Journal: Oncotarget

Article Title: Suppression of ABHD2 , identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9951

Figure Lengend Snippet: a. Seven human ovarian serous adenocarcinoma cell lines and an immortalized human ovarian surface cell line HOSE/E7, all of which do not grow in soft agar, were used. Following transfection of the shRNA library, only OVCA420 cells formed colonies in soft agar. 43 colonies were successfully expanded. shRNAs were amplified by PCR and we reconstructed 69 different shRNA plasmids. Out of the 69 shRNAs in OVCA420 cells, 11 again generated colonies in soft agar. We then measured mRNA expression of these 11 genes using RT-PCR. Of the 11 shRNAs, shRNAs directed against ABHD2, CYB5R3 and ELAC2 suppressed target gene mRNA expression. b. Left: shRNA- ABHD2 transfected OVCA420 cell colony in soft agar. Black bar, 100 μm. Right: normalized ABHD2 / ACTB mRNA expression analyzed by RT-PCR. (n=3, respectively) c. Left: shRNA- ELAC2 transfected OVCA420 cell colony in soft agar. Right: normalized ELAC2 / ACTB mRNA expression. d. Left: shRNA- CYB5R3 transfected OVCA420 cell colony in soft agar. Right: normalized CYB5R3 / ACTB expression.

Article Snippet: Slides were incubated with a rabbit anti-ABHD2 antibody (Abgent) at a 1:40 dilution overnight at 4°C, followed by a one-hour incubation with biotinylated goat anti-rabbit secondary antibodies (Nichirei, Tokyo, Japan) at room temperature.

Techniques: Transfection, shRNA, Amplification, Generated, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Suppression of ABHD2 , identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9951

Figure Lengend Snippet: mRNA expression was evaluated using log2 normalized values. a. Comparison of ABHD2 mRNA expression between ovarian cancer tissues and serous borderline tumors (SBT) using gene expression microarray datasets GSE9891 and GSE2109. b. Comparison of ABHD2 mRNA expression between serous adenocarcinoma and non-serous adenocarcinoma in microarray dataset GSE2109. c. Copy number alterations for ABHD2 in TCGA samples. Del; deletion, Amp; Amplification. d. Correlation between ABHD2 copy number and mRNA expression in TCGA specimens. e. Representative ABHD2 immunohistochemistry staining for HGSOC (intensity 0, 1 and 2), normal fallopian tube and SBT are shown. Comparison of H-scores among HGSOC, fallopian tube and SBT. The H-score is calculated as 2x the percentage of the most strongly stained area plus 1x the percentage of the most weakly stained area, imparting a total score ranging from 0 to 200.

Article Snippet: Slides were incubated with a rabbit anti-ABHD2 antibody (Abgent) at a 1:40 dilution overnight at 4°C, followed by a one-hour incubation with biotinylated goat anti-rabbit secondary antibodies (Nichirei, Tokyo, Japan) at room temperature.

Techniques: Expressing, Microarray, Amplification, Immunohistochemistry, Staining

Journal: Oncotarget

Article Title: Suppression of ABHD2 , identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9951

Figure Lengend Snippet: a. Number of viable control, sh1-OVCA420 and sh2-OVCA420 cells following incubation on ultra-low attachment plates (n=6). b. Representative data showing Annexin V/7-ADD staining following incubation on ultralow attachment plates. c. Comparison of the ratio of the Annexin V(−)/7-ADD(−) fraction (viable cells) and Annexin V(+) fraction (apoptotic cells) between control, sh1 and sh2 cells. d. Number of viable OVCA420-control and OVCA420- ABHD2 cells following incubation on ultra-low attachment plates (n=6). Panels a-c: sh1; sh1-OVCA420, sh2; sh2-OVCA420, control; control-OVCA420; panel d: control; OVCA420-control, ABHD2 ; SKOV3- ABHD2 .

Article Snippet: Slides were incubated with a rabbit anti-ABHD2 antibody (Abgent) at a 1:40 dilution overnight at 4°C, followed by a one-hour incubation with biotinylated goat anti-rabbit secondary antibodies (Nichirei, Tokyo, Japan) at room temperature.

Techniques: Incubation, Staining

Journal: Oncotarget

Article Title: Suppression of ABHD2 , identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9951

Figure Lengend Snippet: a. Number of viable SKOV3-control and SKOV3- ABHD2 cells following incubation on ultra-low attachment plates (n=6). b. Representative data showing Annexin V/7-ADD staining following incubation on ultralow attachment plates. c. Comparison of the ratio of the Annexin V(−)/7-ADD(−) fraction and Annexin V(+) fraction between SKOV3-control and SKOV3- ABHD2 cells. d. Number of viable control- SKOV3, sh1- SKOV3- ABHD2 and sh2-SKOV3- ABHD2 cells following incubation on ultra-low attachment plates (n=6). e. Representative data showing Annexin V/7-ADD staining following incubation on ultralow attachment plates for 48 hours. f. Comparison of the ratio of the Annexin V(−)/7-ADD(−) fraction and Annexin V(+) fraction among control, sh1-SKOV3- ABHD2 and sh2-SKOV3- ABHD2 cells. Panels a-c: control ; SKOV3-control, ABHD2 ; SKOV3- ABHD2 ; panels d-f: control; control-SKOV3- ABHD2, sh1; sh1-SKOV3- ABHD2, sh2 ; sh2-SKOV3- ABHD2 .

