Summary of Study ST003763

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002346. The data can be accessed directly via it's Project DOI: 10.21228/M8CV66 This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST003763
Study Title	Effect of SCD1 or FADS2 knockdown on fatty acid profile of PDAC cells.
Study Summary	In this work we sought to characterise the impact of knockdown of stearoyl-CoA desaturase 1 (SCD1) and fatty acid desaturase 2 (FADS2) on Panc1 and MiaPaca2 PDAC cells by small interfering RNA (siRN). Panc1 cells exhibit significant Δ6 desaturase activity on saturated fatty acids producing n-10 monounsaturated fatty acids, whereas MiaPaCa2 exhibit little to no FADS2 activity. Knockdown of SCD1 (siSCD) in MiaPaCa2 cells produces a significant decrease in 16:1n-7 when compared with an siRNA negative control (siNEG). In Panc1 cells, siSCD results in a large increase in FADS2-derived 16:1n-10, whereas siFADS2 results in a large increase in SCD1-derived 16:1n-7.
Institute	Victor Chang Cardiac Research Institute
Last Name	Hancock
First Name	Sarah
Address	Level 7 Lowy Packer Building, 405 Liverpool Street, Darlinghurst, NSW 2010, Australia
Email	s.hancock@victorchang.edu.au
Phone	+61414537526
Submit Date	2025-02-20
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2025-03-21
Release Version	1

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Project:

Project ID:	PR002346
Project DOI:	doi: 10.21228/M8CV66
Project Title:	Effect of SCD1 or FADS2 knockdown on fatty acid profile of PDAC cells.
Project Summary:	Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with few treatment options and poor survivability. In this work we sought to characterise changes in fatty acid profile following small interfering RNA knockdown of stearoyl-CoA desaturase 1 (SCD1) or fatty acid desaturase 2 (FADS2).
Institute:	Victor Chang Cardiac Research Institute
Last Name:	Hancock
First Name:	Sarah
Address:	Level 7 Lowy Packer Building, 405 Liverpool Street, Darlinghurst, NSW 2010, Australia
Email:	s.hancock@victorchang.edu.au
Phone:	+61414537526

Subject:

