Graphpad v 7

The Kolmogorov-Smirnoff (K-S) test was used to determine data normality and variance heterogeneity, which indicated a normal distribution of data. Differences in nymph immunocompetent capacity between treatments were assessed by a univariate general linear model, having proPO and PO activity as dependent variables, while infection status (Morelos, Chilpancingo and control), intestine region (AMG, PMG and rectum) and incubation temperature (20 °C, 30 °C and 34 °C) were predictive variables. The significance of the whole model, of each separate predictive variable, and of the interactions between infection status with intestine region and temperature was determined. The latter interaction was studied by comparing groups with 95% confidence intervals. This analysis was performed with the software SPSS v.24.0. All data are expressed as mean enzyme activity ± standard error.
The Mantel-Cox log-rank test was used to determine the effect of infection status (Chilpancingo, Morelos or control) and temperature (20 °C, 30 °C or 34 °C) on nymph survival time. Intergroup differences were determined with the Chi-square test between infected and non-infected groups, and between infected groups. This analysis was performed with the software GraphPad v.7.0.

HeLa cells were reverse transfected with siRNA to knock down either VAMP3, VAMP4, VAMP3, and VAMP4 together or nontargeting controls as described above. Knockdown cells then were infected with WT C. trachomatis serovar L2 at an MOI of 0.4 by a rocking infection. At 30 hpi, infected cells were fixed in methanol to enumerate the primary infection or lysed, titrated, and reinfected onto a fresh monolayer of HeLa cells. Secondary infections were fixed in methanol at 28 hpi. All fixed coverslips from the primary and secondary chlamydial infections were processed via indirect immunofluorescence to detect chlamydial organisms (guinea pig anti-L2 antibody). Coverslips were imaged on a Zeiss ApoTome.2 fluorescence microscope at ×40 magnification to enumerate both the primary infection and secondary infection, where a single inclusion represented a single EB. Inclusions were counted and the secondary infection/infectious progeny were normalized to the primary infection by dividing the IFU count of the secondary infection by the average IFU of the primary infection. Progeny counts were graphed and analyzed using GraphPad v.7.0. An ordinary one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons was performed to determine statistical significance, where two asterisks indicates a P value of <0.01 and four asterisks indicates a P value of <0.0001.

Hazard ratios (HRs) and 95% confidence intervals (CIs) were used to assess the risk ratios of survival differences between groups. P < 0.05 was considered to be significantly different between groups. SPSS v.25.0 (IBM, Chicago, IL, USA) and GraphPad v.7.0 (La Jolla, CA, USA) are used for statistical analysis and figures drawing respectively. Figures plotting was performed by R v.3.5.1 and Cytoscape v3.6.1 [20 (link)].