Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Identification of HSPA8 as an interacting partner of MAB21L2 and an important factor in eye development

doi: 10.1002/dvdy.560

Figure Lengend Snippet: Summary of MAB21L2 interacting proteins independently identified in Y2H assays.

Article Snippet: Yeast Two-Hybrid (Y2H) Screen and Interaction Confirmation A full-length N-terminal tagged human MAB21L2 ( NM_006439 ) wild-type (#EX-V1703-M11, Genecopoeia, Rockville, MD, USA) and a mutant human MAB21L2 -p.(Arg51Gly) (all in a pEZ-M11 vector), previously described 2 , as well as zebrafish mab21l2 were used in Y2H analysis 2 .

Techniques: Ubiquitin Proteomics

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Identification of HSPA8 as an interacting partner of MAB21L2 and an important factor in eye development

doi: 10.1002/dvdy.560

Figure Lengend Snippet: Summary of unique interactions identified for MAB21L2-Arg51Gly in Y2H assays.

Article Snippet: Yeast Two-Hybrid (Y2H) Screen and Interaction Confirmation A full-length N-terminal tagged human MAB21L2 ( NM_006439 ) wild-type (#EX-V1703-M11, Genecopoeia, Rockville, MD, USA) and a mutant human MAB21L2 -p.(Arg51Gly) (all in a pEZ-M11 vector), previously described 2 , as well as zebrafish mab21l2 were used in Y2H analysis 2 .

Techniques: Ubiquitin Proteomics

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Identification of HSPA8 as an interacting partner of MAB21L2 and an important factor in eye development

doi: 10.1002/dvdy.560

Figure Lengend Snippet: A, B. Growth plates for 1-by-1 Y2H interaction assay. pB66ø (empty GAL4 DNA-Binding Domain (DBD) vector) was used as a negative control. MAB21L2-WT (A) or MAB21L2-p.(Arg51Gly) (B) was tested against a zebrafish hspa8 fragment; yeast was grown on DO-2 (-tryptophan, -leucine; selects for presence of both bait and prey) and DO-3 selective media (-tryptophan, -leucine, -histidine; selects for interaction of bait and prey). Note the weakened yeast growth on DO-3 medium for MAB21L2-WT (A), in comparison to MAB21L2-p.(Arg51Gly) (B). C, D. Co-immunoprecipitation assay for human full-length FLAG-tagged MAB21L2-WT or MAB21L2-p.(Arg51Gly) and human full length myc-tagged HSPA8 (C) or HSPA5 (D). Cells were transfected with respective constructs, pcDNA empty plasmid was used as a control. Cell lysates were collected, with a portion reserved for ‘input’, and a portion immunoprecipitated with anti-FLAG antibodies. Western blot analysis with anti-myc antibodies for detection of HSPA8/5 and anti-FLAG antibodies for detection of MAB21L2-WT/p.(Arg51Gly) was performed. Both MAB21L2-WT and MAB21L2-p.(Arg51Gly) co-precipitated with HSPA8 and HSPA5.

Article Snippet: Yeast Two-Hybrid (Y2H) Screen and Interaction Confirmation A full-length N-terminal tagged human MAB21L2 ( NM_006439 ) wild-type (#EX-V1703-M11, Genecopoeia, Rockville, MD, USA) and a mutant human MAB21L2 -p.(Arg51Gly) (all in a pEZ-M11 vector), previously described 2 , as well as zebrafish mab21l2 were used in Y2H analysis 2 .

Techniques: Binding Assay, Plasmid Preparation, Negative Control, Comparison, Co-Immunoprecipitation Assay, Transfection, Construct, Control, Immunoprecipitation, Western Blot

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Identification of HSPA8 as an interacting partner of MAB21L2 and an important factor in eye development

doi: 10.1002/dvdy.560

Figure Lengend Snippet: RNA-scope analysis of hspa5 or hspa8 (green) and mab21l2 (yellow) in 24 (A-A” and K-K”), 48-(B-F” and L-O”) and 96-hpf (G-J” and P-S”) wild-type embryos. (l) lens, (r) retina, (mb) midbrain, (opt) optic tectum, (ba) branchial arches, (th) thalamus, (hth) hypothalamus. White arrows indicate the optic fissure (A-B, K-K”) and ciliary marginal zone (C-I and M-R).

Article Snippet: Yeast Two-Hybrid (Y2H) Screen and Interaction Confirmation A full-length N-terminal tagged human MAB21L2 ( NM_006439 ) wild-type (#EX-V1703-M11, Genecopoeia, Rockville, MD, USA) and a mutant human MAB21L2 -p.(Arg51Gly) (all in a pEZ-M11 vector), previously described 2 , as well as zebrafish mab21l2 were used in Y2H analysis 2 .

