Journal: bioRxiv

Article Title: The Balance between Saturated and Unsaturated Fatty Acids Regulates Ovarian Cancer Cell Fate

doi: 10.1101/2022.05.24.493247

Figure Lengend Snippet: (A) XBP1 splicing (u, unspliced transcript; s, spliced transcript) measured by RT-PCR and agarose-gel electrophoresis in OVCAR-5 cells transduced with control shRNA (shCtrl) or shRNAs (1 or 2) targeting SCD (shSCD) and cultured in low serum conditions (1% FBS) for 48 hours. (B) Densitometric analysis of XBP1 splicing products shown in A. Bars represent percent of spliced XBP1 relative to total XBP1 (mean ± SD, n = 3). (C) XBP1 splicing (u, unspliced transcript; s, spliced transcript) in OVCAR-5 cells cultured in low serum conditions and treated with SCD inhibitor CAY10566 for 48 hours. (D) Percent of spliced XBP1 isoform measured by densitometric analysis of PCR products shown in C (mean ± SD, n = 3). (E, F) XBP1 splicing (u, unspliced transcript; s, spliced transcript) (E), and western blot of proteins of the PERK/eIF2α/ATF4 axis (F) in shCtrl and shSCD OVCAR-5 cells cultured under low serum conditions and treated with 50μM palmitic acid for the time periods indicated. (G) XBP1 splicing (u, unspliced transcript; s, spliced transcript) in primary cells from tumors of ovarian cancer patients cultured in low serum conditions and treated with 3μM CAY10566 for 48 hours, or with 1μM CAY10566 and 50μM palmitic acid for 12 hours. (H) Representative SRS images in the C-H and C-D regions of OVCAR-5 shCtrl and shSCD cells cultured in low serum conditions (1% FBS) and treated with 12.5μM palmitic acid-d31 (PA-d31), with or without 52μM oleic acid (OA), or cultured in medium with full serum (10% FBS) and treated with PA-d31 for 24 hr. Yellow arrows indicate rigid ER, gray arrows indicate lipid droplet (LD), and blue arrows indicate cytoplasm. Scale bar: 20 µm. (I) Percentages of shCtrl and shSCD OVCAR-5 cells treated as described in (H) showing C-D SRS signal mainly in rigid ER, lipid droplet (LD), and cytoplasm (n = 139-191). (J) Transmission electron microscopy imaging of smooth ER (red arrows) in OVCAR-5 shCtrl vs shSCD cells cultured in low serum conditions for 48 hours. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: CAY10566 (cat#: HY-15823) and PEG300 (cat#: HY-Y0873) were purchased from MedChemExpress.

Techniques: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Transduction, shRNA, Cell Culture, Western Blot, Transmission Assay, Electron Microscopy, Imaging

Journal: bioRxiv

Article Title: The Balance between Saturated and Unsaturated Fatty Acids Regulates Ovarian Cancer Cell Fate

