Journal: The FASEB Journal

Article Title: PAI‐1 induction during kidney injury promotes fibrotic epithelial dysfunction via deregulation of klotho, p53, and TGF‐β1‐receptor signaling

doi: 10.1096/fj.202002652rr

Figure Lengend Snippet: FIGURE 5 PAI-1 induced p53 upregulation is critical for the subsequent maladaptive response. A, Western blot assessments for total and p-p53 protein levels between CMV-Con and CMV-PAI-1 populations. B-C, Histograms depicting the relative expression of p53 levels (mean ± SD) for three independent studies, n = 3. D-G, Lysates of CMV-PAI-1+Con-shRNA and CMV-PAI-1+p53-shRNA double transductants are immunoblotted for p53 (D, E; P < .001), p21 (D, F; P < .01), p-H3 (D, G; P < .01), fibronectin (H, I; P < .01), collagen-1 (H, J; P < .01). Histograms in (E-G) and (I-J) depict the relative expression (mean ± SD) for indicated proteins from the immunoblots in (D) and (H), shown as biological triplicates for three independent studies (n = 3). K, Confluent monolayers of CMV-PAI-1+Con-shRNA and CMV-PAI-1+p53- shRNA HK2 cultures are serum-starved for 6 days. Phase contrast and crystal violet images are taken on day 0 and day 6 to assess cell monolayer detachment. Scale bar = 400 µm. L, Western blot analysis for FAK and p-ERK1/2 protein levels between CMV-Con and CMV-PAI-1 cultures, with ERK2 serving as a loading control, (n = 3). *P < .05, **P < .01, ***P < .001

Article Snippet: Overnight incubation at 4°C utilized the following primary antibodies; rabbit anti- PAI- 1 (1:3000) and rabbit anti- collagen- 1 (1:5000) as previously described,15 rat anti- klotho (1:5000; Transgenic Inc.- KM2119), rabbit anti- SMAD3 (1:1000; Cell Signaling- 9523), rabbit anti- p- Histone3 (1:1000; Cell Signaling- 9701), rabbit anti- p21 (1:1000; Cell Signaling- 2947), rabbit anti- caspase- 9 (1:1000; Cell Signaling- 9502), rabbit anti- c- caspase- 3 (1:1000; Cell Signaling- 9661), rabbit antiSnail1 (1:1000; Cell Signaling- 38798s), rabbit anti- α- SMA (1:1000; Abcam- ab32575), rabbit anti- phospho- SMAD3 (1:1000; Abcam- ab52903), rabbit anti- fibronectin (1:100,000; Abcam- ab2413), rabbit anti- LOXL2 (1:1000; Abcam- ab96233), rabbit anti- MMP- 9 (1:1000; Abcam- ab38898), rabbit antiMMP- 2 (1:1000; Abcam- ab97779), rabbit anti- VEGF- C (1:1000; Abcam- ab9546), rabbit anti- endothelin- 1 (1:1000; 18201- IBL America, Minneapolis, MN, USA), rabbit antivimentin (1:10,000; Santa Cruz- sc5565), rabbit anti- GAPDH (1:5000; Santa Cruz- sc25778), goat anti- CCN2 (1:500; Santa Cruz- sc14939), rabbit anti- TGF- βRI (1:1000; Santa Cruz- sc9048), rabbit anti- TGF- βRII (1:1000; Santa Cruz- sc220), mouse anti- p53 (1:1000; Santa Cruz- sc126), mouse anti- Ecadherin (1:1000; BD Biosciences- 610181), and rabbit antip- p53 (1:1000; AF1043- R&D systems, Minneapolis, MN, USA).

Techniques: Western Blot, Expressing, shRNA, Control

Journal: The FASEB Journal

Article Title: PAI‐1 induction during kidney injury promotes fibrotic epithelial dysfunction via deregulation of klotho, p53, and TGF‐β1‐receptor signaling

doi: 10.1096/fj.202002652rr

Figure Lengend Snippet: FIGURE 6 Klotho downregulation consequent to PAI-1 induction contributes to epithelial dysfunction. A-C, Immunoblot analysis of CMV- Con and CMV-PAI-1 cell lysates for PAI-1 (A, B; P < .001) and klotho (A, C; P < .01) expression. Histograms in (B-C) represent the relative expression of the indicated proteins as (mean ± SD) for three independent studies (n = 3). D-I, Protein extracts of CMV-PAI-1+CMV-Con and CMV-PAI-1+CMV-Klotho double transgenic cultures are immunoblotted for klotho (D, E; P < .001), CCN2 (D, F; P < .001), p21 (D, G; P < .01), p53 (D, H; P < .001), p-SMAD3 (D, I; P < .01). GAPDH serves as loading control. Histograms in (E-I) depict the relative levels (mean ± SD) of the indicated proteins for three separate experiments (n = 3). **P < .01, ***P < .001