Subject ID:	SU003896
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	source	group1	batch
SA409205	MiaPaCa2_GEMR_siFADS2_p1	Pancreas	MiaPaCa2 cells	siFADS2	1
SA409206	MiaPaCa2_CON_siFADS2_p1	Pancreas	MiaPaCa2 cells	siFADS2	1
SA409207	MiaPaCa2_CON_siFADS2_p2	Pancreas	MiaPaCa2 cells	siFADS2	1
SA409208	MiaPaCa2_CON_siFADS2_p3	Pancreas	MiaPaCa2 cells	siFADS2	1
SA409209	MiaPaCa2_GEMR_siFADS2_p3	Pancreas	MiaPaCa2 cells	siFADS2	1
SA409210	MiaPaCa2_GEMR_siFADS2_p2	Pancreas	MiaPaCa2 cells	siFADS2	1
SA409211	MiaPaCa2_GEMR_siNEG_p1	Pancreas	MiaPaCa2 cells	siNEG	1
SA409212	MiaPaCa2_CON_siNEG_p2	Pancreas	MiaPaCa2 cells	siNEG	1
SA409213	MiaPaCa2_GEMR_siNEG_p3	Pancreas	MiaPaCa2 cells	siNEG	1
SA409214	MiaPaCa2_CON_siNEG_p3	Pancreas	MiaPaCa2 cells	siNEG	1
SA409215	MiaPaCa2_CON_siNEG_p1	Pancreas	MiaPaCa2 cells	siNEG	1
SA409216	MiaPaCa2_GEMR_siNEG_p2	Pancreas	MiaPaCa2 cells	siNEG	1
SA409217	MiaPaCa2_GEMR_siSCD_p1	Pancreas	MiaPaCa2 cells	siSCD	1
SA409218	MiaPaCa2_GEMR_siSCD_p3	Pancreas	MiaPaCa2 cells	siSCD	1
SA409219	MiaPaCa2_CON_siSCD_p3	Pancreas	MiaPaCa2 cells	siSCD	1
SA409220	MiaPaCa2_CON_siSCD_p2	Pancreas	MiaPaCa2 cells	siSCD	1
SA409221	MiaPaCa2_GEMR_siSCD_p2	Pancreas	MiaPaCa2 cells	siSCD	1
SA409222	MiaPaCa2_CON_siSCD_p1	Pancreas	MiaPaCa2 cells	siSCD	1
SA409223	Panc1_CON_siFADS2_p1	Pancreas	Panc1 cells	siFADS2	1
SA409224	Panc1_CON_siFADS2_p3	Pancreas	Panc1 cells	siFADS2	1
SA409225	Panc1_GEMR_siFADS2_p2	Pancreas	Panc1 cells	siFADS2	1
SA409226	Panc1_GEMR_siFADS2_p1	Pancreas	Panc1 cells	siFADS2	1
SA409227	Panc1_CON_siFADS2_p2	Pancreas	Panc1 cells	siFADS2	1
SA409228	Panc1_CON_siFADS2_p4	Pancreas	Panc1 cells	siFADS2	2
SA409229	Panc1_GEMR_siFADS2_p4	Pancreas	Panc1 cells	siFADS2	2
SA409230	Panc1_GEMR_siNEG_p1	Pancreas	Panc1 cells	siNEG	1
SA409231	Panc1_GEMR_siNEG_p2	Pancreas	Panc1 cells	siNEG	1
SA409232	Panc1_CON_siNEG_p3	Pancreas	Panc1 cells	siNEG	1
SA409233	Panc1_CON_siNEG_p2	Pancreas	Panc1 cells	siNEG	1
SA409234	Panc1_CON_siNEG_p1	Pancreas	Panc1 cells	siNEG	1
SA409235	Panc1_GEMR_siNEG_p4	Pancreas	Panc1 cells	siNEG	2
SA409236	Panc1_CON_siNEG_p4	Pancreas	Panc1 cells	siNEG	2
SA409237	Panc1_GEMR_siSCD_p1	Pancreas	Panc1 cells	siSCD	1
SA409238	Panc1_GEMR_siSCD_p2	Pancreas	Panc1 cells	siSCD	1
SA409239	Panc1_CON_siSCD_p2	Pancreas	Panc1 cells	siSCD	1
SA409240	Panc1_CON_siSCD_p3	Pancreas	Panc1 cells	siSCD	1
SA409241	Panc1_CON_siSCD_p1	Pancreas	Panc1 cells	siSCD	1
SA409242	Panc1_GEMR_siSCD_p4	Pancreas	Panc1 cells	siSCD	2
SA409243	Panc1_CON_siSCD_p4	Pancreas	Panc1 cells	siSCD	2
SA409198	Blank_Mia	-	Blank	Blank	1
SA409199	Blank_Panc1	-	Blank	Blank	1
SA409200	Blank_p4	-	Blank	Blank	2
SA409201	FAMEs-mix_02	-	FAMEs	FAMEs	1
SA409202	FAMEs-mix_01	-	FAMEs	FAMEs	1
SA409203	iQC_01	-	pooled QC	iQC	1
SA409204	iQC_02	-	pooled QC	iQC	1

Showing results 1 to 46 of 46

Showing results 1 to 46 of 46

Collection:

Collection ID:	CO003889
Collection Summary:	Samples were obtained from established immortalized adherent human pancreatic ductal adenocarcinoma cells (Panc1 & MiaPaCa2). Cells were cultured in high glucose DMEM containing 4.5 g/L glucose and 4 mM glutamine and were supplemented with 10% FCS. Cells were cultured under 5% CO2 at 37ºC and routinely passaged every 2-3 days (when ~80-90% confluent). Cell lines were regularly screened for mycoplasma infection.
Sample Type:	Pancreas

