Journal: Scientific Reports

Article Title: FADS2 confers SCD1 inhibition resistance to cancer cells by modulating the ER stress response

doi: 10.1038/s41598-024-64043-2

Figure Lengend Snippet: SCD1 inhibitor-resistant cancer cells desaturate palmitic acid to sapienic acid via FADS2. ( A ) Cancer cell lines were treated with the SCD1 inhibitor A939572 (0, 10, 100 nM). Cell viability was assessed by WST-1 assay. ( B , C ) Cancer cells were cultured in RPMI medium or DMEM containing 0.5% serum supplemented with 100 nM A939572 or dimethylsulfoxide (DMSO). ( B ) Total RNA was extracted and FADS2 mRNA was evaluated using qRT-PCR. ( C ) Palmitic, palmitoleic, and sapienic acid levels were evaluated using GC–MS. Data are expressed as means ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. n.s . not significant.

Article Snippet: The membranes were blocked with 3% skim milk (Nacalai Tesque) and probed with the following primary antibodies: mouse or rabbit anti-FADS2 antibody (68026-1-Ig; Proteintech, Rosemont, IL, USA), mouse anti-SCD1 antibody (ab19862; Abcam, Cambridge, UK), rabbit anti-BiP/GRP78 antibody (#3177; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phosphorylated eIF2α (Ser51) antibody (#3398; Cell Signaling Technology), rabbit anti-eIF2α antibody (#9722; Cell Signaling Technology), mouse anti-CHOP antibody (#2895; Cell Signaling Technology), and mouse anti-GAPDH antibody (sc-32233; Santa Cruz Biotechnology, Dallas, TX, USA) at room temperature for 1 h. After washing three times with TBST (25 mM Tris–HCl, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween20, pH7.4), the blots were incubated with goat anti-mouse immunoglobulins/HRP (Dako/Agilent Technologies, Santa Clara, CA, USA) or swine anti-rabbit immunoglobulins/HRP (Dako/Agilent Technologies) secondary antibodies at room temperature for 1 h. Signals were detected using Immobilon Forte Western HRP (Millipore/Merck KGaA) on a Molecular Imager ChemiDoc XRS (Bio-Rad Laboratories, Tokyo, Japan).

Techniques: WST-1 Assay, Cell Culture, Quantitative RT-PCR, Gas Chromatography-Mass Spectrometry

Journal: Scientific Reports

Article Title: FADS2 confers SCD1 inhibition resistance to cancer cells by modulating the ER stress response

doi: 10.1038/s41598-024-64043-2

Figure Lengend Snippet: ER stress state upon SCD1 inhibition differs between SCD1 inhibitor-sensitive and -resistant cancer cells. SCD1 inhibitor-sensitive cancer cells (LK-2) and SCD1 inhibitor-resistant cancer cells (A549) were cultured in RPMI medium containing 0.5% serum supplemented with A939572 or DMSO. Total RNA was extracted from cells and RNA-seq was performed. ( A ) A heat map of ER stress-related genes. ( B ) Comparison of expression levels of genes of interest [ER stress markers (ATF4, ATF6, BID, DDIT3, HSPA5, and XBP1)]. ( C ) The top 20 GO terms of the transcripts upregulated by SCD1 inhibitor treatment in LK-2 cells are shown. Red arrowheads indicate ER stress-related pathways. ( D ) GSEA was used to evaluate the changes in ER stress-related pathways in SCD1 inhibitor-treated LK-2 cells. Data are expressed as means ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. n.s. not significant.

Article Snippet: The membranes were blocked with 3% skim milk (Nacalai Tesque) and probed with the following primary antibodies: mouse or rabbit anti-FADS2 antibody (68026-1-Ig; Proteintech, Rosemont, IL, USA), mouse anti-SCD1 antibody (ab19862; Abcam, Cambridge, UK), rabbit anti-BiP/GRP78 antibody (#3177; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phosphorylated eIF2α (Ser51) antibody (#3398; Cell Signaling Technology), rabbit anti-eIF2α antibody (#9722; Cell Signaling Technology), mouse anti-CHOP antibody (#2895; Cell Signaling Technology), and mouse anti-GAPDH antibody (sc-32233; Santa Cruz Biotechnology, Dallas, TX, USA) at room temperature for 1 h. After washing three times with TBST (25 mM Tris–HCl, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween20, pH7.4), the blots were incubated with goat anti-mouse immunoglobulins/HRP (Dako/Agilent Technologies, Santa Clara, CA, USA) or swine anti-rabbit immunoglobulins/HRP (Dako/Agilent Technologies) secondary antibodies at room temperature for 1 h. Signals were detected using Immobilon Forte Western HRP (Millipore/Merck KGaA) on a Molecular Imager ChemiDoc XRS (Bio-Rad Laboratories, Tokyo, Japan).

Techniques: Inhibition, Cell Culture, RNA Sequencing Assay, Comparison, Expressing

Journal: Scientific Reports

Article Title: FADS2 confers SCD1 inhibition resistance to cancer cells by modulating the ER stress response

doi: 10.1038/s41598-024-64043-2

Figure Lengend Snippet: Knockdown of FADS2 reverses SCD1 inhibitor-induced cell death and fatty acid metabolic profiles. SCD1 inhibitor-resistant A549 cells were transfected with small interfering RNA (siRNA)-FADS2 or siRNA-control (si-negative). ( A ) FADS2 mRNA levels were evaluated by qRT-PCR at 48 h post-transfection. ( B ) FADS2 protein levels were evaluated by western blotting. Original blots are presented in Figure S6. ( C ) A549 cells transfected with siRNA were treated with A939572 (0, 10, 100, 500, 1000 nM). Cell viability was assessed by WST-1 assay. ( D ) The levels of palmitic acid, palmitoleic acid, and sapienic acid in A549 cells transfected with siRNA were evaluated using GC–MS. Data are expressed as means ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n.s. not significant.