Article Snippet: Overnight incubation at 4°C utilized the following primary antibodies; rabbit anti- PAI- 1 (1:3000) and rabbit anti- collagen- 1 (1:5000) as previously described,15 rat anti- klotho (1:5000; Transgenic Inc.- KM2119), rabbit anti- SMAD3 (1:1000; Cell Signaling- 9523), rabbit anti- p- Histone3 (1:1000; Cell Signaling- 9701), rabbit anti- p21 (1:1000; Cell Signaling- 2947), rabbit anti- caspase- 9 (1:1000; Cell Signaling- 9502), rabbit anti- c- caspase- 3 (1:1000; Cell Signaling- 9661), rabbit antiSnail1 (1:1000; Cell Signaling- 38798s), rabbit anti- α- SMA (1:1000; Abcam- ab32575), rabbit anti- phospho- SMAD3 (1:1000; Abcam- ab52903), rabbit anti- fibronectin (1:100,000; Abcam- ab2413), rabbit anti- LOXL2 (1:1000; Abcam- ab96233), rabbit anti- MMP- 9 (1:1000; Abcam- ab38898), rabbit antiMMP- 2 (1:1000; Abcam- ab97779), rabbit anti- VEGF- C (1:1000; Abcam- ab9546), rabbit anti- endothelin- 1 (1:1000; 18201- IBL America, Minneapolis, MN, USA), rabbit antivimentin (1:10,000; Santa Cruz- sc5565), rabbit anti- GAPDH (1:5000; Santa Cruz- sc25778), goat anti- CCN2 (1:500; Santa Cruz- sc14939), rabbit anti- TGF- βRI (1:1000; Santa Cruz- sc9048), rabbit anti- TGF- βRII (1:1000; Santa Cruz- sc220), mouse anti- p53 (1:1000; Santa Cruz- sc126), mouse anti- Ecadherin (1:1000; BD Biosciences- 610181), and rabbit antip- p53 (1:1000; AF1043- R&D systems, Minneapolis, MN, USA).

Techniques: Western Blot, Expressing, Transgenic Assay, Control

Journal: The FASEB Journal

Article Title: PAI‐1 induction during kidney injury promotes fibrotic epithelial dysfunction via deregulation of klotho, p53, and TGF‐β1‐receptor signaling

doi: 10.1096/fj.202002652rr

Figure Lengend Snippet: FIGURE 8 Dysfunction driven by PAI-1 overexpression is independent of TGF-β1 ligand synthesis or release. A, ELISA analysis for active TGF-β1 ligand concentrations in the conditioned media isolated from serum-starved CMV-Con and CMV-PAI-1 cultures. n = 3. B-D, Cytokine protein array analysis of CMV-Con and CMV-PAI-1 conditioned media for active TGF-β1 (B), TGF-β2 (C), and TGF-β3 (D). Graphs depict the relative levels of secreted ligands (mean ± SD), n = 3. E, Immunoblot comparison of fibrotic responses in the cellular lysates of TGF-β1 stimulated or unstimulated CMV-Con and untreated CMV-PAI-1 culture extracted in parallel. F, CMV-Con HK2 cells pretreated with 20 μg/mL of TGF-β1 neutralizing antibody or 20 μg/mL IgY control antisera are stimulated with 2 ng/mL TGF-β1. Cells are harvested after 24 hours and expression of p-SMAD3, fibronectin and E-cadherin are analyzed by western blot. n = 3. G, Equally seeded CMV-PAI-1 HK2s are treated with various concentrations of TGF-β1 neutralizing antibody (0, 20, 40, 60 μg/mL) or 60 μg/mL IgY control antisera for 24 hours prior to western blot analysis of extracts for p-SMAD3, fibronectin, collagen-1, vimentin, p53 and p21, with GAPDH is serving as a loading marker. n = 3, *P < .05, **P < .01, ***P < .001, n.s., not significant. H, Western blot analysis of cell lysate extracts from CMV-PAI-1+Con shRNA and CMV-PAI-1+TGF-β1 shRNA HK2 cells for the indicated fibrotic markers. Cytokine protein array analysis of whole cell lysates (I) and conditioned media (J) used to validate TGF-β1 knockdown