Techniques: RNAscope

Journal: Translational Cancer Research

Article Title: An E7-retinoblastoma protein pathway mechanism may account for the higher carcinogenic ability of HPV16 over HPV58 in cervical cancer

doi: 10.21037/tcr-23-1211

Figure Lengend Snippet: HPV16 E6 and E7 demonstrated a stronger ability to promote cell survival and inhibit cell apoptosis than did HPV58 E6 and E7. (A) 293T and U2OS cells were plated in each well and transfected with the HPV16 and HPV58 E6 and E7 plasmids. CCK8 assay was then performed at the time points indicated. (B) At 48 h after transfection with the HPV16 and HPV58 E6 and E7 plasmids. 293T and U2OS cells were collected for evaluation of apoptosis via flow cytometry. Graph showed the percentage of the early apotosis in 293T and U2OS cells. *, P<0.05; ***, P<0.001. HPV, human papillomavirus; NC, negative control; CCK8, Cell Counting Kit 8.

Article Snippet: We used the U2OS (human osteosarcoma cell) and 293T (human embryonic kidney cell, with wild-type P53 ) cell lines (American Type Culture Collection) that did not have HPV E6 or E7 and could be passed down for cultivation in vitro and a zebrafish model.

Techniques: Transfection, CCK-8 Assay, Flow Cytometry, Negative Control, Cell Counting

Journal: Translational Cancer Research

Article Title: An E7-retinoblastoma protein pathway mechanism may account for the higher carcinogenic ability of HPV16 over HPV58 in cervical cancer

doi: 10.21037/tcr-23-1211

Figure Lengend Snippet: HPV16 E6 and E7 demonstrated a stronger ability to promote cell invasion and cell cycle progression than did HPV58 E6 and E7. (A) At 48 h after transfection with the HPV16 and HPV58 E6 and E7 plasmids. 293T and U2OS cells were collected for Matrigel invasion assays. After 12–24 h of culture, the cells penetrating the Matrigel to the bottom of the chamber were fixed with 100% methanol and stained with 0.5% gentian violet crystal solution for 20 min. Representative images are shown (magnification: 200×). The results were plotted as the average number of invasive cells from five randomly selected fields. Graph shows the average number of invasive cells from five randomly selected fields in 293T and U2OS cells. Data are represented as the mean ± SD of three independent experiments. (B) At 48 h after transfection with the HPV16 and HPV58 E6 and E7 plasmids. 293T and U2OS cells were collected for cell cycle assay under flow cytometry. ***, P<0.001. HPV, human papillomavirus; NC, negative control.

Article Snippet: We used the U2OS (human osteosarcoma cell) and 293T (human embryonic kidney cell, with wild-type P53 ) cell lines (American Type Culture Collection) that did not have HPV E6 or E7 and could be passed down for cultivation in vitro and a zebrafish model.

Techniques: Transfection, Staining, Cell Cycle Assay, Flow Cytometry, Negative Control

Journal: Translational Cancer Research

Article Title: An E7-retinoblastoma protein pathway mechanism may account for the higher carcinogenic ability of HPV16 over HPV58 in cervical cancer

doi: 10.21037/tcr-23-1211

Figure Lengend Snippet: Cells with HPV16 E7 overexpression demonstrated greater Rb loss of function while those with HPV58 E7 overexpression demonstrated greater Rb phosphorylation. (A) At 48 h after the transfection of the HPV16 and HPV58 E7 plasmids into 293T and U2OS cells, the protein level of pRb and Rb was analyzed via Western blotting. (B) At 48 h after transfection of HPV16 and HPV58 E7 plasmids into U2OS cells treated with roscovitine (5 µM, 10 µM, 15 µM, 30 µM), the expression of pRb and Rb was analyzed via Western blotting. (C) At 48 h after transfection of HPV16 and HPV58 E7 plasmids into 293T and U2OS cells treated with roscovitine (30 µM), the expression of pRb and Rb was analyzed via Western blotting. GAPDH was used to quantify the relative levels of pRb and Rb expression. HPV, human papillomavirus; NC, negative control.

Article Snippet: We used the U2OS (human osteosarcoma cell) and 293T (human embryonic kidney cell, with wild-type P53 ) cell lines (American Type Culture Collection) that did not have HPV E6 or E7 and could be passed down for cultivation in vitro and a zebrafish model.

Techniques: Over Expression, Phospho-proteomics, Transfection, Western Blot, Expressing, Negative Control

Journal: Translational Cancer Research

Article Title: An E7-retinoblastoma protein pathway mechanism may account for the higher carcinogenic ability of HPV16 over HPV58 in cervical cancer

doi: 10.21037/tcr-23-1211

Figure Lengend Snippet: Roscovitine restored Rb expression in 293T and U2OS cells and decreased cell activity in the zebrafish model. The activity of 293T (A) and U2OS cells (B) was observed after transfection with HPV16 and HPV58 E7 plasmids and treatment with roscovitine (30 µM). DMSO, dimethyl sulfoxide; HPV, human papillomavirus.

Article Snippet: We used the U2OS (human osteosarcoma cell) and 293T (human embryonic kidney cell, with wild-type P53 ) cell lines (American Type Culture Collection) that did not have HPV E6 or E7 and could be passed down for cultivation in vitro and a zebrafish model.

Techniques: Expressing, Activity Assay, Transfection