Cell culture, transfections, and reagents

HuH7 and HepG2 cells were cultured in RPMI 1640 medium (Sigma-Aldrich, Taufkirchen, Germany), HuH6 cells in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, Taufkirchen, Germany) and M2 cells in Minimum Essential Medium Eagle (MEM; Sigma-Aldrich, Taufkirchen, Germany) all supplemented with 10% fetal bovine serum (FBS; Invitrogen, Karlsruhe, Germany) and 1% Penicillin/Streptomycin (Sigma-Aldrich, Taufkirchen, Germany). For transient transfections of siRNA and plasmids lipofectamine RNAiMAX and lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) were used according to the manufacturer’s instructions. The used siRNA sequences (Sigma-Aldrich, Taufkirchen, Germany, Horizon Discovery Cambridge, United Kingdom) and plasmids are listed in the supplemental information (Tables S1 and S2). For stimulation experiments, the cells were first serum starved in a medium supplemented with 0.2% FBS for 16 h and afterward treated with 20 µM LPA (Sigma-Aldrich, Taufkirchen, Germany). Inhibitor pretreatment with 10 µM HA-100 dihydrochloride (HA-100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or 20 µM Ki-16425 (Sigma-Aldrich, Taufkirchen, Germany) was performed for 45 min before addition of LPA.

DNA cloning and plasmids

The mCherry-FLNA Δ571-866 deletion mutant lacking amino acids 571–866 was generated by using mCherry-FilaminA-N-9 (Addgene, Cambridge, MA, USA) and the primers listed in Supplementary Table 3. PECFP-MRTF-A was produced by sub-cloning the CDS of MRTF-A from p3xFLAG-MRTF-A. The S2152A point mutation was introduced in Myc-FLNA wt by site-directed mutagenesis with the QuickChange II Site-directed mutagenesis kit (Agilent, CA; USA).

F-/G-Actin fractionation assay

Actin lysis buffer (50 m MMES pH 6.8, 50 mM KCl, 1 mM EGTA, 1 mM MgCl2, 0.5% Triton X-100 and protease inhibitor (Merck KGaA, Darmstadt, Germany)) was added to the cells to obtain the G-actin fraction before the cells were harvested and sonicated to receive the F-Actin fraction. Each fraction was subjected to immunoblotting using an anti-actin antibody (Sigma-Aldrich, Merck, Darmstadt, Germany).

RNA extraction, cDNA synthesis, and quantitative real-time PCR analysis

For the mRNA isolation, TRIzol Reagent (Invitrogen, Karlsruhe, Germany) was used according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA with SuperScript II Reverse Transcriptase (Invitrogen, Karlsruhe, Germany). Quantitative real-time PCR (qRT-PCR) was performed with SYBR Green I (Roche, Mannheim, Germany) and gene-specific primers (listed in Supplementary Table 4) at the LightCycler 96 system (Roche, Mannheim, Germany). For the normalization of target genes, the endogenous housekeeping control gene 18S rRNA was used.

Cell proliferation assay

Cells were seeded and treated as described above. After the treatment, the number of cells was determined with a Neubauer counting chamber over a period of 5–6 days at intervals of 24 h.

Immunoblotting

Proteins were mixed with Lämmli-buffer, boiled at 95 °C for 10 min, and separated according to their molecular weight by SDS–polyacrylamide gel electrophoresis (SDS–PAGE). Separated proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck, Darmstadt, Germany), which was blocked with 5% milk powder in TBS-T followed by the incubation with the primary and the respective secondary antibodies listed in Tables S5 and S6. Blots were detected via chemiluminescence in a luminescent imager (ChemiDoc Imaging System, Bio-Rad Laboratories, Hercules, CA, USA).

Senescence-associated ß-galactosidase staining

Cellular senescence was determined using the Senescence-ß-galactosidase staining kit according to the manufacturer’s instructions 5–6 days after treatment (Cell Signaling Technology, Danvers, MA, USA). By counting the number of blue cells per 100 cells in triplicates, the percentage of SA-β-gal-positive cells was calculated.

In situ proximity ligation assay (PLA)

The DuoLink In situ Red Starter kits mouse/rabbit and goat/mouse (Sigma-Aldrich, Merck, Darmstadt, Germany) were used according to the manufacturer’s protocol for Duolink In situ solutions (Sigma-Aldrich, Merck, Darmstadt, Germany). Anti-MRTF-A (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-FLNA (Millipore, Merck, Darmstadt, Germany), anti-LPAR-1 (Abcam, Cambridge, UK), and anti-LPAR-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies were used as primary antibodies. Data were visualized with a confocal microscope (Carl Zeiss, Oberkochen, Germany) or a fluorescence microscope (Motic, Wetzlar, Germany), and data were analyzed by 5–15 randomly selected fields.