Article Snippet: Overnight incubation at 4°C utilized the following primary antibodies; rabbit anti- PAI- 1 (1:3000) and rabbit anti- collagen- 1 (1:5000) as previously described,15 rat anti- klotho (1:5000; Transgenic Inc.- KM2119), rabbit anti- SMAD3 (1:1000; Cell Signaling- 9523), rabbit anti- p- Histone3 (1:1000; Cell Signaling- 9701), rabbit anti- p21 (1:1000; Cell Signaling- 2947), rabbit anti- caspase- 9 (1:1000; Cell Signaling- 9502), rabbit anti- c- caspase- 3 (1:1000; Cell Signaling- 9661), rabbit antiSnail1 (1:1000; Cell Signaling- 38798s), rabbit anti- α- SMA (1:1000; Abcam- ab32575), rabbit anti- phospho- SMAD3 (1:1000; Abcam- ab52903), rabbit anti- fibronectin (1:100,000; Abcam- ab2413), rabbit anti- LOXL2 (1:1000; Abcam- ab96233), rabbit anti- MMP- 9 (1:1000; Abcam- ab38898), rabbit antiMMP- 2 (1:1000; Abcam- ab97779), rabbit anti- VEGF- C (1:1000; Abcam- ab9546), rabbit anti- endothelin- 1 (1:1000; 18201- IBL America, Minneapolis, MN, USA), rabbit antivimentin (1:10,000; Santa Cruz- sc5565), rabbit anti- GAPDH (1:5000; Santa Cruz- sc25778), goat anti- CCN2 (1:500; Santa Cruz- sc14939), rabbit anti- TGF- βRI (1:1000; Santa Cruz- sc9048), rabbit anti- TGF- βRII (1:1000; Santa Cruz- sc220), mouse anti- p53 (1:1000; Santa Cruz- sc126), mouse anti- Ecadherin (1:1000; BD Biosciences- 610181), and rabbit antip- p53 (1:1000; AF1043- R&D systems, Minneapolis, MN, USA).

Techniques: Over Expression, Enzyme-linked Immunosorbent Assay, Isolation, Protein Array, Western Blot, Comparison, Control, Expressing, Marker, shRNA, Knockdown

Journal: The FASEB Journal

Article Title: PAI‐1 induction during kidney injury promotes fibrotic epithelial dysfunction via deregulation of klotho, p53, and TGF‐β1‐receptor signaling

doi: 10.1096/fj.202002652rr

Figure Lengend Snippet: FIGURE 9 Model. PAI-1 upregulation leads to downregulation of klotho, upregulation of p53, and induction of TGF-β1 receptor signaling independent of the TGF-β1 ligand, resulting in expression and secretion of fibrotic markers, downregulation of E-cadherin and upregulation of vimentin, leading to dedifferentiation, and upregulation of p21, p-H3, causing G2/M cell cycle arrest and a propensity to cell death, collectively establishing a role for PAI-1 in tubular epithelial dysfunction. Klotho regulates both p53 and SMAD3 signaling, promoting PAI-1-mediated tubular maladaptive responses

Article Snippet: Overnight incubation at 4°C utilized the following primary antibodies; rabbit anti- PAI- 1 (1:3000) and rabbit anti- collagen- 1 (1:5000) as previously described,15 rat anti- klotho (1:5000; Transgenic Inc.- KM2119), rabbit anti- SMAD3 (1:1000; Cell Signaling- 9523), rabbit anti- p- Histone3 (1:1000; Cell Signaling- 9701), rabbit anti- p21 (1:1000; Cell Signaling- 2947), rabbit anti- caspase- 9 (1:1000; Cell Signaling- 9502), rabbit anti- c- caspase- 3 (1:1000; Cell Signaling- 9661), rabbit antiSnail1 (1:1000; Cell Signaling- 38798s), rabbit anti- α- SMA (1:1000; Abcam- ab32575), rabbit anti- phospho- SMAD3 (1:1000; Abcam- ab52903), rabbit anti- fibronectin (1:100,000; Abcam- ab2413), rabbit anti- LOXL2 (1:1000; Abcam- ab96233), rabbit anti- MMP- 9 (1:1000; Abcam- ab38898), rabbit antiMMP- 2 (1:1000; Abcam- ab97779), rabbit anti- VEGF- C (1:1000; Abcam- ab9546), rabbit anti- endothelin- 1 (1:1000; 18201- IBL America, Minneapolis, MN, USA), rabbit antivimentin (1:10,000; Santa Cruz- sc5565), rabbit anti- GAPDH (1:5000; Santa Cruz- sc25778), goat anti- CCN2 (1:500; Santa Cruz- sc14939), rabbit anti- TGF- βRI (1:1000; Santa Cruz- sc9048), rabbit anti- TGF- βRII (1:1000; Santa Cruz- sc220), mouse anti- p53 (1:1000; Santa Cruz- sc126), mouse anti- Ecadherin (1:1000; BD Biosciences- 610181), and rabbit antip- p53 (1:1000; AF1043- R&D systems, Minneapolis, MN, USA).

Techniques: Expressing

Journal: ROMANIAN BIOTECHNOLOGICAL LETTERS

Article Title: Role of p38-mitogen-activated protein kinase in modulation of the response to therapy in FaDu Human pharyngeal carcinoma cell

doi: 10.25083/rbl/24.1/118.128

Figure Lengend Snippet: Figure 5. Effect of RSV and/or cisplatin on p53 phosphorylation in FaDu cell line. Phosphorylated (phos-) and total p53 protein expression in FaDu cell line treated with RSV 50μM and/or CisPt 10μM for 24h in presence or absence of SB203580 inhibitor (A). The results were analyzed and used to calculate the phospho-p53 /total p53 ratios (B). Results are presented in graphics as mean ± standard errors from three independent experiments.

Article Snippet: The kits consisted of: DuoSet_IC Human Total p53 ELISA [Cat. No DYC1043]; DuoSet_IC Human Phospho-p53 (S15) ELISA [Cat. No DYC1839]; DuoSet_IC Human/Mouse/Rat Total p38 ELISA [Cat. No DYC8691B] DuoSet_IC Human/ Mouse/ Rat Phospho-p38α (T180/Y182) ELISA [Cat. No DYC869B], and were purchased from R&D Systems Inc. (USA).

Techniques: Phospho-proteomics, Expressing

Journal: Oncotarget

Article Title: Bisphenol A induces cell cycle arrest in primary and prostate cancer cells through EGFR/ERK/p53 signaling pathway activation

doi: 10.18632/oncotarget.23360

Figure Lengend Snippet: In (A) , LNCaP cells were left untreated or treated for the indicated times with 50 μM Bisphenol A (BPA) in absence or presence of 10 μM Bicalutamide (Bic), 10 μM ICI 182,780 (ICI) and 20 μM PD 98,059 (PD). Phosphorylation of p53 on residue Ser15 (p-Ser15-p53) was analyzed in lysate proteins using the appropriate antibody. Filters were stripped and re-probed with the anti-p53 antibody and α-Tubulin antibody, as loading control. In (B) , LNCaP cells were stimulated or unstimulated for 5 minutes with 50 μM Bisphenol A (BPA) in absence or presence of 5 μM ZD 1839 (ZD), 10 μM Bicalutamide (Bic) and 10 μM ICI 182,780 (ICI). Lysate proteins were immune-precipitated with EGFR antibody or non specific mouse immunoglobulin (Ctrl Ab) as control. Proteins in immune complexes were detected by Western blot using appropriate antibodies against indicated proteins. In (C) , ERK inhibition rescues the BPA-treated LNCaP cell ability to form colonies. LNCaP cells were seed in 6 well plates and stimulated or unstimulated with 50 μM Bisphenol A (BPA) in absence or presence of 20 μM PD 98,059. After 12 days the cells were fixed, stained and counted as reported in Materials and Methods.

Article Snippet: Anti-p-Ser15-p53 (catalog MAB1839) was from R&D Systems.

Techniques: Phospho-proteomics, Residue, Control, Western Blot, Inhibition, Staining

Journal: Oncotarget

Article Title: Bisphenol A induces cell cycle arrest in primary and prostate cancer cells through EGFR/ERK/p53 signaling pathway activation

doi: 10.18632/oncotarget.23360

Figure Lengend Snippet: Bisphenol A stimulates EGFR phosphorylation and triggers EGFR/AR/ERβ complex assembly. BPA treatment induces ERK activity and the consequent phosphorylation of p53 on residue Ser15 and hence p53 stabilization. This results in increased p27 and p21 expression levels and cyclin D1 protein down regulation, which lead to cell cycle arrest.

Article Snippet: Anti-p-Ser15-p53 (catalog MAB1839) was from R&D Systems.

Techniques: Phospho-proteomics, Activity Assay, Residue, Expressing

Journal: Autophagy

Article Title: RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells

doi: 10.1080/15548627.2018.1427022

Figure Lengend Snippet: DIRAS family expression is downregulated in ovarian cancers and cancer cell lines. (A) Normal ovaries and tumor tissue microarrays with 122 cases represented were analyzed using immunohistochemistry with anti-DIRAS1 and anti-DIRAS2 antibodies and scored 0 to 3. Examples from the tumor array representing the score 0 (no expression), 1 (low expression), 2 (moderate expression) and 3 (high expression) are shown with 10x magnification, and enlarged 40x magnification insets for each antibody. Bar: 100 µm. (B) The fraction of ovarian cancers with DIRAS family expression. (C) The correlation between disease-free survival and DIRAS family member expression. Each circle represents one patient sample. The median disease-free survival time is listed on top. (D) The fraction of ovarian cancers with DIRAS2 expression. (E) The correlation between disease-free survival and overall survival by staining score of DIRAS1. Each circle represents one patient sample. Asterisk denotes significant difference (*p<0.05 or **p<0.01). (F) Kaplan-Meier overall survival analysis of DIRAS1 expression by staining score. (G) Kaplan-Meier overall survival analysis of DIRAS2 expression by staining score. Statistical significance was determined by Mantel-Cox Log-rank analysis of the overall survival. (H) DIRAS1 and DIRAS2 protein expression levels were determined in normal ovarian epithelial scrapings (NOE), 11 ovarian cancer primary tumors and 14 ovarian cancer cell lines using western blot analysis.

Article Snippet: SQSTM1/p62 antibody was purchased from MBL. siRNAs were purchased from Dharmacon and sgRNAs were purchased from Sage Biologicals. pCMV-DIRAS3 Y2, pCMV-ΔNT DIRAS3, plasmids were constructed in our laboratory. pCMV-DIRAS1 and pCMV-DIRAS2 mouse and human plasmids were purchased from Origene (SC100097, SC317295, MC205282 and MC210705) and sequence verification was performed using Sanger-based techniques at our core facility.

Techniques: Expressing, Immunohistochemistry, Staining, Western Blot

Journal: Autophagy

Article Title: RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells

doi: 10.1080/15548627.2018.1427022

Figure Lengend Snippet: Re-expression of DIRAS1 or DIRAS2 inhibits ovarian cancer cell growth in vitro. (A and B) Two distinct subclones of OVCAR8-DIRAS1 or OVCAR8-DIRAS2 cells were seeded at 400 cells/well and doxycycline (1 µg/mL) was added to the media every 48 h for 6 d to induce gene expression. Colonies were allowed to grow for 2 wk and then stained with Coomassie Brilliant Blue. Percentage of growth inhibition was determined for 3 independent experiments all performed with triplicate samples. (C) OVCAR8-Praental cells were seeded at 400 cells/well and doxycycline (1 µg/mL) was added to the media every 48 h for 6 d. Colonies were allowed to grow for 2 wk and then stained with Coomassie Brilliant Blue. Percentage of growth inhibition was determined for 3 independent experiments all performed with triplicate samples. (D–I) Similar experiments were carried out in SKOv3-IP-inducible lines and Hey-A8-inducible lines or their parental control cells, seeded at 800 and 600 cells per well, respectively. Asterisk denotes significant difference (*p<0.05 or **p<0.01).

