Fv1000 system

The developing vegetative apices of young stems of each group (CK, MeJA36, MeJA48) were treated as described previously. The samples were embedded in paraffin and sliced into 4 μm-thick sections. Before incubation with antibody, the samples were soaked in 10 mM citrate buffer (PH 6.0) at 95 °C three times for 10 min, then the sections were post-fixed in 4% paraformaldehyde for 20 min and treated with 1% Triton X-100 30 min, finally blocked in 3% bovine serum albumin (BSA) solution for 1 h at room temperature. After removing excess of BSA solution, the sections were incubated in anti-ATG8a antibody (dilution: 1:1000) overnight at 4 °C and 30 min at room temperature, and washed with PBST for three times. Subsequently, the sections were incubated with fluorescent isothiocyanate-conjugated goat anti-rabbit IgG (Alexa Fluor 488; Abcam) at a dilution of 1:1000 for 2.5 h, and 0.05 mM Monodansylcadaverine (MDC, Sigma) for 30 min respectively. It is noteworthy that the sections were washed with PBST for three times at each incubation interval. All confocal fluorescence images were collected using an Olympus FV1000 system. Finally, we quantified the mean fluorescence intensity in laticifers using the previous method [26 (link)].

WT OsPSS-1 was amplified with primer pair OsPSS-1-GFP-BamHI-F/R (S1 Table). The fragment was cloned into the BamHI and BglII sites of vector PA7-GFP to produce constructs OsPSS-1-GFP and GFP-OsPSS-1, respectively. A LactC2 fragment was amplified from plasmid p416-GFP-LactC2 (Haematologic Technologies) using primers C2 domain-F/R (S1 Table) and inserted into the BglII site of PA7-GFP. OsPSS-1-GFP and GFP-LactC2 were co-transformed into rice or Arabidopsis protoplasts with various organelle markers and markers of the endomembrane system. Transient expression using Arabidopsis protoplasts from cell suspensions and rice protoplasts from rice leaf sheaths was described previously [68 (link),69 (link)].
Confocal images were acquired at specific time points after transformation using an Olympus FV1000 system. For each experiment, more than 20 individual cells were imaged. For statistical analysis, the PSC colocalization plug-in [70 (link)] for ImageJ [71 ] was used to calculate the linear Pearson correlation coefficient (r_p) of red and green fluorescent signals. We changed red fluorescent signal to magenta to make it easy for colorblind individuals to interpret the data in figures.

iCD103 DCs and BMDMs were infected with M. bovis BCG GFP to check the infection rate. After 24 h, Hoechst 33342 was used to stain nuclei and cells were loaded in VI 0.5 μ-slides (Ibidi). Confocal microscopic images were taken on an Olympus FV1000 system using a 60× oil objective. All images were equally adjusted using Fiji software (NIH).

MC3T3-E1 cells were plated in culture dishes specialized for immunofluorescence at a density of 1 × 10⁴/ml. PTH was administered as described above. Next, the cells were fixed with ice cold 4% paraformaldehyde for 20 min, permeabilized with 0.25% Triton X-100 for 5 min, and blocked with 3% bovine serum albumin for 30 min at 37°C. The cells were then incubated with primary antibodies (CST, USA) overnight at 4°C, followed by secondary conjugated antibodies (CST, USA) for 45 min at 37°C after several rinses with PBS. The images were obtained using a laser scanning confocal microscope (FV1000 System, Olympus, Japan).

MDCK-pTR GFP-RasV12 cells were mixed with MDCK or MDCK ADAMDECA1-shRNA1 cells at a ratio of 1:50 and cultured on the collagen matrix. Type-I collagen (Cellmatrix Type I-A) was obtained from Nitta Gelatin (Osaka, Japan) and neutralized on ice to a final concentration of 2 mg ml⁻¹ according to the manufacturer’s instructions. The mixture of cells was incubated for 8–12 h until they formed an epithelial monolayer, followed by tetracycline treatment for 16 h for analyses of accumulation of filamin or EPLIN or 24 h for those of apical extrusion. Cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.5% Triton X-100 in PBS, followed by blocking with 1% BSA in PBS. Primary or secondary antibodies were incubated for 2 h or 1 h, respectively. Immunofluorescence images were analyzed by the Olympus FV1000 system and Olympus FV10-ASW software. Quantification of immunofluorescence intensity of filamin or EPLIN was performed as previously described^{13 (link),14 (link)}. For quantification of apical extrusion, more than 100 RasV12 cells were analyzed for each experimental condition. RasV12 cells that have been completely detached from the basal matrix are defined as apically extruded cells.