Dylight 594

Immunofluorescence centrosome staining was done as previously established with modification (20 (link), 46 (link)). HeLa, A2EN, or primary cervical cells were infected with the appropriate strains of C.t. at an MOI of 1 by centrifugation at 700 × g for 30 min. At 36 h postinfection, cells were fixed on ice with cold methanol for 6 min and blocked for 2 h at room temperature in 0.1% Triton-X in PBS with 2% FBS. Cells were stained with anti-pericentrin (abcam) and anti-Chlamydia HSP60 (Millipore Sigma). Dylight-488 and Dylight-594 (Thermo Fisher Scientific) secondaries were used along with DAPI (Thermo Fisher Scientific) to stain the nuclei. Images were captured using a Leica DFC7000T confocal microscope equipped with Leica software. At least 10 images were collected per coverslip, with three technical replicates per biological replicate, with at least 2 biological replicates.

EVT were plated on coverslips and fixed with 4% PFA, then permeabilized with 0.3% Triton X‐100, and finally blocked with 5% BSA. Then, the cells were incubated with primary antibodies against CK7 (ET1609‐62; HuaBio; 1:300) and HLG‐A (ab7758; Abcam; 1:200) at 4°C overnight. After incubation with DyLight 488‐ or DyLight 594‐labelled secondary antibodies (1:1000, Thermo Fisher), the nuclei were counterstained with DAPI (Sigma). Images were acquired using a laser scanning confocal microscope (Olympus).

Murine CCL19 was purchased from R&D systems, USA. Biotinylated, truncated murine CCL21 (mCCL21 24–98 bio) was synthesized by ALMAC (Craigavon, UK). Chemokines were reconstituted as follows: Desiccated chemokines were reconstituted to a concentration of 25 μg/mL in PBS and stored at −20 °C. Prior to use, mCCL21 24–98 bio was diluted to a working concentration of 250 ng/mL in PBS.
DyLight 594 (Thermo Fisher Scientific) labeled mCCL21 24–98 bio was prepared following the manufacturers protocol. Briefly, 100 μg mCCL21 24–98 bio were reconstituted in 100 μL phosphate buffer containing 0.1 M Na₂HPO₄, 0.15 M NaCl adjusted to pH 7.2–7.5. 65 μg DyLight 594 NHS ester (Thermo Fisher Scientific) were added and the mixture was allowed to react for 1 h at room temperature. Subsequently, excessive DyLight 594 NHS ester was quenched for 1 h at room temperature by adding 1.4 mL of Tris/HCl pH 7.6. mCCL21 24–98 bio DL595 was purified using MW10 kDa spin columns (Amicon Ultra-2 Centrifugal Filter devices, Millipore) and stored at −20 °C.

cDNA of full length and truncated BPIFA1 was transformed in BL21-Codon Plus competent cells (Agilent Technologies), and purified as previously described24 (link). S18 peptide was synthesized and purified by the UNC Microprotein Sequencing and Peptide Synthesis Facility, as described previously17 (link). Fluorescent labelling was done using DyLight 594 or DyLight 633 NHS ester (Thermo Fisher) by following the manufacturer's instructions.

U0126 (catalog no. S1901) and EGF (catalog no. P5552) were purchased from Beyotime Biotechnology. Mitoxantrone (catalog no. HY-13502) was purchased from MCE. TureColor three-color pre-stained protein Marker (catalog no. C510010) was purchased from Sangon Biotech. BSA (catalog no. B7004M) was purchased from US EVERBRIGHT. UltraSignal ECL (catalog no. 4AW011-100) was purchased from 4A Biotech Co., Ltd. Antibodies information: anti-USP11 (Santa Cruz, catalog no. sc-365528/ Abcam, catalog no. ab109232); anti-p21 (Santa Cruz, catalog no. sc-397/ Cell Signaling Technology, catalog no. 2947S); anti-Flag (MBL, catalog no. M185-3L); anti-Myc (MBL, catalog no. M192-3); anti-HA (MBL, catalog no. M180-3); anti-ERK1/2 (Cell Signaling Technology, catalog no. 4695S); anti-phospho-ERK1/2 (Cell Signaling Technology, catalog no. 4376S); anti-phospho-MAPK/CDK Substrates (Cell Signaling Technology, catalog no. 2325S); anti-phosphoserine/threonine (BD Transduction Laboratories, catalog no. M180-3); anti-GAPDH (COOLRUN Life Science, catalog no. AT0002); Dylight 488 (Thermo Fisher Scientific, catalog no. #35502); Dylight 594 (Thermo Fisher Scientific, catalog no. #35560).

For immunofluorescence staining, cultured cells and organoids were fixed for 20 min, permeabilized with 0.1% Triton X-100 for 15 min, washed thrice with PBS for 5 min, and blocked with 10% bovine serum albumin for 1 h. Cells were incubated with the primary antibody anti-SCUBE3 (ab189955; Abcam, Cambridge, UK), and the organoids were incubated with anti-DSPP (sc-73632; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight. Sections of tooth root fragments were stained with DSPP (sc-73632; Santa Cruz) and α-tubulin (sc-8035; Santa Cruz). After washing thrice with PBS for 5 min, cells and organoids were, respectively, incubated with Dylight 594 (35560; Thermo Fisher Scientific, Waltham, UK) and CoraLite 488-conjugated (CL488-66122; ProteinTech Group, Chicago, IL, USA) secondary antibodies for 1 h in the dark. DAPI (Sigma-Aldrich) was used to stain the nuclei. Finally, the cells were observed under a Leica DMI4000 B fluorescence microscope (Leica Imaging Systems, Cambridge, UK). Organoids were transferred to observation dishes specified for confocal laser microscopy and examined with a confocal microscope (Carl Zeiss, Göttingen, Germany).