Prism version 9

Prism version 9 (Graphpad) was used unless other software is specified. Where two groups were compared a student's t-test was used and for more than two groups a one-way ANOVA was used as indicated in the figure legends. Cytokine and flow cytometry data (but not cell count data) were logarithmically transformed prior to analysis. Differential expression analysis for the RNA-seq data was performed by Novogene using DESeq2 (open source software), based on a model using the negative binomial distribution with p values adjusted for false-discovery using the method of Benjamini and Hochberg. In Metascape [17 (link)], p-values were calculated based on the cumulative hypergeometric distribution and q-values are calculated using the Benjamini-Hochberg procedure to account for multiple testings. Analysis was performed using differentially expressed gene lists for each monocyte donor and separately and for a list produced by merging lists from the three monocyte donors. For analysis of the Nanostring data, Log2 data were analysed in Prism version 9 (Graphpad) using multiple unpaired t tests with Benjamini, Krieger, and Yekutieli's correction for false discovery.

Statistical analysis was performed using Prism Version 9 (GraphPad Software Inc., San Diego, La Jolla, USA). Differences between the means were examined using one-way analyses of variance (ANOVA, α = 0.05) and considered significant if p-values were less than 0.05 (*p < 0.05). Any correlation between time of infusion and blood sampling with the diameter of the inhibition zone (IZ) in the agar diffusion test was determined with the Pearson-test. Descriptive statistics were also analysed with Prism Version 9 (GraphPad Software Inc., San Diego, La Jolla, USA).The resulting data is expressed as means and standard deviations.

Rhythmicity Analysis Incorporating Non-parametric methods (RAIN) [108 (link)] was used to determine rhythmicity and phase of protein occupancy in ChIP assays, protein expression, and mRNA expression. Differences in daily rhythmicity were assessed using Detection of Differential Rhythmicity (DODR) [109 (link)] and differences in overall expression of rhythmic data (MESOR and amplitude) was measured using CircaCompare [110 (link)]. RAIN, DODR, and CircaCompare were performed using R version 4.0.3. To analyze the differences between treatments at each time point, two-way ANOVA followed by Sidak’s multiple comparisons test was used. Comparisons between only two conditions was determined using One sample t and Wilcoxon test to a hypothetical mean value corresponding to the normalization condition. One-way ANOVA followed by Dunnett’s multiple comparison tests was performed to analyze the comparisons of a control and experimental conditions, e.g. in S1B Fig. Two-way ANOVA, One sample t, and one-way ANOVA were performed using GraphPad Prism Version 9.3.1 (GraphPad Software, La Jolla, California, USA).

Mean ± standard error (SE) was used for data presentation. The differences between groups were examined for statistical significance by one-way analysis of variance (ANOVA) followed by Tukey’s analysis or Student’s T-test for multiple and two-group comparisons, respectively. The time-point experiments and survival analysis were analyzed by the repeated-measures ANOVA and the log-rank test, respectively. All statistical analyses were performed with SPSS 11.5 software (SPSS, IL, USA) and GraphPad Prism version 9.3.1 software (La Jolla, CA, USA). A p-value of <0.05 was considered statistically significant.

Statistical analysis and graph design was performed with SPSS software Version 28.0.1.0 (IBM, Ehningen, Germany) and GraphPad PRISM Version 9.3.1 (GraphPad Software, Inc., La Jolla, CA, USA), respectively. First, data distribution was tested with the Kolmogorov–Smirnov test and with the Shapiro–Wilk test. For non-normally distributed data, the Mann–Whitney-U test was used, while, for normally distributed data, the independent t-test was used. For correlation analysis of M1 and M2 macrophages, Pearson’s correlation test was used due to normal data distribution. These data are plotted as a correlation graph with listed Pearson’s correlation coefficient r in the figure description. StxB::555 measurements, cell counting, and qRT PCR data are visualized as boxplots containing the median value with the upper and lower 25% and 75% quartile. STxB::555 ratio analysis is plotted as a bar graph depicting the mean value with standard deviation. Behavioral data of young and old WT mouse groups were pooled due to no age differences, as previously shown [31 (link)] (referred to as “pooled WT”). Significance was considered at a p value of <0.05.

For our statistical analysis, we utilized SPSS version 28.0 (IBM Corp. Released 2021. IBM SPSS Statistics for Windows, Version 28.0. Armonk, NY, USA: IBM Corp.) Overall-survival (OS) Kaplan–Meier curves were plotted and compared using the log-rank test. For medians of OS, 95% confidence intervals (CI) were calculated. Cox proportional-hazards regression model was used for the calculation of univariate and multivariate hazard ratios for OS. Univariate variables with a trend towards significance (p < 0.10) and after testing of the proportional hazards assumption were used for analysis in the multivariate model. For the analysis of intratumoral heterogeneity of IC expression, we averaged the respective IC expression of all spots located in the periphery and in the center of the respective tumor and performed a paired t-test to test for differences. A p-value of <0.05 was considered a statistically significant difference. Graphics were designed using CorelDRAW^® Graphic Suite 2021 (Corel Corporation, Ottawa, ON, Canada) and GraphPad Prism Version 9.3.1 (GraphPad Software, San Diego, CA, USA).