Transfectin reagent

HEK293 cells (ATCC, CRL-11268) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Transient transfections were performed with TransFectin reagent (Bio-Rad, #1703352). Antibody used for LUMIER experiments was mouse anti-FLAG (Sigma-Aldrich, #F3165). The expression constructs were cloned using cDNA as PCR templates for amplifying the inserts (CDK6: Gene ID 12571, p16: Gene ID 12578), digestion with restriction enzymes and ligation into a Flag or NLuc vector.

HEK293T, HEK293 β2AR [36 (link)] cells and U87-MG cells (ATCC^® HTB-14) were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The colon cancer cell lines SW480 (ATCC^® CCL-228) and SW620 (ATCC^® CCL-227) were grown in RPMI-1640 media supplemented with 10% FBS. Cells were grown in water-saturated, 5% CO₂ atmosphere. Transient transfections were performed with Transfectin reagent (Bio-Rad, #1703352). Forskolin was purchased from MCE Med Chem Express (#HY-15371), Isoproterenol was purchased from Sigma (#I6504), KT5720 was purchase from Enzo Life Sciences (BML-EI199-0100), and employed with indicated concentrations and time frames. Primary antibodies used were the mouse anti-GFP antibody (Roche, #11814460001, Germany), the rabbit polyclonal Phospho-PKA Substrate (RRXS*/T*) antibody (Cell Signaling, #9624), the rabbit polyclonal TAF15 antibody (Cell Signaling, #13150), the mouse monoclonal (mAb) Anti- PKA RIα (D54D9) antibody (BD Biosciences, #610166), and the mouse monoclonal Anti-PKAc antibody (BD Biosciences, #610981). Lamin A/C (Cell Signaling, #4777) Mouse mAb, GAPDH (Cell Signaling, #2118) Rabbit mAb, Phospho-VASP (Ser157) (Cell Signaling, #84519) Rabbit mAb.

U2OS, COS-1, MRC-5 and CV-1 cells (American Type Culture Collection, Manassas, VA) were transfected with either an EGFP-C1 plasmid or a tandem dimeric EGFP (EGFP₂) plasmid as described previously [8] (link). These mammalian cells were maintained in a mixture of DMEM medium and 10% fetal bovine serum (Hyclone Laboratories, Logan, UT). U2OS, CV-1, COS-1 and MRC-5 cells were transfected using TransFectin reagent (Bio-Rad, Hercules, CA) according to the manufacturer's instructions 24 hours before measurement. All cells were subcultured into eight-well coverglass chamber slides (Nalge Nunc International, Rochester, NY) with the media replaced by Leibovitz L15 medium (Gibco, Auckland, NZ) immediately before measurement. FFS measurements on cells were performed as previously described [14] (link). For photodepletion experiments, cells were exposed repeatedly for short time intervals to epifluorescence light. After each exposure the instrument performed a short two-photon FFS measurement to record the brightness and the photodepletion fraction.

The SAS, FaDu, OECM1 OSCC cell lines, and H1299 NSCLC cell lines were cultured as previously described [2 , 24 (link)]. The HEK293T cell line was utilized for virus production for infection. Oral mucosal fibroblast-1 (OMF-1) was a kind gift from Prof. Li, W.C. All cell lines have been authenticated using Short Tandem Repeats (STR) profiling within three years. All cultivated conditions were mycoplasma-free. mirVana miR-432-5p mimic (Cat No. #4,464,066) and scr control were purchased from ThermoFisher Scientific (Waltham, MA, USA). Protein secretion was inhibited by BFA (Befeldin A; Sigma-Aldrich, St Louise, MO, USA) [39 (link)]. The Transfectin™ Reagent (BioRad, Hercules, CA, USA) was employed for transfection. The cell viability was assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or trypan blue (both reagents purchased from Sigma-Aldrich). The fold changes in viability across different time points were plotted and transformed into semi-log graphs. The differences in growth curves and population doubling time (PDT) in the exponential growth phase were analyzed. Cell migration, invasion, and co-culture were analyzed using transwell (Millicell Hanging Cell Culture Insert; Merck, Darmstadt, Germany) assays [2 ]. Unless specified, all other materials were purchased from Sigma-Aldrich.

Short interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). siRNAs were dissolved in transfection diluent according to the manufacturer’s protocols. Transformed CT1 fibroblasts and WHCO1 cancer cells were transfected with COL1A1 and Fibronectin siRNAs using Transfectin reagent (BioRad, Hercules, CA, USA) and ECM synthesis continued for the indicated period. To maintain knockdown efficiency, subsequent transfections were carried out every 3 days for the duration of the ECM synthesis. In a separate experiment, type I collagen knockdown was achieved by removing ascorbic acid during ECM synthesis with a similar result compared to the use of COL1A1 siRNA. Cells were cultured in 6-well plates with or without the addition of ascorbic acid and ECM synthesis was continued for the duration indicated above [218 (link)]. Confirmation of type I collagen and fibronectin knockdowns was done using immunoblot and SDS PAGE analysis.

N2A cells were cultured in DMEM medium with high glucose, FBS and PenStrep. These cells were transfected with expression constructs at 60–70% confluency with Transfectin reagent (BioRad, 1703352). Protein lysates were isolated after 48 hours and processed for immunoprecipitation using α-Flag antibody (Sigma, F3165). Dynabeads were used for the IP and proteins were eluted in Lämmli-buffer containing 2-mercaptoethanol, boiled for 10′. Protein samples were separated using 12% SDS-poly-acrylamide gels and transferred to Nitrocellulose membranes (Protan, GE). Primary antibody α-HA antibody (Cell Signalling, 3724) was incubated with the membrane overnight at 4 °C. Secondary antibody horse radish peroxidase conjugated α-rabbit-Ig (Jackson Immunoresearch Labs, 711-035-152) incubation was performed for 1h at RT. Detection was done by chemiluminescence (ECL, GE Healthcare).

HT29 (Lot. 09K003) and HT29-MTX-E12 (Lot. 18K206) cell lines were obtained from ECACC. HT29 cells were cultured in RPMI (Merck) containing 10% fetal bovine serum (v/v) (Gibco), 1% sodium pyruvate (Gibco), 2 g/l sodium carbonate (Sigma), and 1% penicillin/streptomycin (Gibco). For differentiating into mucin-secreting goblet-like cells, HT29 cells were grown in glucose-free DMEM (Gibco), supplemented with galactose (250 mM) for 3 d. HT29-MTX-E12 cells were grown in DMEM GlutaMAX medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (v/v), 1% sodium pyruvate, and 1% penicillin/streptomycin. Plasmid transfections and siRNA-mediated knockdowns were done, along with cell seeding using Transfectin reagent (Bio-Rad) and Dharmafect reagent (Horizon Discovery), respectively. To inhibit lysosomal and proteasomal degradation, cells were treated with 100 nm Bafilomycin (Sigma) and 10 µM MG132 (Sigma) for 6 hr and 8 hr, respectively.