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Cell Biology International (2010) 34, 737–746 (Printed in Great Britain)

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A new protocol for functional analysis of adipogenesis using reverse transfection technology and time-lapse video microscopy

Elke Grönniger¹, Sonja Wessel¹, Sonja Christin Kühn, Jörn Söhle, Horst Wenck, Franz Stäb and Marc Winnefeld²

Research and Development, Research Special Skincare, Beiersdorf AG, Unnastrasse 48, 20245 Hamburg, Germany

¹These authors contributed equally to this work.

²To whom correspondence should be addressed (email marc.winnefeld@beiersdorf.com).

This supplementary data is also available as a PDF

SUPPLEMENTARY ONLINE DATA

1. Material and methods

1.1. Conventional transfection of human preadipocytes

One day prior to transfection, human preadipocytes (1.5×10⁵) were seeded in six-well cavities. Before transfection, cells were washed with DPBS (Dulbecco’s phosphate-buffered saline; Cambrex), and 2 ml OPTI-MEM (Gibco BRL) was added. Then, 100 pmol of siRNA (final concentration, 30 nM) was diluted into OPTI-MEM to a final volume of 250 μl. Next, 5 μl of Lipofectamine 2000 (Invitrogen) was mixed into 245 μl of OPTI-MEM and incubated for 5 min. These solutions were mixed and incubated for another 20 min at room temperature. The transfection solution was then added to the 2 ml of OPTI-MEM covering the cells. After 8 h, 2.5 ml of growth medium (Cambrex) containing 2 mM l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin and 20% (v/v) fetal calf serum (Cambrex) was added to the medium.

1.2. Determination of PPARγ knock-down efficiency

Human preadipocytes (1.5×10⁵) were cultured in six-well cavities and transfected using PPARγ–siRNA as described above. After incubation in basal growth medium for 4 days, differentiation into adipocytes was initiated as described above. Cells were harvested on day 2 after induction of differentiation and the illustra RNAspin Mini RNA Isolation Kit (GE Healthcare) was used to isolate total RNA following the manufacturer’s protocol. RNA concentration and purity were assessed spectrophotometrically using NanoDrop® ND-1000 in combination with the software NanoDrop® V3.3.0 (peQLab). Total RNA was reverse transcribed into cDNA using Oligo (dT)-Primer (Invitrogen). The resulting cDNA was analysed for PPARγ mRNA expression by Real-Time TaqMan®-PCR using the 7900HT Fast-Real-Time PCR System (Applied Biosystems). FAM-labelled primers for the qRT-PCR (Applied Biosystems) were as follows: Inventoried TaqMan Assays for the internal control GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Hs99999905_m1) and for the target RNA PPARγ (Hs00234592_m1). PCR conditions were as follows: 95°C for 20 s followed by 40 cycles at 95°C for 1 s and 60°C for 20 s. Real-time PCR data were analysed utilizing the Sequence detector version 2.3 software supplied with the 7900HT Fast-Real-Time PCR System (Applied Biosystems). Quantification was achieved using the method, which calculates the relative changes in gene expression of the target normalized to an endogenous reference (GAPDH) and relative to a calibrator that serves as the control group.

1.3. Software used to prepare Supplementary Figure 1 and Supplementary Movies

For Supplementary Figure S1, Excel and PowerPoint. For Supplementary Movies S1–S5, ScanˆR (Olympus), ImageJ 1.40g and PowerPoint.

2. Results

2.1. PPARγ knock-down efficiency

To determine the knock-down efficiency of the PPARγ–siRNA, cell populations were conventionally transfected with scrambled siRNA or PPARγ–siRNA respectively. Subsequently, mRNA levels were analysed by qRT-PCR. Compared with controls, the mRNA levels of PPARγ clearly decreased 6 days after transfection with PPARγ-specific siRNA (Supplementary Figure 1). The conventional transfection strategy was used because no efficient PPARγ-specific antibody was commercially available, and therefore a knock-down following reverse transfection could not be shown in cell culture. However, the results clearly confirm the functionality of the PPARγ-specific siRNA used for our experiments.

Figure S1 Specific down-regulation of PPARγ mRNA levels after siRNA treatment

Preadipocytes were ‘conventionally’ transfected with PPARγ–siRNA or scrambled siRNA in six-well plates. Differentiation was initiated after 4 days and PPARγ–mRNA levels normalized to GAPDH were determined by qRT-PCR 6 days post-transfection. The PPARγ level of control cells (without siRNA) was set as 100%. Four independent experiments were prepared (n = 4). Results are depicted as means±S.D.

Movie S1

Live-cell imaging using time-lapse video microscopy showing fat accumulation during siRNA treatment

Pre-adipocytes were grown on Lab-Tek chamber slides, cover Permanox spotted with scrambled-siRNA (2 μl spot volume). After 4 days, differentiation was initiated. After 3 additional days, cell plates were transferred to an incubation chamber (humidified atmosphere of 5% CO2 at 37°C) associated with a ScanˆR microscope (Olympus) and video microscopy was started. Bright-field images were acquired for 4 days with a time-lapse interval of 15 min.

Movie S2

Live-cell imaging using time-lapse video microscopy showing fat accumulation during PPARγ knock-down in human (pre)adipocytes

Preadipocytes were grown on Lab-Tek chamber slides, cover Permanox spotted with PPARγ-siRNA (2 μl spot volume). After 4 days, differentiation was initiated. After 3 additional days, cell plates were transferred to an incubation chamber (humidified atmosphere of 5% CO2 at 37°C) associated with a ScanˆR microscope (Olympus) and time-lapse video microscopy was started. Bright-field images were acquired for 4 days with a time-lapse interval of 15 min.

Movie S3

Real-time fluorescent microscopy showing the incorporation of fluorescently labelled fatty acids into (pre)adipocytes reverse transfected with scrambled-siRNA

Pre-adipocytes were reverse transfected with scrambled-siRNA and after 3 additional days differentiation was induced. Fluorescence intensity was time-lapse recorded with an interval of 60 min per image for 93 h starting on day 7 after initiation of differentiation. Incorporation of fatty acids into cells was measured using the ScanˆR microscope.

Movie S4

Real-time fluorescent microscopy showing the incorporation of fluorescently labelled fatty acids into (pre)adipocytes reverse transfected with PPARγ-siRNA

Pre-adipocytes were reverse transfected with PPARγ-siRNA and after 3 additional days differentiation was induced. Fluorescence intensity was time-lapse recorded with an interval of 60 min per image for 93 h starting on day 7 after initiation of differentiation. Incorporation of fatty acids into cells was measured using the ScanˆR microscope.

Movie S5

Real-time fluorescent microscopy showing the incorporation of fluorescently labelled fatty acids into (pre)adipocytes reverse transfected with TIP60-siRNA

Pre-adipocytes were reverse transfected with TIP60-siRNA and after 3 additional days differentiation was induced. Fluorescence intensity was time-lapse recorded with an interval of 60 min per image for 93 h starting on day 7 after initiation of differentiation. Incorporation of fatty acids into cells was measured using the ScanˆR microscope.

Received 9 October 2009/11 February 2010; accepted 1 April 2010

Published as Cell Biology International Immediate Publication 1 April 2010, doi:10.1042/CBI20090299

© The Author(s) Journal compilation © 2010 Portland Press Limited