1. Supplementary figure 1. (A) (B) 108 481 126 837 OKF6 91.5 241 TIGK 104
Notch-1
108
481
126
837
Isotype Ctrl.
Notch1
Surface
Intracellular
OKF6
(B)
Notch-1
91.5
241
Isotype Ctrl.
Notch1
TIGK
104
281
Surface
Intracellular
Supplementary figure 1. Oral epithelial cells express Notch-1 receptor. Cell surface and intracellular Notch-1 expression were determined by flow cytometry in (A) OKF6 and (B) TIGK cells as described in methods. Cells were either, incubated with a human Notch-1 monoclonal IgG1 conjugated with PE or an IgG1 isotype control. A representative dot plot of cell size (FSC) and granularity (SSC) obtained by FACS analysis of 10,000 events, as well as histograms depicting the Mean fluorescence intensities of cells positive for Notch-1 (blue line and values) or incubated with the isotype control (gray line and values) are shown.

2. Supplementary figure 2.
Supplementary figure 2. Effect of Notch-1 inhibitors on cell viability of oral epithelial cells. Pg-challenged OKF6 cells incubated with the solvent for Notch-1 inhibitors (DMSO) were used as control. Percentage of viable cells was determined by Trypan blue for each experimental condition using the automated cell counter (Countess II).

3. Supplementary figure 3. # *
Supplementary figure 3. Effect of Notch-1 silencing in IL-8 expression. Levels of IL-8 (mRNA) induced by P. gingivalis [1:100] for 48h in OKF6 cells transfected with siRNA-Notch-1 or the siRNA-negative control. Normalized mRNA levels to the levels of OKF6 cells neither transfected with siRNAs nor challenged with P. gingivalis are shown. The mean ± SD of triplicates from each treatment group from a representative experiment reproduced at least twice are shown. *p≤0.01 in cells transfected with Notch-1 siRNA vs. cells transfected with a negative siRNA control in both Mock and P. gingivalis groups. #p≤0.01 in Mock vs. P. gingivalis for each, Ctrl. siRNA and Notch-1 siRNA.

4. Supplementary Figure 4. (A) TLR2 TLR4 THP1 OKF6 TIGK 66.9 568 126 89.3
Isotype Ctrl.
TLR-2
66.9
568
126
TLR-4
THP1
89.3
197
86.9
OKF6
TIGK
125
722
123
Supplementary Figure 4.
Supplementary Figure 4. Toll-like receptors (TLR) 2 and 4 are not involved in P. gingivalis-induced PLA2-IIA expression or Notch-1 activation. (A) Expression analysis of TLR2 and TLR4 in oral epithelial cells (OKF6 and TIGK) by flow cytometry (FACS). Representative dot plots and histograms obtained by FACS analysis of 10,000 events are shown. THP-1 cells were used as a positive control for the TLRs expression. 2.5μL purified monoclonal antibodies against TLR2, TLR4 or an IgG isotype control (R&D Systems) were mixed with 1x105 cells in 100μL FACS buffer suspension and incubated for 30 minutes on ice. Unbound antibodies were washed off by adding 2mL FACS buffer and centrifuged at 1200rpm for 5 min, and further Allophycocyanin (APC) goat-anti-mouse IgG secondary antibody (diluted 1:200) (BioLegend) was added in 100μL PBS for 30 minutes on ice. At least 10,000 events were acquired on an LSR-II flow cytometer (BD, Franklin Lakes, NJ) for each experimental condition and receptor expression analysis was performed using FlowJo software. The mean of geometric mean fluorescence intensity (gMFI) is shown inside each histogram. For neutralization experiments, 1x105 TIGK cells/well were pre-incubated 30 minutes with either a purified IgG monoclonal anti-human TLR2 or an IgG isotype control antibody (R&D) diluted at 1µg/mL, 5µg/mL, and 10µg/mL in Ker-SFM for 30 minutes at 37°C before challenge with 1ml Ker-SFM containing Pg [MOI 1:50] for 48h. Then, the media was aspirated and the cells washed with 0.5mL sterile PBS. RNA was extracted using Ambion™ PureLink™ RNA Mini Kit (Thermo) and was used for further qRT-PCR analysis for (B) PLA2G2A and (C) HES-1 gene expression. (D) As a control for neutralizing activity of anti-TLR2 antibody, THP-1 cells (1x105) in RPMI1640 supplemented with 10% FBS were pre-incubated with 10µg/mL of anti-TLR2 or its correspondent isotype control for 30 minutes and further exposed to the Pg triple mutant for gingipains [MOI 1:50] for 24h. Supernatants were harvested and centrifuged at 3000rpm for 10min for IL-8 determination by ELISA. The mean ± SD of nine replicates from each treatment group from a representative experiment are shown. *p≤0.01 when P. gingivalis challenge was done in THP-1 cells pre-incubated with an anti-TLR2 vs. cells incubated with its corresponding isotype control.

5. Supplementary Figure 4. (C) (B) (D) THP-1 *
Supplementary Figure 4. Toll-like receptors (TLR) 2 and 4 are not involved in P. gingivalis-induced PLA2-IIA expression or Notch-1 activation. (A) Expression analysis of TLR2 and TLR4 in oral epithelial cells (OKF6 and TIGK) by flow cytometry (FACS). Representative dot plots and histograms obtained by FACS analysis of 10,000 events are shown. THP-1 cells were used as a positive control for the TLRs expression. 2.5μL purified monoclonal antibodies against TLR2, TLR4 or an IgG isotype control (R&D Systems) were mixed with 1x105 cells in 100μL FACS buffer suspension and incubated for 30 minutes on ice. Unbound antibodies were washed off by adding 2mL FACS buffer and centrifuged at 1200rpm for 5 min, and further Allophycocyanin (APC) goat-anti-mouse IgG secondary antibody (diluted 1:200) (BioLegend) was added in 100μL PBS for 30 minutes on ice. At least 10,000 events were acquired on an LSR-II flow cytometer (BD, Franklin Lakes, NJ) for each experimental condition and receptor expression analysis was performed using FlowJo software. The mean of geometric mean fluorescence intensity (gMFI) is shown inside each histogram. For neutralization experiments, 1x105 TIGK cells/well were pre-incubated 30 minutes with either a purified IgG monoclonal anti-human TLR2 or an IgG isotype control antibody (R&D) diluted at 1µg/mL, 5µg/mL, and 10µg/mL in Ker-SFM for 30 minutes at 37°C before challenge with 1ml Ker-SFM containing Pg [MOI 1:50] for 48h. Then, the media was aspirated and the cells washed with 0.5mL sterile PBS. RNA was extracted using Ambion™ PureLink™ RNA Mini Kit (Thermo) and was used for further qRT-PCR analysis for (B) PLA2G2A and (C) HES-1 gene expression. (D) As a control for neutralizing activity of anti-TLR2 antibody, THP-1 cells (1x105) in RPMI1640 supplemented with 10% FBS were pre-incubated with 10µg/mL of anti-TLR2 or its correspondent isotype control for 30 minutes and further exposed to the Pg triple mutant for gingipains [MOI 1:50] for 24h. Supernatants were harvested and centrifuged at 3000rpm for 10min for IL-8 determination by ELISA. The mean ± SD of nine replicates from each treatment group from a representative experiment are shown. *p≤0.01 when P. gingivalis challenge was done in THP-1 cells pre-incubated with an anti-TLR2 vs. cells incubated with its corresponding isotype control.