Treatment:

Treatment ID:	TR003905
Treatment Summary:	Gemcitabine (GEMC) resistant (GEMR) cells were produced by serial treatment with escalating doses of GEMC for approximately 12 weeks. The final concentration of GEMC for Panc1 cells was 150 nM, resulting in a half-maximal inhibitory concentration of 720.5 nM (approximately 2.5 times increase over control (CON) cells, 273.3 nM). The final concentration of GEMC for MiaPaCa2 cells was 64 nM, resulting in a half-maximal inhibitory concentration of 92.2 nM (approximately 3x increase over control (CON) cells, 30.6 nM). Knockdown experiments were performed in adherent cells grown in 6 well plates for 72 h. Cells were seeded at 1-3105 cells/well and allowed to adhere overnight. Cells were transfected with Silencer Select siRNA targeting ACACA (s883), FASN (s5030), SCD (s12505,s12504) FADS2 (s18025,s18023) or negative control (Silencer Select Negative Control No. 1) using Lipofectamine RNAiMAX transfection reagent as per the manufacturer's instructions (ThermoFisher Scientific). Sample groups include source - blank, fatty acid methyl ester (FAMEs) reference standard, pooled internal quality controls (iQC), MiaPaCa2 & Panc1 cells group1 - siRNA knockdown groups, negative control (siNEG), stearoyl-CoA desaturase 1 (siSCD) and fatty acid desaturase 2 (siFADS2). group2 - control (CON) or gemcitabine-resistant (GEMR) cells batch - two different batches were process to ensure a minimum n>2

Treatment ID:

TR003905

Treatment Summary:

Gemcitabine (GEMC) resistant (GEMR) cells were produced by serial treatment with escalating doses of GEMC for approximately 12 weeks. The final concentration of GEMC for Panc1 cells was 150 nM, resulting in a half-maximal inhibitory concentration of 720.5 nM (approximately 2.5 times increase over control (CON) cells, 273.3 nM). The final concentration of GEMC for MiaPaCa2 cells was 64 nM, resulting in a half-maximal inhibitory concentration of 92.2 nM (approximately 3x increase over control (CON) cells, 30.6 nM). Knockdown experiments were performed in adherent cells grown in 6 well plates for 72 h. Cells were seeded at 1-3105 cells/well and allowed to adhere overnight. Cells were transfected with Silencer Select siRNA targeting ACACA (s883), FASN (s5030), SCD (s12505,s12504) FADS2 (s18025,s18023) or negative control (Silencer Select Negative Control No. 1) using Lipofectamine RNAiMAX transfection reagent as per the manufacturer's instructions (ThermoFisher Scientific). Sample groups include source - blank, fatty acid methyl ester (FAMEs) reference standard, pooled internal quality controls (iQC), MiaPaCa2 & Panc1 cells group1 - siRNA knockdown groups, negative control (siNEG), stearoyl-CoA desaturase 1 (siSCD) and fatty acid desaturase 2 (siFADS2). group2 - control (CON) or gemcitabine-resistant (GEMR) cells batch - two different batches were process to ensure a minimum n>2

Sample Preparation:

