Journal: Lipids in health and disease

Article Title: The protumorigenic enzyme GPAT2 inhibits arachidonic acid-triggered apoptosis in breast cancer.

doi: 10.1186/s12944-024-02344-1

Figure Lengend Snippet: Fig. 1 Concentration-dependent induction of AA-related cell death in GPAT2-silenced cells. scr-MDA and sh-MDA cells were grown in 10% FBS DMEM supplemented with 50 or 100 µM AA for 24–48 h before the cell proliferation rate was measured via an MTT proliferation assay. The values represent the means ± SDs of 3 independent experiments (***P< 0.001, **P< 0.01)

Article Snippet: Cell lines stably expressing a small hairpin RNA targeting GPAT2 mRNA (shRNA-GPAT2) (HuSH−29 plasmid, OriGene) or a non-silencing scrambled RNA (shRNA-scr) were generated in our laboratory, as we previously reported [1], to generate sh-MDA (reduced GPAT2 expression) and scr-MDA (retaining GPAT2 expression) cell lines.

Techniques: Concentration Assay, Proliferation Assay

Journal: Lipids in health and disease

Article Title: The protumorigenic enzyme GPAT2 inhibits arachidonic acid-triggered apoptosis in breast cancer.

doi: 10.1186/s12944-024-02344-1

Figure Lengend Snippet: Fig. 2 AA induces apoptosis only in GPAT2-silenced cells. scr-MDA and sh-MDA cells were treated with 100 µM AA for 48 h, and the percentage of apoptotic cells was determined by counting the number of apop totic and nonapoptotic cells via a TUNEL assay and hematoxylin staining (***P< 0.001)

Article Snippet: Cell lines stably expressing a small hairpin RNA targeting GPAT2 mRNA (shRNA-GPAT2) (HuSH−29 plasmid, OriGene) or a non-silencing scrambled RNA (shRNA-scr) were generated in our laboratory, as we previously reported [1], to generate sh-MDA (reduced GPAT2 expression) and scr-MDA (retaining GPAT2 expression) cell lines.

Techniques: TUNEL Assay, Staining

Journal: Lipids in health and disease

Article Title: The protumorigenic enzyme GPAT2 inhibits arachidonic acid-triggered apoptosis in breast cancer.

doi: 10.1186/s12944-024-02344-1

Figure Lengend Snippet: Fig. 3 AA increases the activity of several caspases in GPAT2-silenced cells. sh-MDA cells were treated with 100 µM AA for 48 h, and the activity of cas pases 1, 2, 3, 5, 6, 8 and 9 was quantified using the colorimetric assay kit described in the Materials and Methods section. Staurosporine (ST) was used as a positive control for caspase-3 activation. The results are expressed as the mean ± SD of 3 independent experiments (***P< 0.001)

Article Snippet: Cell lines stably expressing a small hairpin RNA targeting GPAT2 mRNA (shRNA-GPAT2) (HuSH−29 plasmid, OriGene) or a non-silencing scrambled RNA (shRNA-scr) were generated in our laboratory, as we previously reported [1], to generate sh-MDA (reduced GPAT2 expression) and scr-MDA (retaining GPAT2 expression) cell lines.

Techniques: Activity Assay, Colorimetric Assay, Positive Control, Activation Assay

Journal: Lipids in health and disease

Article Title: The protumorigenic enzyme GPAT2 inhibits arachidonic acid-triggered apoptosis in breast cancer.

doi: 10.1186/s12944-024-02344-1

Figure Lengend Snippet: Fig. 5 GPAT2 silencing alters the mRNA expression of genes involved in arachidonic acid metabolism. Total RNA was extracted from scr-MDA and sh- MDA cells, subjected to cDNA synthesis, and amplified via qRT‒PCR with primers for human PTGS2, ALOX5, AKR1C3 and EPHX2. The values represent the means ± SDs of 3 independent experiments (***P< 0.001, **P< 0.01)

Article Snippet: Cell lines stably expressing a small hairpin RNA targeting GPAT2 mRNA (shRNA-GPAT2) (HuSH−29 plasmid, OriGene) or a non-silencing scrambled RNA (shRNA-scr) were generated in our laboratory, as we previously reported [1], to generate sh-MDA (reduced GPAT2 expression) and scr-MDA (retaining GPAT2 expression) cell lines.

Techniques: Expressing, cDNA Synthesis, Amplification

Journal: Lipids in health and disease

Article Title: The protumorigenic enzyme GPAT2 inhibits arachidonic acid-triggered apoptosis in breast cancer.

doi: 10.1186/s12944-024-02344-1

Figure Lengend Snippet: Fig. 6 Schematic model of arachidonic acid-induced apoptosis in GPAT2-silenced cells. The cellular level of unesterified arachidonic acid is a general mechanism by which apoptosis is regulated, and GPAT2 promotes carcinogenesis by decreasing this level. In scr-MDA cells, GPAT2 esterifies arachidonoyl- CoA to glycerol−3-phosphate, allowing these cells to survive AA treatment. Instead, sh-MDA cells overexpress eicosanoid-producing enzymes, and AA increases BNIP3 expression, which leads to apoptosis in GPAT2-silenced cells. (cyt. C: cytochrome C, AA: arachidonic acid)

Article Snippet: Cell lines stably expressing a small hairpin RNA targeting GPAT2 mRNA (shRNA-GPAT2) (HuSH−29 plasmid, OriGene) or a non-silencing scrambled RNA (shRNA-scr) were generated in our laboratory, as we previously reported [1], to generate sh-MDA (reduced GPAT2 expression) and scr-MDA (retaining GPAT2 expression) cell lines.

