecipitation and western blot analysis as described in ( A ). Proximity Ligation Assays (PLA) were performed to determine the number of direct interactions between endogenous Dock7 and either ( C ) endogenous AKT or ( D ) phospho-AKT in HeLa cells, respectively. ( E ) Cirspr-Cas9 Dock7 KO and WT HeLa cells were seeded and allowed to recover for 24 h. Media was then changed to either full serum media or serum-free media for 24 h. Serum-starved cells were then treated with either vehicle control DMSO (10 µl), Okadaic Acid (10 nM), or Calyculin A (50 nM) for 1 h before being collected for Western blot analysis. ( F ) Proximity ligation assay was performed on Cirspr-Cas9 Dock7 KO and WT HeLa cells to measure the number of complexes formed between AKT and PHLPP in the presence and absence of Dock7.

Techniques Used: Stable Transfection, Expressing, Control, Immunoprecipitation, SDS Page, Western Blot, Ligation, Proximity Ligation Assay

( A and B ) Dock7 was semi-stably knocked down using shRNA and either an empty vector or vectors containing the limit DHR domains were expressed using lentiviral system. Cells were then seeded in either ( A ) soft agar suspension and counted two weeks later or ( B ) in 100 mm plates for protein analysis using western blotting. ( C ) Lentiviral transduction system was used to overexpress the specified domains and their mutants in HeLa WT cells. Cells expressing each plasmid were selected with antibiotics for 3-5 days, seeded, and allowed to recover for 24 h. Media was changed to serum-free media and cells were collected after 24 h for Western blot analysis. ( D ) Semi-stable HeLa cells overexpressing either an empty vector, DHR1L, or its C2 mutant were seeded, allowed to recover, and then allowed to grow for four days in serum-free conditions. Cells were then trypsin-treated and counted. ( E - G ) Dock7 was knocked down using shRNA in MDA-MB-231 cells, and then the specified constructs were overexpressed. Cells were then either grown in complete media or starved for 20-24 h. Proximity ligation assays (PLA) were used determine the number of interactions between endogenous AKT or phospho-AKT and each Dock7 construct. Western blots are representative of three separate biological replicates. Survival assays were performed in triplicates. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in (DG) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01, ∗p < 0.1, and not significant (n.s.)

Figure Legend Snippet: ( A and B ) Dock7 was semi-stably knocked down using shRNA and either an empty vector or vectors containing the limit DHR domains were expressed using lentiviral system. Cells were then seeded in either ( A ) soft agar suspension and counted two weeks later or ( B ) in 100 mm plates for protein analysis using western blotting. ( C ) Lentiviral transduction system was used to overexpress the specified domains and their mutants in HeLa WT cells. Cells expressing each plasmid were selected with antibiotics for 3-5 days, seeded, and allowed to recover for 24 h. Media was changed to serum-free media and cells were collected after 24 h for Western blot analysis. ( D ) Semi-stable HeLa cells overexpressing either an empty vector, DHR1L, or its C2 mutant were seeded, allowed to recover, and then allowed to grow for four days in serum-free conditions. Cells were then trypsin-treated and counted. ( E - G ) Dock7 was knocked down using shRNA in MDA-MB-231 cells, and then the specified constructs were overexpressed. Cells were then either grown in complete media or starved for 20-24 h. Proximity ligation assays (PLA) were used determine the number of interactions between endogenous AKT or phospho-AKT and each Dock7 construct. Western blots are representative of three separate biological replicates. Survival assays were performed in triplicates. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in (DG) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01, ∗p < 0.1, and not significant (n.s.)

Techniques Used: Stable Transfection, shRNA, Plasmid Preparation, Suspension, Western Blot, Transduction, Expressing, Mutagenesis, Construct, Ligation

( A ) In normal tissues, chronic stress will promote cell death. ( B ) When Dock7 is overexpressed in cancer cells and cells are challenged with stress, Dock7 will be able to maintain AKT phosphorylated to inhibit apoptosis and promote survival. Created with BioRender.com

Figure Legend Snippet: ( A ) In normal tissues, chronic stress will promote cell death. ( B ) When Dock7 is overexpressed in cancer cells and cells are challenged with stress, Dock7 will be able to maintain AKT phosphorylated to inhibit apoptosis and promote survival. Created with BioRender.com

Techniques Used:

CRISPR:

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells
Article Snippet: .. A solution of 1 μg of CRISPR (Santa Cruz Biotechnology, sc-404461) and DOCK7 HDR (h) (Santa Cruz Biotechnology, sc-404461-HDR) plasmid DNA was added to 150 μl Plasmid Transfection Medium (Santa Cruz Biotechnology, sc-108062) was prepared and mixed. .. A separate solution of 5 μl of UltraCruz Transfection Reagent (Santa Cruz Biotechnology, sc-395739) in 150 μl Plasmid Transfection Medium was also prepared and mixed, then both solutions were incubated for 5 min.

