Data Available Statement, manuscript: Cell survival and differentiation with nanocrystalline glass-like carbon using substantia nigra dopaminergic cells derived from transgenic mouse embryos. Rodriguez-Losada et al

1 The DATA AVAILABLE STAMENTS: ( According to Plos One policy) Points: 1.- SUMMARY STATISTIC of experiments 2.- DATA POINTS BEHIND MEANS 3.- MEDIANS 4.- VARIANCE MEASURES Figure 2………………………………………………..page 2 Figure 3……………………………………………......page 5 Figure 4……………………………………………......page 13 Figure 5……………………………………………......page 15 Figure 7……………………………………………......page 13 SS1…………………………………………………......page 21 2 Figure 2 : Fig. 2 E. Average branch length (A) RAW DATA (B) ANOVA Analysis Repeated measures ANOVA summary Assume sphericity? No F 16,31 P value 0,0003 P value summary *** Statistically significant (P < 0.05)? Yes Geisser-Greenhouse's epsilon 0,4829 R square 0,5762 Was the matching effective? F 0,1929 P value 0,998 P value summary ns Is there significant matching (P < 0.05)? No R square 0,02652 ANOVA table SS DF MS F (DFn, DFd) P value Treatment (between columns) 28862 3 9621 F (1,449, 17,39) = 16,31 P = 0,0003 Individual (between rows) 1365 12 113,7 F (12, 36) = 0,1929 P = 0,9980 Residual (random) 21230 36 589,7 Total 51457 51 MEAN SEM N MEAN SEM N MEAN SEM N MEAN SEM N 0 0 0 10 10 10 20 20 20 50 50 50 4,076923 1,909697 13 32,06538 2,946563 13 69,41615 9,464513 13 52,30846 6,512528 13 3,846154 1,746227 13 50,542 6,122102 10 75,37308 8,02756 13 59,73231 9,548361 13 5,538462 1,910213 13 30,57462 5,849475 13 71,63 10,53237 7 103,2831 13,20914 13 3 Data summary Number of treatments (columns) 4 Number of subjects (rows) 13 (C) Comparison Compare column means (main column effect) Number of families 1 Number of comparisons per family 6 Alpha 0,05 Tukey's multiple comparisons test Mean Diff, 95% CI of diff, Significant? Summary 0 vs. 10 -32,17 -47,56 to - 16,79 Yes **** 0 vs. 20 -67,75 -83,49 to - 52,00 Yes **** 0 vs. 50 -67,29 -82,36 to - 52,21 Yes **** 10 vs. 20 -35,57 -51,62 to - 19,53 Yes **** 10 vs. 50 -35,12 -50,50 to - 19,73 Yes **** 20 vs. 50 0,4578 -15,29 to 16,20 No ns Test details Mean 1 Mean 2 Mean Diff, SE of diff, N1 N2 q DF 0 vs. 10 4,487 36,66 -32,17 5,915 39 36 7,692 135 0 vs. 20 4,487 72,23 -67,75 6,053 39 33 15,83 135 0 vs. 50 4,487 71,77 -67,29 5,796 39 39 16,42 135 10 vs. 20 36,66 72,23 -35,57 6,168 36 33 8,156 135 10 vs. 50 36,66 71,77 -35,12 5,915 36 39 8,395 135 20 vs. 50 72,23 71,77 0,4578 6,053 33 39 0,107 135 4 (D) Two-wat ANOVA analysis Two-way ANOVA Ordinary Alpha 0,05 Source of Variation % of total variation P value P value summary Significant? Interaction 7,685 0,0003 *** Yes Row Factor 1,854 0,0416 * Yes Column Factor 50,51 < 0,0001 **** Yes ANOVA table SS DF MS F (DFn, DFd) P value Interaction 