Article Snippet: SQSTM1/p62 antibody was purchased from MBL. siRNAs were purchased from Dharmacon and sgRNAs were purchased from Sage Biologicals. pCMV-DIRAS3 Y2, pCMV-ΔNT DIRAS3, plasmids were constructed in our laboratory. pCMV-DIRAS1 and pCMV-DIRAS2 mouse and human plasmids were purchased from Origene (SC100097, SC317295, MC205282 and MC210705) and sequence verification was performed using Sanger-based techniques at our core facility.

Techniques: Expressing, In Vitro, Staining, Inhibition

Journal: Autophagy

Article Title: RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells

doi: 10.1080/15548627.2018.1427022

Figure Lengend Snippet: Re-expression of DIRAS family members induces autophagy. OVCAR8-inducible cells were treated with or without doxycycline for 18–48 h with or without the presence of chloroquine (5 µM). (A) Cell lysate was collected and western blot analysis was performed to examine the conversion of LC3-I to LC3-II, SQSTM1/p62 levels, and expression of DIRAS1 or DIRAS2. ACTB/β-actin was used as a loading control. Upon re-expression of DIRAS1 or DIRAS2 we observed decreased SQSTM1 levels and increased conversion to LC3-II, which was further enhanced by the addition of chloroquine. (B and C) Immunofluorescence staining of DIRAS1 or DIRAS2 (green) and LC3B (red) was performed to document the formation of autophagosomes. Scale bar: 10 µm. (D and E) Fluorescence microscopy of OVCAR8-DIRAS1 or OVCAR8-DIRAS2 ovarian cancer cells with or without doxycycline treatment (1 µg/mL), for 18–30 h to induce gene expression, 24 h post infection with GFP-mCherry-LC3B-expressing lentivirus to visualize autophagic flux. Yellow puncta indicate the presence of GFP and mCherry signal. Red puncta indicate the fusion of the autophagosome with the lysosome and quenching of GFP. Scale bar: 10 µm. Average puncta counts were determined for each time point and condition using ImageJ and plotted with GraphPad Prism. Data were collected over 3 independent experiments and more than 50 cells were analyzed per condition. Asterisk denotes significant difference as determined by Student t test (*p<0.05 or **p<0.01). (F) TEM images of induced or noninduced OVCAR8-DIRAS1 or DIRAS2 cells. Red arrows indicate typical double-membrane autophagosomes. Scale bars: 5 µm and 5 nm for close-up image. Data were collected over 3 independent experiments. Asterisk denotes significant difference (*p<0.05 or **p<0.01).

Article Snippet: SQSTM1/p62 antibody was purchased from MBL. siRNAs were purchased from Dharmacon and sgRNAs were purchased from Sage Biologicals. pCMV-DIRAS3 Y2, pCMV-ΔNT DIRAS3, plasmids were constructed in our laboratory. pCMV-DIRAS1 and pCMV-DIRAS2 mouse and human plasmids were purchased from Origene (SC100097, SC317295, MC205282 and MC210705) and sequence verification was performed using Sanger-based techniques at our core facility.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence, Microscopy, Infection

Journal: Autophagy

Article Title: RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells

doi: 10.1080/15548627.2018.1427022

Figure Lengend Snippet: DIRAS1 and DIRAS2 growth inhibition is dependent upon functional autophagy. Using CRISPR/Cas9 double-nickase control plasmid or ATG7 sgRNA, OVCAR8-DIRAS1 and OVCAR8-DIRAS2 inducible cells were generated from single-cell clones. (A and B) Western blot analysis revealed that control cells induced autophagy upon re-expression of DIRAS1 or DIRAS2 whereas ATG7−/− cells did not. Knockout efficiency was observed by western blot analysis of ATG7. (C and D) Clonogenic assays were performed with these sublines, documenting that growth inhibition seen by re-expressing DIRAS1 or DIRAS2 is dependent upon functional autophagy. Experiments were completed in triplicate. Asterisk denotes significant difference (*p<0.05 or **p<0.01).