Plasmid Preparation:

Transfection:

Image Search Results

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) The Cancer Genome Atlas (TGCA) expression profile for Dock-C family members, Dock6, Dock7, and Dock8, in triple-negative breast cancer and normal tissue. ( B ) Protein expression of Dock7 in a breast cancer cell-line cohort. ( C ) Dock7 mRNA levels across patients with either receptor-positive (BC) or triple-negative breast cancers (TNBC). ( D and E ) Quantification of colonies formed in soft agar suspension for SKRB3 and MCF7, respectively. (Below) Western blot showing Dock7 protein expression after either control or Dock7 KD for each cell line. ( F ) Dock7 expression profile for MDA-MB-231 cells that were used for either (G) soft agar assay or ( H ) survival in serum-free media for 4 days. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in ( C ), ( D - E ), and ( G - H ) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01

Article Snippet: A solution of 1 μg of CRISPR (Santa Cruz Biotechnology, sc-404461) and DOCK7 HDR (h) (Santa Cruz Biotechnology, sc-404461-HDR) plasmid DNA was added to 150 μl Plasmid Transfection Medium (Santa Cruz Biotechnology, sc-108062) was prepared and mixed.

Techniques: Expressing, Suspension, Western Blot, Control, Soft Agar Assay

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A and B ) Soft agar colony formation assay and survival in serum-free media assays for HeLa cells and A549 cells, respectively, where Dock7 has been knocked down using shRNA. Western blot analysis shows Dock7 protein expression for each cell line. ( C ) Kaplan– Meier survival plot from The Protein Atlas showing a correlation between Dock7 mRNA expression and survival in liver cancer patients. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in ( A - B ) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01, and not significant (n.s.)

Techniques: Soft Agar Assay, shRNA, Western Blot, Expressing

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) Either Myc-tagged TSC1, Flag-tagged TSC2, or ( B ) Myc-mTOR were transiently expressed in HEK293T cells and isolated with anti-Myc, or anti-Flag beads. Immunoprecipitated endogenous Dock7 protein was separated and analyzed by Western blot. ( C ) HeLa cells were transiently transfected with either WT Dock7 or Rheb for 48 h after which, the cells were starved overnight and collected for analysis. Heregulin treated (1 nmol/L, 30 mins) and mock-transfected cells were used as positive and negative controls, respectively. Western blots are representative of three separate biological replicates.

Techniques: Isolation, Immunoprecipitation, Western Blot, Transfection

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) Cells were either mock-transfected or transiently transfected with plasmids to overexpress HA-tagged WT Rheb or the active forms of Cdc42 and Rac. Heregulin treatment (1 nmol/L for 1 h) was used as a control to confirm mTORC1 signaling stimulation. ( B ) HA-tagged Cdc42 was overexpressed, and different Rac1 siRNAs were used to knock-down Rac protein. ( C ) HA-tagged Rac1 was overexpressed, and different Cdc42 siRNAs were used to knock-down Cdc42 protein. ( D ) Cells were either mock-transfected or transiently transfected with plasmids to overexpress either V5-tagged Dock7 WT or its GEF-defective mutant (GDM). ( E ) V5-tagged Dock7 was transiently overexpressed, while either Cdc42 or Rac was knocked down using siRNA. All experiments were performed in HeLa cells. After the genetic manipulation specified, cells were serum starved overnight (16-20 h) and collected for Western blot analysis. Western blots are representative of three separate biological replicates, and numbers under western blots represent fold difference between phosphorylation states when normalized to endogenous protein levels.

Techniques: Transfection, Control, Knockdown, Mutagenesis, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: CRISPR-Cas9 with Dock7 gene guiding sequence was expressed in cells and antibiotic resistance was applied. Once cells were selected, Dock7 protein expression was determined using western blotting ( A ) and then these cells were used as follows: ( B and C ) Cells were seeded and allowed to grow in full media or ( B ) in soft agar suspension for two weeks ( C ). Colonies formed in soft agar suspension were counted two weeks after seeding. ( D ) Cells were seeded and allowed to recover for a day before changing media to serum-free media for 24 h. Cells were then either collected (Lane 1 and 4), treated with 100 nM insulin for 1 h (Lane 3 and 6), or media was changed to HBBS to remove amino acids for 1 h (Lane 2 and 5) before being collected and used for Western blot analysis. ( E ) Cells were grown in either serum-free or glutamine-free media for two days before they were trypsinized and counted. ( F ) Cells were grown in either complete media or serum-free conditions for 20-24 h and then treated with EdU (10 μm) for 4 h. Cells were then fixed and EdU incorporation was determined. ( G ) Dock7 KO and WT HeLa cells were seeded, starved for 24 h, then stained with TUNEL and cleaved caspase 3 antibody to determine apoptotic index. DAPI stain was used to normalize for cell number. Western blots are representative of three separate biological replicates, and numbers under western blots represent fold difference between phosphorylation states when normalized to endogenous protein levels. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in (C and E-G) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01, ∗p < 0.1, and not significant (n.s.)