17676 6 2946 F (6, 135) = 4,498 P = 0,0003 Row Factor 4264 2 2132 F (2, 135) = 3,255 P = 0,0416 Column Factor 116180 3 38727 F (3, 135) = 59,13 P < 0,0001 Residual 88423 135 655 Number of missing values 9 5 Figure 3. Figure 3. A. MTT Assay Figure 3 A. Figure MTT Assay (A) Raw data 24h 3days 7days MEAN SEM N MEAN SEM N MEAN SEM N control 98,26897 1,372357 8 99,22726 2,771767 8 116,2627 10,72751 8 1 194,0968 8,510856 8 102,0682 13,18118 8 116,2627 10,72751 8 5 79,02796 3,731295 8 93,02271 2,416775 8 147,9425 30,02064 8 10 61,07412 2,649979 8 132,4772 18,26153 8 112,6832 9,661859 8 20 39,43631 4,616852 8 93,4091 4,682454 8 219,3066 24,22078 8 50 25,65468 4,005649 8 80,63636 3,701685 8 144,1939 8,716076 8 6 (B) Statistical Analysis Two-way ANOVA Ordinary Alpha 0,05 Source of Variation % of total variation P value P value summary Significant? Interaction 40,36 < 0,0001 **** Yes Row Factor 8,38 < 0,0001 **** Yes Column Factor 19,84 < 0,0001 **** Yes ANOVA table SS DF MS F (DFn, DFd) P value Interaction 185370 10 18537 F (10, 126) = 16,19 P < 0,0001 Row Factor 38487 5 7697 F (5, 126) = 6,722 P < 0,0001 Column Factor 91135 2 45568 F (2, 126) = 39,80 P < 0,0001 Residual 144277 126 1145 Number of missing values 0 7 (C) Multiple Comparison Within each column, compare rows (simple effects within columns) Number of families 3 Number of comparisons per family 15 Alpha 0,05 Tukey's multiple comparisons test Mean Diff, 95% CI of diff, Significant? Summary 24h control vs. 1 -95,83 -144,8 to - 46,86 Yes **** control vs. 5 19,24 -29,72 to 68,21 No ns control vs. 10 37,19 -11,77 to 86,16 No ns control vs. 20 58,83 9,867 to 107,8 Yes ** control vs. 50 72,61 23,65 to 121,6 Yes *** 1 vs. 5 115,1 66,10 to 164,0 Yes **** 1 vs. 10 133 84,06 to 182,0 Yes **** 1 vs. 20 154,7 105,7 to 203,6 Yes **** 1 vs. 50 168,4 119,5 to 217,4 Yes **** 5 vs. 10 17,95 -31,01 to 66,92 No ns 5 vs. 20 39,59 -9,374 to 88,56 No ns 5 vs. 50 53,37 4,408 to 102,3 Yes * 10 vs. 20 21,64 -27,33 to 70,60 No ns 10 vs. 50 35,42 -13,55 to 84,38 No ns 20 vs. 50 13,78 -35,18 to 62,75 No ns 8 3days control vs. 1 -2,841 -51,81 to 46,12 No ns control vs. 5 6,205 -42,76 to 55,17 No ns control vs. 10 -33,25 -82,22 to 15,72 No ns control vs. 20 5,818 -43,15 to 54,78 No ns control vs. 50 18,59 -30,37 to 67,56 No ns 1 vs. 5 9,045 -39,92 to 58,01 No ns 1 vs. 10 -30,41 -79,37 to 18,56 No ns 1 vs. 20 8,659 -40,31 to 57,62 No ns 1 vs. 50 21,43 -27,53 to 70,40 No ns 5 vs. 10 -39,45 -88,42 to 9,511 No ns 5 vs. 20 - 0,3864 -49,35 to 48,58 No ns 5 vs. 50 12,39 -36,58 to 61,35 No ns 10 vs. 20 39,07 -9,897 to 88,03 No ns 10 vs. 50 51,84 2,875 to 100,8 Yes * 20 vs. 50 12,77 -36,19 to 61,74 No ns 7days control vs. 1 0 -48,97 to 48,97 No ns control vs. 5 -31,68 -80,65 to 17,29 No ns control vs. 