doi: 10.1101/2022.05.24.493247

Figure Lengend Snippet: (A, B) XBP1 splicing (u, unspliced transcript; s, spliced transcript) measured by RT-PCR and agarose gel electrophoresis in OVCAR-5 cells transduced with control shRNA (shCtrl) or shRNAs (1 or 2) targeting SCD (shSCD), cultured in medium containing low serum, and treated with indicated doses of oleic acid for 48 hours (A), or with 50μM palmitic acid and indicated doses of oleic acid for 12 hours (B). (C, D) Western blot of SCD and proteins of the PERK/eIF2α/ATF4 axis in shCtrl and shSCD cells cultured in low serum medium, and treated with different doses of oleic acid for 48 hours (C), or with 50μM palmitic acid and indicated doses of oleic acid for 12 hours (D). (E, F) XBP1 splicing (u, unspliced transcript; s, spliced transcript) in OVCAR-5 cells cultured in medium containing low serum and treated with 21.6nM CAY10566 and indicated doses of oleic acid for 48 hours (E), or with 8.1nM CAY10566, 50μM palmitic acid and indicated doses of oleic acid for 12 hours. (G) Proteins of the PERK/eIF2α/ATF4 pathway measured by western blot in OVCAR-5 cells treated as described in (E). (H) Western blot of proteins of the PERK/eIF2α/ATF4 pathway in OVCAR-5 cells treated as described in (F). Arrows indicate the band of interest. (I, J) Verification of SCD overexpression by qRT-PCR (I) and western blotting (J) in OVCAR-5 cells transduced with a SCD expression vector (pLenti-SCD). Cells transduced with empty vector (pLenti-Ctrl) served as control. Bars represent means ± SD, n = 3. (K) XBP1 splicing (u, unspliced transcript; s, spliced transcript) in pLenti-SCD vs pLenti-Ctrl cells cultured in low serum conditions and treated with 50μM palmitic acid for 12 hours. (M) Percent of spliced isoform (mean ± SD, n = 3) calculated by densitometric analysis of PCR products shown in (L). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: CAY10566 (cat#: HY-15823) and PEG300 (cat#: HY-Y0873) were purchased from MedChemExpress.

Techniques: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Transduction, shRNA, Cell Culture, Western Blot, Over Expression, Quantitative RT-PCR, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: The Balance between Saturated and Unsaturated Fatty Acids Regulates Ovarian Cancer Cell Fate

doi: 10.1101/2022.05.24.493247

Figure Lengend Snippet: (A-B) Time-lapse imaging of Annexin V staining to measure apoptosis in OVCAR-5 cells transduced with control shRNA (shCtrl) or shRNA targeting SCD (shSCD), cultured in low serum conditions, and treated with 52μM oleic acid (A), or with 50μM palmitic acid alone or in combination with 52μM oleic acid (B). (C-D) Time-lapse of Annexin V imaging to measure apoptosis in OVCAR-5 cells cultured in low serum conditions and treated with 21.6nM CAY10566, 52μM oleic acid or combination (C), or with 8.1nM CAY10566, 50μM palmitic acid, 52μM oleic acid, or combinations (D). (E) Western blot of full-length and cleaved caspase-3 in shSCD vs shCtrl cells cultured under low serum medium and treated with 50μM palmitic acid and indicated doses of oleic acid for 12 hours. (F) Western blot of full-length and cleaved caspase-3 in OVCAR-5 cells cultured in low serum medium and treated with 8.1nM CAY10566, 50μM palmitic acid and indicated doses of oleic acid for 12 hours. (G) Time-lapse of Annexin V imaging to determine apoptosis in OVCAR-5 cells overexpressing SCD (pLenti-SCD) and control cells (pLenti-Ctrl), cultured in medium containing low serum and treated with 50μM palmitic acid. Values in panels A-D & G are means ± SD, n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: CAY10566 (cat#: HY-15823) and PEG300 (cat#: HY-Y0873) were purchased from MedChemExpress.

Techniques: Imaging, Staining, Transduction, shRNA, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: The Balance between Saturated and Unsaturated Fatty Acids Regulates Ovarian Cancer Cell Fate

doi: 10.1101/2022.05.24.493247

Figure Lengend Snippet: (A-C) Total tumor weight (A), total tumor volume (B) and total number of metastases (C) in athymic nude mice intraperitoneally injected with OVCAR-5 cells transduced with control shRNA (shCtrl) or shRNA targeting SCD (shSCD), and evaluated after 28 days (values are means ± SE, n = 14 per group). (D) qRT-PCR measurements of SCD expression (mean ± SD, n = 6) in a random sample of tumor xenografts described in (A). (E, F) Agarose gel electrophoresis of XBP1 splicing products (u, unspliced transcript; s, spliced transcript) (E), and percent spliced XBP1 isoform estimated by image analysis of transcript bands (mean ± SD, n = 6) (F) in a random sample of tumor xenografts described in (A). (G, H) Ascites volume (G) and total number of metastases (H) in athymic nude mice intraperitoneally injected with OVCAR-5 cells, fed with a palmitic acid-rich diet or control diet, and treated with SCD inhibitor CAY10566 or vehicle for 28 days. Values are means ± SE, n = 10. (I-L) XBP1 splicing products (u, unspliced transcript; s, spliced transcript) (I, K), and percent intensity of the spliced transcript estimated by image analysis (J, L) in a random sample (n = 5) of tumor xenografts described in (F). Values are means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: CAY10566 (cat#: HY-15823) and PEG300 (cat#: HY-Y0873) were purchased from MedChemExpress.