Immunofluorescence (IF)

4% paraformaldehyde in PBS solution was used (10 min at RT) for the fixation of the cells, followed by permeabilization with 0.2% Triton X-100 in PBS for 7 min at RT. After blocking with 1% BSA in PBS for 30 min at 37 °C the cells were incubated with primary antibodies for 1 h at room temperature followed by the addition of the respective secondary antibodies labeled with Alexa Fluor 488/555/647 (Invitrogen, Karlsruhe, Germany). Used antibodies are listed in Tables S7/S8 in the supplementary material. F-actin filaments were stained with phalloidin coupled to Alexa Fluor 488/555 (Invitrogen, Karlsruhe, Germany), focal adhesions with anti-paxillin antibody, and nuclei with 4’,6-diamidino 2-phenylindole (DAPI; Sigma-Aldrich, Taufkirchen, Germany). Images were taken using a confocal microscope (Carl Zeiss, Oberkochen, Germany) and a fluorescence microscope (Motic, Wetzlar, Germany).

FRET acceptor photobleaching fluorescence resonance energy transfer (FRET)

Cells were seeded on round 30 mm diameter glass slides and transiently transfected with the method of choice. 48 h after transfection media was changed to 0.2% FBS media and cells were starved for 18 h. After starvation, LPA was added to the media for a final concentration of 20 µM and cells were incubated for 45 min at 37 °C (control cells were incubated with water). After stimulation, cells were fixed for 30 min with 4% paraformaldehyde in Hanks’ Balanced Salt Solution (HBSS). Imaging was performed in HBSS at room temperature. The FRET acceptor dsRed was excited with a 514 nm Argon laser, and the emission was detected in the 525–650 nm range. The FRET donor ECFP was excited with a 405 nm laser, and the emission was detected in the 450–490 nm range. Imaging was performed by sequential acquisition with FRET AB-Wizard with a Leica TCS SP5II AOBS confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with an HCX PL APO ×63/1.20 Lambda blue water immersion objective and controlled by the LAS AF software (version 2.7.3.9723, Leica Microsystems). DsRed photobleaching in whole cells was obtained with 10 sequential illuminations at 514 nm (zoom factor 8×). FRET efficiency was calculated using the following formula:

ECFPpre and ECFPpost refer to the ECFP intensity before and after dsRed photobleaching, respectively, and were determined in the entire bleaching region of interest (ROI).

Co-Immunoprecipitation (CoIP)

Cells were lysed in 500 µl immunoprecipitation buffer (containing 0.2% phenylmethylsulfonyl fluoride, 150 mM NaCl, 50 mM Tris–HCl, protease inhibitor cocktail (Merck KGaA, Darmstadt, Germany) and 10% glycerol) and placed on ice for 45 min. Afterward, the samples were centrifuged at 12,700×g for 15 min at 4 °C. Meanwhile the Dynabeads (Invitrogen, Carlsbad, USA) were coupled to the respective antibodies. Used antibodies are listed in Table S9. 55 µl of the bead solution were coupled with 5 µl of the respective antibody by rotating for 30 min at RT and added to the supernatant of the lysed cells. The mixture was rotated overnight at 4 °C, washed with immunoprecipitation buffer, and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis according to the description above.

Human liver biopsies

Tumorous and non-tumorous liver samples were extracted from routine surgical specimens respectively from the same patients and delivered to the Institute of Pathology, Greifswald Medical School. Ethical approval was received from the local Ethics Committee of the Greifswald Medical School (no. BB67/10).

Nuclear and cytoplasmic fractionation assay

Cells were harvested with trypsin and the NE-PER Nuclear and Cytoplasmic Extraction kit was used according to the manufacturer’s protocol (Thermo Scientific, Waltham, USA).

Organoid culture

Biopsies were washed three times with cold PBS and incubated with 2 mM EDTA in PBS for 30 min at 4 °C on a rotating wheel. The supernatant was restored with fresh, cold PBS, centrifuged (200×g, 5 min, 4 °C), and resuspended in Matrigel (Corning, New York, USA). Then 250 µl medium (Organoid Growth Medium, Stem cell, Vancouver, Canada) was added and organoids were cultured at 37 °C in a 5% CO2 atmosphere. The medium was changed every 2–3 days and organoids were passaged once a week. To obtain organoid monolayers for the PLA assay, the organoid medium was removed, and replaced with Cell recovery solution (Corning, New York, USA), and the plate was put on ice for 20 min to dissolve the Matrigel. The organoids were collected with cold PBS in a 15 mL tube and centrifugated at 200×g for 5 min at 4 °C. The pellet was washed with 5 ml cold PBS and again centrifugated, resuspended in 500 µl TripLE Express (Thermo Scientific, Waltham, USA), and incubated at 37 °C for 15 min. Organoids were dissociated into single cells by pipetting vigorously. The single cells were centrifuged at 200×g for 5 min, 4 °C. The pellet was resuspended in organoid medium, and cells were seeded into precoated (Matrigel, PBS 1:50; 1 h; 37 °C) chamber slides (Thermo Scientific, Waltham, USA).

Statistical analysis

Statistical analysis was carried out using Student’s t-test (two-sided) or ANOVA. Unless otherwise indicated, data from three independent experiments were analyzed and values were presented as mean and standard deviation (mean ± SD). Values are considered significant with *p < 0.05, **p < 0.01, and ***p < 0.001.