Gentamicin

The HT-29 human colorectal adenocarcinoma cell line was obtained from the American Type Culture Collection (ATCC #HTB-38) and was maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 0.05 mg/mL gentamicin (Sigma-Aldrich, MO, USA) and were grown in 75 cm² tissue culture flasks (Greiner-Bio-One, NC, USA) at 37°C, 5% CO₂ in a humidified incubator (Thermo Fisher, MA, USA). Cells were passaged every 3 days or when cells reached ~90% confluency. Cell passages 12–28 were used for all assays.
The non-transformed rat IEC-6 (ATCC #CRL-1592) small IEC line was maintained in DMEM supplemented with 10% FBS and 0.05 mg/mL gentamicin, and cell cultures were maintained in a similar manner to HT-29 cells. Cell passages 6–15 were used for all assays.

Human epithelial HL cells of respiratory tract origin [26] (link) were grown in RPMI 1640 (BioWhittaker, Lonza, Basel, Switzerland), supplemented with 7.5% fetal bovine serum (FBS) (BioWhittaker, Lonza, Basel, Switzerland), 2 mM L-glutamine (BioWhittaker, Lonza, Basel, Switzerland) and 20 µg/ml of gentamicin (Fluka, Buchs, Switzerland). HeLa-229 cells (CCL-2.1; ATCC, Manassas, VA, USA) were maintained in RPMI1640 supplemented with 20 mM HEPES (pH 8.0), 10% FBS, 2 mM L-glutamine, 8 µg/ml gentamicin and 1 µg/ml amphotericin B. The cells were cultured in standard cell culture protocols at 37°C and 5% CO_2. For all infections with C. pneumoniae, HL cells were seeded into 24-well plates with coverslips at density 4×10⁵ cells per well and incubated overnight before used for infection.
The C. pneumoniae clinical isolate K7 [27] (link) was propagated as described in Alvesalo et al.[23] and stored in SPG buffer in −70°C. C. trachomatis serovars K (VR-887, ATCC) and L2 (VR-902B; ATCC) were propagated in HeLa 229 cells and handled as described in [28] (link).

DXMa was purchased from LA COOPER (Melun, France). Hydroxypropyl-γ-cyclodextrin (HPγCD, W8HP, DS = 0.6, and Mw = 1576 Da) was a kind gift from ASHLAND (Schaffhausen, Switzerland) and hydroxypropyl-β-cyclodextrin (HPβCD, KLEPTOSE DS = 0.63 and Mw = 1391 Da) was obtained from ROQUETTE (Lestrem, France). CELLUVISC^® (sodium carboxymethylcellulose) and VISMED^® (sodium hyaluronate) are marketed gels used for the treatment of dry eye syndrome. DEXAFREE^® (DXM sodium phosphate 1% solution eye drops), MAXIDEX^® (DXM 0.1% suspension eye drops) and BSS^® (Alcon Laboratories, Rueil-Malmaison, France) are human authorized ocular medicines. Normal human primary corneal epithelial cells (ATCC PCS 700-010), medium (ATCC PCS-700-030), growth kit (ATCC PCS-700-040), PBS (ATCC 30-2200), trypsin EDTA (ATCC PCS-999-003 and 005), and antibiotics (gentamicin, streptomycin, and amphotericin BATC PCS-999-002) were obtained from LGC standard - ATCC^® (Molsheim, France). Thioglycollate with resazurine medium and Tryptic soy broth were obtained from BIOMERIEUX (Craponne, France). ALAMARBLUE^® was purchased from BIO-RAD (Marnes-la-Coquette, France) and DMSO from SIGMA-ALDRICH (Lyon, France). Purified water was prepared by DIRECT-Q^®3UV water purifier (MILLIPORE, Molsheim, France). All other solvents and chemicals were of HPLC and analytical grade, respectively.

Human epithelium cells (INT407, ATCC CCL-6) were purchased from ATCC and cultured at standard condition (37°C, 5% CO₂, 95% humidity) in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS and 100 μg/mL gentamicin (HyClone Laboratories Inc., Logan, UT, United States). The cultured cells were seeded at approximately 2 × 10⁵ cells/mL/well into 24-well tissue culture plates (BD Falcon, Franklin Lakes, NJ, United States) to reach 80–90% confluence monolayer at standard condition for cell adhesion assay. The post-confluent INT-407 cell monolayers were rinsed with PBS and stabilized in antibiotic-free DMEM for 1 h prior to the invasion assay.
Human macrophage cell line (U937, ATCC CRL3253) was purchased from ATCC and grown at standard condition in RPMI-1640 Medium supplemented with 10% FBS and 100 μg/mL gentamicin. An aliquot of 6 mL cell suspension containing 1 × 10⁶ cells were transferred into 25 cm² flask (Greiner Bio-One, Monroe, NC, United States) and cultured at standard condition for 24–30 h. After time, the cell monolayer was washed for three times with RPMI for further bacterial infection.

The human embryonic kidney HEK-293T cell line, human hepatoma-derived HuH-7.5 cell line, and human alveolar basal epithelial A549 cell line were maintained in DMEM supplemented with 10% fetal bovine serum (Sigma F8067) and gentamicin (Gibco). Human bone osteosarcoma epithelial cells (U2OS, ATCC HTB-96) were grown in McCoy's 5a Medium Modified (ATCC 30–2007)/10% FCS/gentamicin. All cell lines used in this study were monitored by SYBR Green real-time PCR RT assay periodically to ensure the absence of retroviral contamination and were stained with DAPI to test for mycoplasma contamination.

Primary human aortic smooth muscle cells (hASMCs) (ATCC PCS-100-012, American Type Culture Collection, ATCC, Manassas, VA, USA) were maintained in a humidified atmosphere of 37 °C, with 5% CO₂ in VSMC basal medium (without glucose and phenol red) (ATCC^® PCS-100-030™) supplemented with recombinant human basic fibroblast growth factor (5 ng/mL), rhInsulin (5 µg/mL), recombinant human epidermal growth factor (5 ng/mL), L-glutamine (10 mM), ascorbic acid (50 µg/mL), fetal bovine serum (5%), gentamicin (10 µg/mL), penicillin (10 Units/mL), streptomycin (10 µg/mL), amphotericin B (0.28 µg/mL), and phenol red (33 µM) (ATCC). To induce clinically hyperglycemic condition, we stimulated the cells with 25 mM (450 mg/dL) of glucose. Based on our previous study for cell viability [27 (link)], we used 1 and 10 μg/mL concentration of C. turczaninowii extract in this study.