Techniques: CRISPR, Sequencing, Expressing, Western Blot, Suspension, Staining, TUNEL Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) WT HeLa cells were seeded and culturedfor one day before media was changed to serum-free media containing either vehicle DMSO, Rapamycin (1 nM) or Torin (100 nM). Cells were treated for 20 h, then collected, and lysed for Western blot analysis. ( B ) WT HeLa cells were seeded in soft agar suspension and allowed to recover for a day before treatment began. Cells were then treated with 200 μl of complete media containing either vehicle DMSO, Rapamycin (1 nM), Torin (100 nM), or MK2206 (10 μM) on day 2, and every 3 days subsequently. Colonies were quantified 2 weeks after first drug treatment. ( C ) Either Rictor or Raptor was semi-stably knocked down with shRNA, and then full-length V5-tagged Dock7 protein was overexpressed. After 48 h, cells were starved overnight and collected for Western blot. ( D ) Dock7-V5 containing plasmid was transiently transfected in WT HeLa cells and cells were treated with serum-free media either containing vehicle control (DMSO, 1:100) or Rapamycin (1 nM). Cells were then collected and used for Western blot analysis. ( E ) V5-tagged Dock7 was transiently overexpressed in HeLa cells, and Rheb was knocked down using siRNA the next day. After 48 h, cells were starved overnight and collected for Western blot analysis. ( F ) S6 Kinase was knocked down using siRNA, and cells were seeded in soft agar suspension. Colonies formed were counted two weeks after. S6K protein levels were determined using western blotting. Western blots are representative of three separate biological replicates, and numbers under western blots represent fold difference between phosphorylation states normalized to endogenous protein levels. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in ( B and F ) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01.

Techniques: Western Blot, Suspension, Stable Transfection, shRNA, Plasmid Preparation, Transfection, Control, Phospho-proteomics

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) Flag-tagged AKT was transiently overexpressed in HEK293T cells semi-stably expressing either V5-DHR1L or V5-DHR2. Cells were then either treated with vehicle control (DMSO, 1:1000) or with MK2206 (10 μM) for 16 h before being collected and lysed. Immunoprecipitation was performed using anti-V5 beads and complexes were resolved on an SDS-PAGE gel for Western analysis. ( B ) HEK293T cells semi-stably expressing either V5-DHR1L or V5-DHR2 were grown in full-serum or serum starved for 16 h, collected, lysed then used for immunoprecipitation and western blot analysis as described in ( A ). Proximity Ligation Assays (PLA) were performed to determine the number of direct interactions between endogenous Dock7 and either ( C ) endogenous AKT or ( D ) phospho-AKT in HeLa cells, respectively. ( E ) Cirspr-Cas9 Dock7 KO and WT HeLa cells were seeded and allowed to recover for 24 h. Media was then changed to either full serum media or serum-free media for 24 h. Serum-starved cells were then treated with either vehicle control DMSO (10 µl), Okadaic Acid (10 nM), or Calyculin A (50 nM) for 1 h before being collected for Western blot analysis. ( F ) Proximity ligation assay was performed on Cirspr-Cas9 Dock7 KO and WT HeLa cells to measure the number of complexes formed between AKT and PHLPP in the presence and absence of Dock7.

Techniques: Stable Transfection, Expressing, Control, Immunoprecipitation, SDS Page, Western Blot, Ligation, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A and B ) Dock7 was semi-stably knocked down using shRNA and either an empty vector or vectors containing the limit DHR domains were expressed using lentiviral system. Cells were then seeded in either ( A ) soft agar suspension and counted two weeks later or ( B ) in 100 mm plates for protein analysis using western blotting. ( C ) Lentiviral transduction system was used to overexpress the specified domains and their mutants in HeLa WT cells. Cells expressing each plasmid were selected with antibiotics for 3-5 days, seeded, and allowed to recover for 24 h. Media was changed to serum-free media and cells were collected after 24 h for Western blot analysis. ( D ) Semi-stable HeLa cells overexpressing either an empty vector, DHR1L, or its C2 mutant were seeded, allowed to recover, and then allowed to grow for four days in serum-free conditions. Cells were then trypsin-treated and counted. ( E - G ) Dock7 was knocked down using shRNA in MDA-MB-231 cells, and then the specified constructs were overexpressed. Cells were then either grown in complete media or starved for 20-24 h. Proximity ligation assays (PLA) were used determine the number of interactions between endogenous AKT or phospho-AKT and each Dock7 construct. Western blots are representative of three separate biological replicates. Survival assays were performed in triplicates. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in (DG) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01, ∗p < 0.1, and not significant (n.s.)

Techniques: Stable Transfection, shRNA, Plasmid Preparation, Suspension, Western Blot, Transduction, Expressing, Mutagenesis, Construct, Ligation

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) In normal tissues, chronic stress will promote cell death. ( B ) When Dock7 is overexpressed in cancer cells and cells are challenged with stress, Dock7 will be able to maintain AKT phosphorylated to inhibit apoptosis and promote survival. Created with BioRender.com

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dock7 hdr h (Santa Cruz Biotechnology)

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