Article Snippet: SQSTM1/p62 antibody was purchased from MBL. siRNAs were purchased from Dharmacon and sgRNAs were purchased from Sage Biologicals. pCMV-DIRAS3 Y2, pCMV-ΔNT DIRAS3, plasmids were constructed in our laboratory. pCMV-DIRAS1 and pCMV-DIRAS2 mouse and human plasmids were purchased from Origene (SC100097, SC317295, MC205282 and MC210705) and sequence verification was performed using Sanger-based techniques at our core facility.

Techniques: Inhibition, Functional Assay, CRISPR, Plasmid Preparation, Generated, Clone Assay, Western Blot, Expressing, Knock-Out

Journal: Autophagy

Article Title: RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells

doi: 10.1080/15548627.2018.1427022

Figure Lengend Snippet: DIRAS1 and DIRAS2 induce autophagy by inhibiting PI3K and regulating the FOXO3 transcription factor. (A) OVCAR8-inducible cells were treated with or without doxycycline for 24 h to induce DIRAS1 or DIRAS2 gene expression. Cell lysate was collected and western blot analysis was performed. Re-expression of DIRAS1 and DIRAS2 resulted in a significant decrease in p-AKT (S473) and p-MAPK/ERK (42/44). p-MTOR (S2488), a downstream target of AKT and key inhibitor of autophagy, was also decreased following re-expression of DIRAS1 or DIRAS2. (B) ImageJ quantification was performed for 3 independent western blot analyses to determine the relative expression of p-AKT, p-MAPK/ERK and p-MTOR following re-expression of DIRAS1 or DIRAS2 (*p<0.05; **p<0.01). (C) Cell lysate was collected and western blot analysis was performed to examine the nuclear localization of FOXO3 or TFEB. PARP and TUBA/α-tubulin were used as nuclear and cytoplasm extraction controls. The numbers below the blots correspond to ImageJ densitometry of the blot. (D) Immunofluorescence staining of FOXO3 and TFEB nuclear localization following re-expression of DIRAS1 or DIRAS2. Scale bars: 20 µm. (E and F) OVCAR8-inducible cell lines were treated with or without doxycycline for 24 h to induce gene expression of DIRAS1 or DIRAS2. mRNA expression of LC3B, ULK1, RAB7, GABARAP, BECN1, and LAMP1 was measured by qRT-PCR. (E) Re-expression of DIRAS1 in OVCAR8 ovarian cancer cells increased mRNA expression of ULK1, RAB7, GABARAP, BECN1 and LAMP1. (F) Re-expression of DIRAS2 in OVCAR8 increases mRNA expression of LC3B, ULK1, RAB7, GABARAP, and BECN1.

Article Snippet: SQSTM1/p62 antibody was purchased from MBL. siRNAs were purchased from Dharmacon and sgRNAs were purchased from Sage Biologicals. pCMV-DIRAS3 Y2, pCMV-ΔNT DIRAS3, plasmids were constructed in our laboratory. pCMV-DIRAS1 and pCMV-DIRAS2 mouse and human plasmids were purchased from Origene (SC100097, SC317295, MC205282 and MC210705) and sequence verification was performed using Sanger-based techniques at our core facility.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Autophagy

Article Title: RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells

doi: 10.1080/15548627.2018.1427022

Figure Lengend Snippet: Transient expression of murine DIRAS1 and DIRAS2 inhibit murine ovarian cancer cell clonogenic growth and induce autophagy. Long-term clonogenic growth was assessed following transient overexpression of murine DIRAS1 and DIRAS2 in ID8 (A), IG10 (B), TBR2 (C) and TKOOV10 (D) murine ovarian cancer cell lines. Selection was performed with G418 for 2 wk until colonies reached at least 50 cells in size. Columns represent the mean and bars represent the S.D. The experiment was performed at least 3 independent times. Asterisk denotes significant difference (**p<0.01). ID8 (A), IG10 (B), TBR2 (C) and TKOOV10 (D) murine ovarian cancer cells were seeded at 0.3 × 106 cells per well and transfected with empty vector, DIRAS1- or DIRAS2-encoding plasmid DNA and treated with or without chloroquine (5 µm) for 24 h prior to lysing and immunoblotting as indicated. Densitometry was measured with ImageJ and experiments were performed at least 3 times. Autophagic flux was determined by the ratio of LC3-II:LC3-I and normalized to the loading control, ACTB/β-actin.