Sampleprep ID:	SP003902
Sampleprep Summary:	Lipids were extracted from cells grown to 80-90% confluency in 6 well plates using a modified methyl tert-butyl ether (MTBE) method. Adherent cells were washed with ice-cold PBS and scraped into methanol containing 0.01% butylated hydroxytoluene and 100 pmol each of internal standards (phosphatidylcholine 19:0/19:0, phosphatidylethanolamine 17:0/17:0, phosphatidylserine 17:0/17:0, lysophosphatidylcholine 17:0, lysophosphatidylethanolamine 14:0, phosphatidylglycerol 17:0/17:0, phosphatidic acid 17:0/17:0, ceramide d18:1/17:0, dihydrosphingomyelin 12:0, cholesteryl ester 22:1, diacylglycerol 17:0/17:0, D5-triacylglycerol 16:0/16:0/16:0 and cardiolipin 14:0/14:0/14:0/14:0, and 20 nmol of methyl nonadecanoate). An empty well was also scraped for use in background subtraction. To this, MTBE was added at a ratio of 3:1 v/v, and extracts were rotated overnight at 4ºC. Following overnight rotation, 1 part of ice-cold 150 mM ammonium acetate was added, samples were vortexed vigorously and centrifuged at 2000 x g for 5 min. The upper organic phase containing lipids was transferred into a 1.5 mL autosampler vial and dried under N2 at 37ºC Dried lipids were reconstituted in chloroform:methanol:water (60:30:4.5 v/v/v), transferred to a sleeved vial, and stored at -20ºC until analysis. The aqueous phase containing the protein pellet was dried under N2 and digested with 1M NaOH overnight at 4ºC. Digested protein was diluted 1:2 v/v with water and protein concentration was determined using a Pierce BCA assay kit (ThermoFisher Scientific) Following MTBE extraction of lipids, an aliquot was taken and hydrolyzed in 0.6 M KOH in 75% v/v methanol at 60ºC for 30 min. Samples were then cooled to room temperature and neutralized with 25% v/v acetic acid. Water and n-hexane (3:2 v/v) were added separately and samples were vortexed vigorously before being centrifuged at 2000 x g for 5 min. The upper phase containing hydrolyzed fatty acids was removed, and the aqueous phase was washed with a second volume of n-hexane and combined. The combined organic phase was then dried under N2 at 37°C. Dried hydrolyzed fatty acids were derivatized with an AMP+ MaxSpec kit (Cayman Chemical, Ann Arbor, MI, USA) as per manufacturer instructions. Following this, AMP-derivatized fatty acids were re-extracted using MTBE:water (1:1 v/v), and the upper MTBE phase was dried under N2 before being resuspended in methanol. Samples were stored at -20°C until analysis. A 37-component fatty acid methyl ester standard (Merck Life Sciences) containing an additional 3.184 nmol of methyl nonadecanoate was concurrently subjected to the same derivatization procedure and used as an external quality control for fatty acid identification by LC-MS.

Sampleprep ID:

SP003902

Sampleprep Summary:

Lipids were extracted from cells grown to 80-90% confluency in 6 well plates using a modified methyl tert-butyl ether (MTBE) method. Adherent cells were washed with ice-cold PBS and scraped into methanol containing 0.01% butylated hydroxytoluene and 100 pmol each of internal standards (phosphatidylcholine 19:0/19:0, phosphatidylethanolamine 17:0/17:0, phosphatidylserine 17:0/17:0, lysophosphatidylcholine 17:0, lysophosphatidylethanolamine 14:0, phosphatidylglycerol 17:0/17:0, phosphatidic acid 17:0/17:0, ceramide d18:1/17:0, dihydrosphingomyelin 12:0, cholesteryl ester 22:1, diacylglycerol 17:0/17:0, D5-triacylglycerol 16:0/16:0/16:0 and cardiolipin 14:0/14:0/14:0/14:0, and 20 nmol of methyl nonadecanoate). An empty well was also scraped for use in background subtraction. To this, MTBE was added at a ratio of 3:1 v/v, and extracts were rotated overnight at 4ºC. Following overnight rotation, 1 part of ice-cold 150 mM ammonium acetate was added, samples were vortexed vigorously and centrifuged at 2000 x g for 5 min. The upper organic phase containing lipids was transferred into a 1.5 mL autosampler vial and dried under N2 at 37ºC Dried lipids were reconstituted in chloroform:methanol:water (60:30:4.5 v/v/v), transferred to a sleeved vial, and stored at -20ºC until analysis. The aqueous phase containing the protein pellet was dried under N2 and digested with 1M NaOH overnight at 4ºC. Digested protein was diluted 1:2 v/v with water and protein concentration was determined using a Pierce BCA assay kit (ThermoFisher Scientific) Following MTBE extraction of lipids, an aliquot was taken and hydrolyzed in 0.6 M KOH in 75% v/v methanol at 60ºC for 30 min. Samples were then cooled to room temperature and neutralized with 25% v/v acetic acid. Water and n-hexane (3:2 v/v) were added separately and samples were vortexed vigorously before being centrifuged at 2000 x g for 5 min. The upper phase containing hydrolyzed fatty acids was removed, and the aqueous phase was washed with a second volume of n-hexane and combined. The combined organic phase was then dried under N2 at 37°C. Dried hydrolyzed fatty acids were derivatized with an AMP+ MaxSpec kit (Cayman Chemical, Ann Arbor, MI, USA) as per manufacturer instructions. Following this, AMP-derivatized fatty acids were re-extracted using MTBE:water (1:1 v/v), and the upper MTBE phase was dried under N2 before being resuspended in methanol. Samples were stored at -20°C until analysis. A 37-component fatty acid methyl ester standard (Merck Life Sciences) containing an additional 3.184 nmol of methyl nonadecanoate was concurrently subjected to the same derivatization procedure and used as an external quality control for fatty acid identification by LC-MS.