Techniques: Injection, Transduction, shRNA, Quantitative RT-PCR, Expressing, Agarose Gel Electrophoresis

Journal: eLife

Article Title: Reducing lipid bilayer stress by monounsaturated fatty acids protects renal proximal tubules in diabetes

doi: 10.7554/eLife.74391

Figure Lengend Snippet: ( A, C, E ) Quantitative RT-PCR detection of ER stress markers in induced renal epithelial cells (iRECs) treated 16 hr with several combinations of BSA-bound fatty acids ( A ), palmitic acid (PA) plus ER stress signaling inhibitors ( B ), PA plus SCD1 inhibitor ( C ), and tunicamycin (10 µM) and thapsigargin (1 µM) plus oleic acid (OA) ( E ). ( B ) Cytotoxicity in iRECs at 36 hr treatment with PA 250 µM plus the inhibitors of PERK, ATF6, and IRE1α. Fold representation of object counts/confluence. ( D ) Cytotoxicity in iRECs at 24 hr treatment with PA 250 µM plus the SCD1 inhibitor. Fold representation of object counts/confluence. Data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = nonsignificant; two-way ANOVA and Holm–Sidak’s multiple comparisons test; ( D ) n = 4; ( A–C, E ) n = 3.

Article Snippet: Desaturation of fatty acids was inhibited by the SCD1 inhibitor CAY10566 (MedChemExpress, #HY-15823-1mg).

Techniques: Quantitative RT-PCR

Journal: eLife

Article Title: Reducing lipid bilayer stress by monounsaturated fatty acids protects renal proximal tubules in diabetes

doi: 10.7554/eLife.74391

Figure Lengend Snippet: ( A ) Representative images of LDs stained using BODIPY in induced renal epithelial cells (iRECs) treated for 16 hr with bovine serum albumin (BSA), palmitic acid (PA) 250 µM, oleic acid (OA) 250 µM, PA 250 µM plus OA 125 µM, BSA plus the SCD1 inhibitor CAY10556 (2.5 µM) and PA plus CAY10556 (2.5 µM). Scale bars: 10 μm. ( B–E ) Quantification of LD number ( B, D ) and LD average volume ( C, E ) in iRECs treated for 16 hr with BSA, PA 250 µM, OA 250 µM, PA 250 µM plus OA 125 µM, BSA plus the SCD1 inhibitor CAY10556 (2.5 µM) and PA plus CAY10556 (2.5 µM). Every dot represents the measurement in one single cell. Data information: in ( B–E ), data are presented as the mean + all values. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Kruskal–Wallis plus Dunn’s multiple comparisons test. ( B–E ) 10 cells per field from three fields were analyzed for three independent biological replicates.

Article Snippet: Desaturation of fatty acids was inhibited by the SCD1 inhibitor CAY10566 (MedChemExpress, #HY-15823-1mg).

Techniques: Staining

Journal: eLife

Article Title: Reducing lipid bilayer stress by monounsaturated fatty acids protects renal proximal tubules in diabetes

doi: 10.7554/eLife.74391

Figure Lengend Snippet:

Article Snippet: Desaturation of fatty acids was inhibited by the SCD1 inhibitor CAY10566 (MedChemExpress, #HY-15823-1mg).

Techniques: Enzyme-linked Immunosorbent Assay, Sequencing