NIH 3T3 Cells

The PH domains of PLCδ₁ (1–170), Bruton's tyrosine kinase (1–177), Akt protein kinase (1–167), and dynamin (508–652) were amplified with the Advantage Klentaq polymerase mix (CLONTECH Labs, Inc., Palo Alto, CA) from human cDNAs (marathon cDNA from brain and K562 leukemia cells; CLONTECH Labs, Inc.) with the following primer pairs:
PLCδ: 5′-GGCATGGACTCGGGCCGGGACTTCCTG-3′, 5′-AAGATCTTCCGGGCATAGCTGTCG-3′;
Btk: 5′-CCAAGTCCTGGCATCTCAATGCATCTG-3′,
5′-TGGAGACTGGTGCTGCTGCTGGCTC-3′;
Akt: 5′-GTCAGCTGGTGCATCAGAGGCTGTG-3′,
5′-CACCAGGATCACCTTGCCGAAAGTGCC-3′;
Dyn: 5′-ATGCTCAGCAGAGGAGCAACCAGATG-3′,
5′-GAGTCCACAAGATTCCGGATGGTCTC-3′.
The amplified products were subcloned into the PGEM-Easy T/A cloning vector (Promega Corp., Madison, WI) and sequenced with dideoxy sequencing (thermosequenase; Amersham Corp.). A second amplification reaction was performed from these plasmids with nested primers that contained restriction sites for appropriate cloning into the pEGFP-N1 (PLCδ, Btk, and Akt) or pEGFP-C1 (dynamin) plasmids (CLONTECH Labs, Inc.) to preserve the reading frame. Plasmids were transfected into COS-7 cells or NIH-3T3 cells and cell lysates were resolved by SDS-PAGE followed by Western blot analysis for the presence of the GFP fusion proteins using a polyclonal antibody against GFP (CLONTECH Labs, Inc.).
Mutations were created in the PH_PLCδ–GFP fusion plasmid by the QuickChange™ mutagenesis kit (Stratagene, La Jolla, CA). For practical purposes, a SalI site was introduced into the PH domain sequence which changed S34 to a T but this substitution did not change any characteristic compared with the wild-type protein. All mutations were confirmed by dideoxy sequencing and the expression of the fusion protein by Western blot analysis.

L929, NIH 3T3, and HeLa-B cells were grown in DME medium (GIBCO) supplemented with 10% fetal calf serum.
Centrin cDNA was sub-cloned in the pEGFP-N1 vector (Clontech) and cells were transformed by electroporation. Stable clones expressing the centrin/GFP fusion protein were then isolated from each of the three parental cell lines, by using the limited dilution method in the presence of 500 μg/ml G418. Multiple independent clones, expressing ∼20 times the endogenous level of centrin, were kept for each of the three parental cell lines. The centrioles in all of the clones exhibited a similar behavior to that described here.
Synchronization in early G₁ was accomplished by a single thymidine block of 20 h (5 mM thymidine for L929 and 2 mM for HeLa). Mitotic cells were then collected by shakeoff 8 or 20 h after releasing the block (for L929 and HeLa, respectively). Cells were then replated on coverslips and used 2 h later. Synchronization at the G₁/S border was accomplished using the double-thymidine block technique. To determine the duration of S and G₂ we incubated cells at various times after thymidine washout with 30 μM BrdU for 15 min and then analyzed them by FACS^®. The maximum content of S cells (by BrdU incorporation) was reached 1–4 h after release. The maximum number of G₂ cells (double DNA content and no BrdU incorporation) was observed ∼5 h after release in L929 cells and 9–10 h in HeLa cells.

NIH3T3 cells (RRID: CVCL_M025) were cultured in a medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution (HyClone) in a humidified atmosphere of 5% CO₂ at 37°C, and the medium was changed every 2 days. Cell cytotoxicity was measured using the MTT assay. Before the MTT assay, all samples were prepared in 48-well plates. The cells were seeded onto the MNs in the 48-well plates. After culturing for 1, 3, and 7 days, 400 μl of MTT solution (5 μg/ml) was added to each well, and the cells were incubated at 37°C for 4 hours. After the medium was removed, 400 μl of dimethyl sulfoxide was added to each well, and the plates were shaken for 15 min on a shaking table. The supernatant fluid was assessed using a SpectraMax i3 platform (Molecular Devices, CA, USA). To align with the antimicrobial experimental protocol, all the MN samples used in the in vitro cell experiments were also exposed to ultrasound stimulation (1.0 MHz, 1.5 W cm⁻², and 50% duty cycle) for 15 min on the day 1 of cell culture and the culture continued until day 7. The cell viability was assessed on days 1, 3, and 7 by MTT assay. At day 1, the cells were washed thrice with PBS (pH 7.4), fixed in 4% formaldehyde solution for 10 min at room temperature, and rinsed thoroughly with PBS. Afterward, the samples were stained with fluorescein isothiocyanate–phalloidin (YiSen, Shanghai) at room temperature in darkness for 30 min and further stained with 4′,6-diamidino-2-phenylindole (YiSen, Shanghai) for 30 s in darkness. The cell morphology of different samples was examined using an IFM (Olympus, IX73). For the transcriptome sequencing test and qRT-PCR analysis, 2 ml of NIH3T3 cell suspensions (1 × 10⁵ cells/ml) was cultured in six-well plates with different samples for 72 hours. The cell lysates were then stored at −80°C before sequencing using a triazolo reagent (Beyotime Biotechnology). RNA sequencing was performed using Illumina HiSeq X10 (Illumina, USA). The value of the gene expression was converted to log₁₀ (transcript per million readings + 1). The data were analyzed by the online Majorbio cloud platform. The qRT-PCR primer sequences are listed in table S2. For the ELISA assay, the concentrations of cytokines in cell growth medium and cells were measured by an ELISA assay kit (Shanghai Jianglai Biotechnology Co. Ltd., Shanghai Enzyme-linked Biotechnology Co. Ltd.).