Techniques: Expressing

Journal: Genome integrity

Article Title: UHRF1 is a genome caretaker that facilitates the DNA damage response to gamma-irradiation.

doi: 10.1186/2041-9414-1-7

Figure Lengend Snippet: Figure 1 Characterization of UHRF1-depleted HeLa cell lines. (A) Stable depletion of UHRF1 by shRNA in HeLa cells as shown by Western analysis (UHRF1 shRNA clones 1-3) compared to clones expressing a nonspecific shRNA (control shRNA clones 1-3). Tubulin levels are shown as loading controls. (B) Representative indirect immunofluorescence of H3K9me3 and H4K20me3 in control and UHRF1 shRNA cells at 40 × magnification. (C-D) Representative indirect immunofluorescence deconvolution of H3K9me3 and H4K20me3 in two UHRF1 shRNA and two control shRNA cell lines. Line traces are shown on the right (blue = DAPI, red = H3K9me3 or H3K20me3) for representative photomicrographs labeled with Greek characters.

Article Snippet: Human HeLa cells (ATCC) with diminished expression of UHRF1 were selected for resistance to puromycin following stable transfection with either HuSH 29mer short hairpin RNA (shRNA) constructs against UHRF1 (5’-AGG AGA CGT TCC AGT GTA TCT GCT GTC AG-3’ and ‘5-TTC GTG GAC GAA GTC TTC AAG ATT GAG CG-3’) or with a control shRNA plasmid (pRS-shGFP, non-effective, Origene), together with a vector conferring puromycin resistance.

Techniques: shRNA, Western Blot, Clone Assay, Expressing, Control, Immunofluorescence, Labeling

Journal: Genome integrity

Article Title: UHRF1 is a genome caretaker that facilitates the DNA damage response to gamma-irradiation.

doi: 10.1186/2041-9414-1-7

Figure Lengend Snippet: Figure 3 Cell cycle analysis in UHRF1-depleted cells following g-irradiation. UHRF1 or control shRNA-expressing cells were pulsed with BrdU and then treated with 1 Gy (A) or 5 Gy (B) of g-irradiation. Cells were harvested at the indicated times and stained with PI and for BrdU. For each time point, representative DNA content histograms (PI stain) are shown on the left with corresponding BrdU-PI bivariate plots on the right. Numerical values indicate percentage of G1, S and G2/M cells for each time point. The percentage change of cells in either G1 phase (C), S phase (D) or G2/M phases (E) compared to non-irradiated cells are plotted as mean ± S.D. of three independent experiments (shown for 1 Gy).

Article Snippet: Human HeLa cells (ATCC) with diminished expression of UHRF1 were selected for resistance to puromycin following stable transfection with either HuSH 29mer short hairpin RNA (shRNA) constructs against UHRF1 (5’-AGG AGA CGT TCC AGT GTA TCT GCT GTC AG-3’ and ‘5-TTC GTG GAC GAA GTC TTC AAG ATT GAG CG-3’) or with a control shRNA plasmid (pRS-shGFP, non-effective, Origene), together with a vector conferring puromycin resistance.

Techniques: Cell Cycle Assay, Irradiation, Control, shRNA, Expressing, Staining

Journal: Genome integrity

Article Title: UHRF1 is a genome caretaker that facilitates the DNA damage response to gamma-irradiation.

doi: 10.1186/2041-9414-1-7

Figure Lengend Snippet: Figure 4 Decreased irradiation-induced gH2AX in UHRF1-depleted cells. UHRF1 and control shRNA-expressing cells were exposed to either 1 Gy (A) or 5 Gy (B) g-irradiation and harvested following 0, 1, 3, 6, 12 or 24 h. Representative histograms plot gH2AX expression as measured by gH2AX-FITC intensity/cell (y-axis) vs. DNA content (x-axis) (left hand side of panel). Numbers indicate the percentage of cells showing elevated gH2AX levels. The percentage of cells with elevated gH2AX expression plotted is the mean ± S.D. of three independent experiments (right hand side of panel). (C) The decreased accumulation of gH2AX in UHRF1-depleted cells was confirmed by indirect immunofluorescence 12 h after 5 Gy g-irradiation.

Article Snippet: Human HeLa cells (ATCC) with diminished expression of UHRF1 were selected for resistance to puromycin following stable transfection with either HuSH 29mer short hairpin RNA (shRNA) constructs against UHRF1 (5’-AGG AGA CGT TCC AGT GTA TCT GCT GTC AG-3’ and ‘5-TTC GTG GAC GAA GTC TTC AAG ATT GAG CG-3’) or with a control shRNA plasmid (pRS-shGFP, non-effective, Origene), together with a vector conferring puromycin resistance.

Techniques: Irradiation, Control, shRNA, Expressing, Immunofluorescence