Combined analysis:

Analysis ID	AN006178
Chromatography ID	CH004690
MS ID	MS005883
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Agilent 1290 Infinity II
Column	Thermo Accucore C30 (150 x 2.1 mm, 2.6 μm)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Agilent 6560 Ion Mobility
Ion Mode	POSITIVE
Units	nmol/mg protein

Chromatography:

Chromatography ID:	CH004690
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Thermo Accucore C30 (150 x 2.1 mm, 2.6 μm)
Column Temperature:	30°C
Flow Gradient:	0-3.25 min increase from 50% B to 56% B, 3.25-6.5 min increase to 58% B, 6.5-7.5 min increase to 80% B, 7.5-9.5 min increase to 100% B, hold at 100% B for 1.5 mins, re-equilibrated at 50% B for 4.5 mins.
Flow Rate:	0.4 mL/min
Solvent A:	100% Water; 0.1% Formic acid
Solvent B:	100% Acetonitrile; 0.1% Formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS005883
Analysis ID:	AN006178
Instrument Name:	Agilent 6560 Ion Mobility
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	AMP-derivatized fatty acids were detected as positive ions in QTOF-only mode on an Agilent 6560 IM-MS QTOF mass spectrometer (Agilent Technologies). Source conditions were as follows: capillary voltage of 3.5 kV, nozzle voltage of 1 kV and fragmentor at 360 V. Nitrogen was used as the sheath and collision gas, with drying gas set at 5 L/min and sheath gas at 12 L/min (both gases heated to 300°C) and the nebulizer pressure set at 20 psi. Ions were acquired in Auto MS/MS mode at an MS1 mass range of m/z 100-1000 and MS2 mass range of m/z 50-600. Spectra were acquired in MS1 mode at 5 spectra/s and 200 ms/spectrum, and in MS2 mode at 10 spectra/s and 100 ms/spectrum. Precursor ions were isolated using a window of 1.3 Th, acquisition time of 50 ms/spectra, and collision energy of 45 eV. Calibration mix A was infused throughout and masses were automatically detected and corrected using a 100 ppm window and minimum height of 1x10^3 counts to ensure 5 ppm mass accuracy, with m/z 121.050873 and 922.009798 used as reference ions. AMP-derivatized fatty acids as [M+H]+ were aligned using Mass Profinder v10.0 (Agilent Technologies) with an RT tolerance of 0.5 min and MS1 tolerance of 10 ppm. Lipids were identified using a custom personal compound database and library (PCDL) generated from the RT of external quality control samples (attached) and MS/MS spectra were manually inspected to ensure correct MUFA isomer identification. Using this method, FA 18:1 isomers were only partially separated and so these data were not analyzed further. Data were exported, blank subtracted and quantified from internal standards before being normalized to total protein.
Ion Mode:	POSITIVE