Article Snippet: The membranes were blocked with 3% skim milk (Nacalai Tesque) and probed with the following primary antibodies: mouse or rabbit anti-FADS2 antibody (68026-1-Ig; Proteintech, Rosemont, IL, USA), mouse anti-SCD1 antibody (ab19862; Abcam, Cambridge, UK), rabbit anti-BiP/GRP78 antibody (#3177; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phosphorylated eIF2α (Ser51) antibody (#3398; Cell Signaling Technology), rabbit anti-eIF2α antibody (#9722; Cell Signaling Technology), mouse anti-CHOP antibody (#2895; Cell Signaling Technology), and mouse anti-GAPDH antibody (sc-32233; Santa Cruz Biotechnology, Dallas, TX, USA) at room temperature for 1 h. After washing three times with TBST (25 mM Tris–HCl, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween20, pH7.4), the blots were incubated with goat anti-mouse immunoglobulins/HRP (Dako/Agilent Technologies, Santa Clara, CA, USA) or swine anti-rabbit immunoglobulins/HRP (Dako/Agilent Technologies) secondary antibodies at room temperature for 1 h. Signals were detected using Immobilon Forte Western HRP (Millipore/Merck KGaA) on a Molecular Imager ChemiDoc XRS (Bio-Rad Laboratories, Tokyo, Japan).

Techniques: Transfection, Small Interfering RNA, Quantitative RT-PCR, Western Blot, WST-1 Assay, Gas Chromatography-Mass Spectrometry

Journal: Scientific Reports

Article Title: FADS2 confers SCD1 inhibition resistance to cancer cells by modulating the ER stress response

doi: 10.1038/s41598-024-64043-2

Figure Lengend Snippet: Knockdown of FADS2 enhances the ER stress response induced by SCD1 inhibitor. Total RNA was extracted from A549 cells transfected with siRNA-FADS2 or siRNA-control and treated with SCD1 inhibitor, and RNA-seq was performed. ( A ) A heat map of ER stress-related genes. ( B ) Comparison of expression levels of genes of interest [ER stress markers (ATF4, ATF6, BID, DDIT3, HSPA5, and XBP1)].

Article Snippet: The membranes were blocked with 3% skim milk (Nacalai Tesque) and probed with the following primary antibodies: mouse or rabbit anti-FADS2 antibody (68026-1-Ig; Proteintech, Rosemont, IL, USA), mouse anti-SCD1 antibody (ab19862; Abcam, Cambridge, UK), rabbit anti-BiP/GRP78 antibody (#3177; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phosphorylated eIF2α (Ser51) antibody (#3398; Cell Signaling Technology), rabbit anti-eIF2α antibody (#9722; Cell Signaling Technology), mouse anti-CHOP antibody (#2895; Cell Signaling Technology), and mouse anti-GAPDH antibody (sc-32233; Santa Cruz Biotechnology, Dallas, TX, USA) at room temperature for 1 h. After washing three times with TBST (25 mM Tris–HCl, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween20, pH7.4), the blots were incubated with goat anti-mouse immunoglobulins/HRP (Dako/Agilent Technologies, Santa Clara, CA, USA) or swine anti-rabbit immunoglobulins/HRP (Dako/Agilent Technologies) secondary antibodies at room temperature for 1 h. Signals were detected using Immobilon Forte Western HRP (Millipore/Merck KGaA) on a Molecular Imager ChemiDoc XRS (Bio-Rad Laboratories, Tokyo, Japan).

Techniques: Transfection, RNA Sequencing Assay, Comparison, Expressing

Journal: Scientific Reports

Article Title: FADS2 confers SCD1 inhibition resistance to cancer cells by modulating the ER stress response

doi: 10.1038/s41598-024-64043-2

Figure Lengend Snippet: Schematic of the proposed mechanism. SCD1 inhibitor-resistant cancer cells escape ER stress-dependent cell death by avoiding palmitic acid accumulation via FADS2.

Article Snippet: The membranes were blocked with 3% skim milk (Nacalai Tesque) and probed with the following primary antibodies: mouse or rabbit anti-FADS2 antibody (68026-1-Ig; Proteintech, Rosemont, IL, USA), mouse anti-SCD1 antibody (ab19862; Abcam, Cambridge, UK), rabbit anti-BiP/GRP78 antibody (#3177; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phosphorylated eIF2α (Ser51) antibody (#3398; Cell Signaling Technology), rabbit anti-eIF2α antibody (#9722; Cell Signaling Technology), mouse anti-CHOP antibody (#2895; Cell Signaling Technology), and mouse anti-GAPDH antibody (sc-32233; Santa Cruz Biotechnology, Dallas, TX, USA) at room temperature for 1 h. After washing three times with TBST (25 mM Tris–HCl, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween20, pH7.4), the blots were incubated with goat anti-mouse immunoglobulins/HRP (Dako/Agilent Technologies, Santa Clara, CA, USA) or swine anti-rabbit immunoglobulins/HRP (Dako/Agilent Technologies) secondary antibodies at room temperature for 1 h. Signals were detected using Immobilon Forte Western HRP (Millipore/Merck KGaA) on a Molecular Imager ChemiDoc XRS (Bio-Rad Laboratories, Tokyo, Japan).

Techniques: