Journal: Biochemical and biophysical research communications

Article Title: Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2

doi: 10.1016/j.bbrc.2017.06.195

Figure Lengend Snippet: Proteins identified as targets of KT195

Article Snippet: Clones isolated by limiting dilution were screened for levels of ABHD2 protein on Western blots and mRNA by real-time PCR (probe Mm00502098_m1, ThermoFisher Scientific), as previously described [ 18 ].

Techniques:

Journal: Biochemical and biophysical research communications

Article Title: Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2

doi: 10.1016/j.bbrc.2017.06.195

Figure Lengend Snippet: Subcellular fractions (cytosol (C), ER, MAM, mitochondria (M)) and whole cell lysates (L) were prepared from IMLF and analyzed for protein markers by Western blot analysis using antibodies to ABHD2, fatty acid CoA ligase 4 (FACL4), oxidative phosphorylation complex V (OxPhos), MEK1/2 and GRP78.

Article Snippet: Clones isolated by limiting dilution were screened for levels of ABHD2 protein on Western blots and mRNA by real-time PCR (probe Mm00502098_m1, ThermoFisher Scientific), as previously described [ 18 ].

Techniques: Western Blot, Phospho-proteomics

Journal: Biochemical and biophysical research communications

Article Title: Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2

doi: 10.1016/j.bbrc.2017.06.195

Figure Lengend Snippet: IMLF were transfected with lentiviruses containing ABHD2 shRNA or control vector and stable clones generated. (A) LDH release was measured from ABHD2 shRNA-knockdown IMLF (triangles) and vector controls (circles) 30 min after stimulation with the indicated concentrations of A23187 (n=3, *P<0.05). A Western blot (inset) shows that ABHD2 shRNA-knockdown IMLF express less ABHD2 than vector controls. G-CEPIAer or CEPIAmt was expressed in vector control (solid line) and ABHD2 shRNA-knockdown IMLF (hatched line) to measure relative changes in ER (B) and mitochondrial (C) calcium, respectively. Calcium transients were measure after addition of A23187 (1 μg/ml) by live-cell fluorescent imaging. The data represent the average of three independent experiments from analysis of at least 5 cells/experiment.

Article Snippet: Clones isolated by limiting dilution were screened for levels of ABHD2 protein on Western blots and mRNA by real-time PCR (probe Mm00502098_m1, ThermoFisher Scientific), as previously described [ 18 ].

Techniques: Transfection, shRNA, Control, Plasmid Preparation, Clone Assay, Generated, Knockdown, Western Blot, Imaging

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

Journal: The Journal of General Physiology

Article Title: The epithelial potassium channel Kir7.1 is stimulated by progesterone

doi: 10.1085/jgp.202112924

Figure Lengend Snippet: Expression of steroid receptors in the CP of adult female and male wild-type mice, analyzed by mRNA sequencing

Article Snippet: The C57BL/6N (Charles River) mice and the Abhd2 knockout mice of similar background were kept at the Animal Facility of the University of California, Berkeley, in a room with controlled light (14 h light, 10 h darkness) and temperature (23 ± 0.5°C).

Techniques: Expressing, Membrane

Journal: The Journal of General Physiology

Article Title: The epithelial potassium channel Kir7.1 is stimulated by progesterone

doi: 10.1085/jgp.202112924

Figure Lengend Snippet: The relative gene expression of the lateral CPs isolated from Abhd2 +/+ and Abhd2 −/− male and female mice. Number of mRNA-sequencing reads from corresponding CPs mapped to the mouse genome. Abhd2 and Kcnj13 expression levels are shown for wild-type (A) and knockout (B). Red dotted lines represent an expected number of sequencing reads for genes with similar expression levels between two samples. Signals <10 reads are within statistical noise and therefore scored as nonexpressed sequences. Data are averaged from triplicate experiments for each genotype and sex.

Article Snippet: The C57BL/6N (Charles River) mice and the Abhd2 knockout mice of similar background were kept at the Animal Facility of the University of California, Berkeley, in a room with controlled light (14 h light, 10 h darkness) and temperature (23 ± 0.5°C).

Techniques: Gene Expression, Isolation, Sequencing, Expressing, Knock-Out

Journal: The Journal of General Physiology

Article Title: The epithelial potassium channel Kir7.1 is stimulated by progesterone

doi: 10.1085/jgp.202112924

Figure Lengend Snippet: Expression of Abhd2 in murine CPs. (A, B, and F) ISH shows strong expression of Abhd2 in the lateral (A, solid arrow; and B), third (A, open arrow), and fourth ventricle CP (F) of Abhd2 +/+ mice. (D, E, H, and I) The adult Abhd2 −/− mice display similar background staining when using either the antisense (D and H) or the sense (E and I) probe against Abhd2 . (J) Although the antisense probe failed to bind to Abhd2 mRNA in the Abhd2 −/− animals in ISH experiments, a product lacking either Abhd2 exon 6 or both exon 6 and exon 7 was detected in the kidney of Abhd2 −/− mice. However, this mRNA product would lead to a frameshift and results in a truncated nonfunctional form of the protein. (K, K′, and M) ABHD2 protein was detected by immunohistochemistry and Western blot in the CP of Abhd2 +/+ mice (K and M), with a localization on the apical side of the epithelium (K′). (L and L′) ABHD2 was not detected in CP of Abhd2 −/− mice (L′). (M) ABHD2 was not detected in the other regions of the brain, such as the olfactory bulb (OB), cortex (COR), hippocampus (HIP), and cerebellum (CER). However, it was exclusively found in both the lateral (LCP) and the fourth (4CP) ventricle CP of Abhd2 +/+ animals. Abhd2 −/− animals display a strong expression of Kir7.1 even in the absence of ABHD2. Actin was used as a loading control.

Article Snippet: The C57BL/6N (Charles River) mice and the Abhd2 knockout mice of similar background were kept at the Animal Facility of the University of California, Berkeley, in a room with controlled light (14 h light, 10 h darkness) and temperature (23 ± 0.5°C).

Techniques: Expressing, Staining, Immunohistochemistry, Western Blot, Control

Journal: The Journal of General Physiology

Article Title: The epithelial potassium channel Kir7.1 is stimulated by progesterone

doi: 10.1085/jgp.202112924

Figure Lengend Snippet: Wild-type and Abhd2 −/− murine CPECs show similar potentiation of Kir7.1 by progesterone (P4). (A and B) Immunohistochemical staining of the lateral ventricle CP of ABHD2 in Abhd2 +/+ (A) and Abhd2 −/− (B) adult male mice. (C and D) Immunocytochemical staining of cultured CPECs shows the presence of Kir7.1 in cells of both genotypes ( Abhd2 +/+ and Abhd2 −/− ), although the latter lack ABHD2. (E) Representative recordings from adult male CPECs from Abhd2 −/− mice show potentiation of the Kir7.1 current similar to that of cells from Abhd2 +/+ animals when 10 µM P4 was added to the bath solution. Whole-cell recordings were performed using Cs + bath and pipette solutions, as indicated. (F) The fold change of I Kir7.1 at −80 mV was calculated from recordings of 6 female and 10 male wild-type cells and 8 female and 16 male Abhd2 −/− cells, as shown. Although the Abhd2 knockout female cells show a slightly smaller current fold increase, the difference is not statistically significant when compared with that of wild-type cells.

Article Snippet: The C57BL/6N (Charles River) mice and the Abhd2 knockout mice of similar background were kept at the Animal Facility of the University of California, Berkeley, in a room with controlled light (14 h light, 10 h darkness) and temperature (23 ± 0.5°C).

Techniques: Immunohistochemical staining, Staining, Cell Culture, Transferring, Knock-Out