Article Snippet: SQSTM1/p62 antibody was purchased from MBL. siRNAs were purchased from Dharmacon and sgRNAs were purchased from Sage Biologicals. pCMV-DIRAS3 Y2, pCMV-ΔNT DIRAS3, plasmids were constructed in our laboratory. pCMV-DIRAS1 and pCMV-DIRAS2 mouse and human plasmids were purchased from Origene (SC100097, SC317295, MC205282 and MC210705) and sequence verification was performed using Sanger-based techniques at our core facility.

Techniques: Expressing, Over Expression, Selection, Transfection, Plasmid Preparation, Western Blot

Journal: Autophagy

Article Title: RAS-related GTPases DIRAS1 and DIRAS2 induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells

doi: 10.1080/15548627.2018.1427022

Figure Lengend Snippet: Murine DIRAS1 and DIRAS2 are essential for starvation-induced autophagy. Mouse ovarian cancer cells were transfected with control, murine Diras1 or murine Diras2 siRNA for 72 h prior to autophagy induction by serum starvation for 4–16 h. (A) Western-blot analysis was performed as indicated and documented a decrease in serum starvation-induced autophagic flux following knockdown of DIRAS1 or DIRAS2. (B) Immunofluorescence staining of IG10, TBR2, and TKOOV10 cells for LC3 puncta following autophagy induction by serum starvation for 16 h was performed. Quantification of puncta/cell was calculated for at least 100 cells per experiment, for 3 experiments. Columns indicate the mean and bars represent the S.D. Significance denoted by the asterisk (*p<0.05, **p<0.01). (C) qRT-PCR analysis of murine Diras1 and murine Diras2 mRNA expression following knockdown with siRNA. Columns indicate the mean and bars represent the s.d. Significance denoted by the asterisk (*p<0.05, **p<0.01).

Article Snippet: SQSTM1/p62 antibody was purchased from MBL. siRNAs were purchased from Dharmacon and sgRNAs were purchased from Sage Biologicals. pCMV-DIRAS3 Y2, pCMV-ΔNT DIRAS3, plasmids were constructed in our laboratory. pCMV-DIRAS1 and pCMV-DIRAS2 mouse and human plasmids were purchased from Origene (SC100097, SC317295, MC205282 and MC210705) and sequence verification was performed using Sanger-based techniques at our core facility.

Techniques: Transfection, Western Blot, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: CRISPR Screening in Tandem with Targeted mtDNA Damage Reveals WRNIP1 Essentiality

doi: 10.1101/2023.10.03.560559

Figure Lengend Snippet: (a) Schematic representation of the proposed cellular response to mtDox damage. mtDNA damage caused by mtDox leads to mitochondrial herniation followed by release of mitochondrial contents into the cytoplasm, sensed through RIG-I innate immune pathway. Functional WRNIP1 transports activated RIG-I to MAVS which stimulates immune response activation, leading to nuclear recognition of damage. (b) Digitonin based extraction followed by qPCR measurement of mtDNA released into the cytoplasm over time following 8 μM mtDox treatment (n = 4, * p < 0.0011, ** p < 0.013). (c) Digitonin based extraction followed by RT-qPCR measurement of mtRNA released into the cytoplasm over time (n = 4, * p < 0.013, ** p < 0.037). (d) Western blot of RIG-I-FLAG co-immunoprecipitation displaying time-course interaction of RIG-I with WRNIP1 following mtDox treatment. Anti-FLAG M2 magnetic beads were used to pull down RIG-I-FLAG transfected lysates, and blot was counter-stained with antibodies for FLAG and WRNIP1 (n = 3). (e) Time-course Western blot for phosphorylated IRF3 (p-IRF3) indicating downstream innate immune activation. Performed with parent (WT) and WRNIP1 knockout (KO) cells lines (n = 3). (f) Clonogenic analysis of relative survival following mtDox treatment between LacZ, cGAS, and MAVS knockout (n = 4). All p -values determined using unpaired t -test. Data represented as mean ± s.d.

Article Snippet: HCT116 TP53 (-/-) cells were transfected with a FLAG-tagged RIG-I expression vector purchased from Origene (RC217615) using Lipofectamine 3000 in six 150 mm dishes plated to 90% confluency the day before.

Techniques: Functional Assay, Activation Assay, Extraction, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Magnetic Beads, Transfection, Staining, Knock-Out