Article Snippet: Slides were incubated with a rabbit anti-ABHD2 antibody (Abgent) at a 1:40 dilution overnight at 4°C, followed by a one-hour incubation with biotinylated goat anti-rabbit secondary antibodies (Nichirei, Tokyo, Japan) at room temperature.

Techniques: Incubation, Staining

Journal: Oncotarget

Article Title: Suppression of ABHD2 , identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9951

Figure Lengend Snippet: All experiments were performed in triplicate. a. Phosphorylated p38 (P-P38) and phosphorylated ERK1/2 (P-ERK1/2) increased following knockdown of ABHD2 (sh1 and sh2) in OVCA420 cells. On the contrary, P-P38 and P-ERK1/2 decreased following overexpression of ABHD2 in SKOV3 and OVCA420 cells. b. Resistance of OVCA420 cells to anoikis on ultra-low attachment dishes was inhibited by GSK1120212, a specific inhibitor of the ERK1/2 pathway. Reduction of P-ERK1/2 following treatment with GSK1120212 was confirmed by Western blotting. DMSO, vehicle control. Cells were treated with differing doses of GSK1120212 as indicated. c. Resistance of OVCA420 cells to anoikis was inhibited following treatment with SB203580, a specific inhibitor of the the p38MAPK pathway. d. Levels of P-P38 and P-ERK1/2 increased following knockdown of ABHD2 (sh1 and sh2) in SKOV3- ABHD2 cells. e. Resistance to anoikis in sh1-SKOV3- ABHD2 and sh2-SKOV3- ABHD2 cells was inhibited following treatment with 100nM GSK1120212 (GSK) and 30μM SB203580 (SB).”

Article Snippet: Slides were incubated with a rabbit anti-ABHD2 antibody (Abgent) at a 1:40 dilution overnight at 4°C, followed by a one-hour incubation with biotinylated goat anti-rabbit secondary antibodies (Nichirei, Tokyo, Japan) at room temperature.

Techniques: Over Expression, Western Blot

Journal: Oncotarget

Article Title: Suppression of ABHD2 , identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9951

Figure Lengend Snippet: ABHD2 expression levels and clinicopathological factors

Article Snippet: Slides were incubated with a rabbit anti-ABHD2 antibody (Abgent) at a 1:40 dilution overnight at 4°C, followed by a one-hour incubation with biotinylated goat anti-rabbit secondary antibodies (Nichirei, Tokyo, Japan) at room temperature.

Techniques: Expressing

Journal: Oncotarget

Article Title: Suppression of ABHD2 , identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9951

Figure Lengend Snippet: a. Differences in survival based on ABHD2 immunohistochemical scores (H-score) in HGSOC. b. Differences in survival based on ABHD2 mRNA expression in GSE9891 (n=285, mostly HGSOC) and GSE3149 (n=146, mostly HGSOC) datasets. Samples were divided into high (greater than the median value) and low (less than the median) expression cases. c. Analysis of HGSOC patients (n=36) from KOV-75 based on ABHD2 mRNA expression. d. Analysis of non-HGSOC patients (n=39) from KOV-75 based on ABHD2 mRNA expression. n.s., not significant.

Article Snippet: Slides were incubated with a rabbit anti-ABHD2 antibody (Abgent) at a 1:40 dilution overnight at 4°C, followed by a one-hour incubation with biotinylated goat anti-rabbit secondary antibodies (Nichirei, Tokyo, Japan) at room temperature.

Techniques: Immunohistochemical staining, Expressing

Journal: Oncotarget

Article Title: Suppression of ABHD2 , identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

doi: 10.18632/oncotarget.9951

Figure Lengend Snippet: a. Representative data showing 7-ADD staining following 24 hour incubation with 10 μM cisplatin. b. The ratio of the 7-AAD negative live OVCA420 cells markedly increased following suppression of ABHD2 (sh1 and sh2) as compared to the control after 24 hour incubation with 10 μM cisplatin (n=3). c. Dose-response curves following incubation of OVCA420 cells with the indicated concentrations of cisplatin for 72 hours (n=6). d. Cisplatin IC50 values increased following suppression of ABHD2 in OVCA420 cells. e. Representative data showing 7-ADD staining following 24 hour incubation with 100 μM Carboplatin. f. The ratio of 7-AAD negative live OVCA420 cells markedly increased following suppression of ABHD2 (sh1 and sh2) as compared to the control following a 24 hour incubation with 100 μM carboplatin (n=3).

Article Snippet: Slides were incubated with a rabbit anti-ABHD2 antibody (Abgent) at a 1:40 dilution overnight at 4°C, followed by a one-hour incubation with biotinylated goat anti-rabbit secondary antibodies (Nichirei, Tokyo, Japan) at room temperature.

Techniques: Staining, Incubation