10 3,579 -45,39 to 52,54 No ns control vs. 20 -103 -152,0 to - 54,08 Yes **** control vs. 50 -27,93 -76,90 to 21,03 No ns 1 vs. 5 -31,68 -80,65 to 17,29 No ns 1 vs. 10 3,579 -45,39 to 52,54 No ns 1 vs. 20 -103 -152,0 to - 54,08 Yes **** 9 1 vs. 50 -27,93 -76,90 to 21,03 No ns 5 vs. 10 35,26 -13,71 to 84,22 No ns 5 vs. 20 -71,36 -120,3 to - 22,40 Yes *** 5 vs. 50 3,749 -45,22 to 52,71 No ns 10 vs. 20 -106,6 -155,6 to - 57,66 Yes **** 10 vs. 50 -31,51 -80,48 to 17,45 No ns 20 vs. 50 75,11 26,15 to 124,1 Yes *** Test details Mean 1 Mean 2 Mean Diff, SE of diff, N1 N2 q DF 24h control vs. 1 98,27 194,1 -95,83 16,92 8 8 8,01 126 control vs. 5 98,27 79,03 19,24 16,92 8 8 1,608 126 control vs. 10 98,27 61,07 37,19 16,92 8 8 3,109 126 control vs. 20 98,27 39,44 58,83 16,92 8 8 4,918 126 control vs. 50 98,27 25,65 72,61 16,92 8 8 6,07 126 1 vs. 5 194,1 79,03 115,1 16,92 8 8 9,618 126 1 vs. 10 194,1 61,07 133 16,92 8 8 11,12 126 1 vs. 20 194,1 39,44 154,7 16,92 8 8 12,93 126 1 vs. 50 194,1 25,65 168,4 16,92 8 8 14,08 126 5 vs. 10 79,03 61,07 17,95 16,92 8 8 1,501 126 5 vs. 20 79,03 39,44 39,59 16,92 8 8 3,309 126 5 vs. 50 79,03 25,65 53,37 16,92 8 8 4,461 126 10 vs. 20 61,07 39,44 21,64 16,92 8 8 1,809 126 10 vs. 50 61,07 25,65 35,42 16,92 8 8 2,961 126 20 vs. 50 39,44 25,65 13,78 16,92 8 8 1,152 126 3days control vs. 1 99,23 102,1 -2,841 16,92 8 8 0,2375 126 control vs. 5 99,23 93,02 6,205 16,92 8 8 0,5186 126 control vs. 10 99,23 132,5 -33,25 16,92 8 8 2,779 126 control vs. 20 99,23 93,41 5,818 16,92 8 8 0,4863 126 control vs. 50 99,23 80,64 18,59 16,92 8 8 1,554 126 16 95% confidence interval 1,662 to 3,900 R square 0,7541 F test to compare variances F,DFn, Dfd P value P value summary Significantly different? (P < 0.05) Figure 5 B. TH analysis control NGLC-50 Number of values 6 6 Minimum 100 226,5 25% Percentile 100 226,5 Median 100 373,4 75% Percentile 100 439,6 Maximum 100 439,6 Mean 100 346,5 Std. Deviation 0 109 Std. Error of Mean 0 62,96 Lower 95% CI of mean 100 75,64 Upper 95% CI of mean 100 617,4 Sum 300 1040 Table Analyzed TH Column B NGLC-50 vs. vs, Column A control Unpaired t test P value 0,0173 P value summary * Significantly different? (P < 0.05) Yes Oneor two-tailed P value? Two-tailed t, df t=3,916 df=4 How big is the difference? Mean ± SEM of column A 100,0 ± 0,0 N=3 17 Mean ± SEM of column B 346,5 ± 62,96 N=3 Difference between means 246,5 ± 62,96 95% confidence interval 71,72 to 421,3 R square 0,7931 F test to compare variances F,DFn, Dfd P value P value summary Significantly different? (P < 0.05) 18 Figure 7 : figure 7 F. control 5 nm 20 nm 80 nm BTTN Number of values 9 9 9 9 6 Minimum 0 0,4 0,66 0,535 -0,001 25% Percentile 0 0,6185 0,745 0,7775 -0,0003 Median 0 0,7 0,862 0,826 0,299 75% Percentile 0 0,91 0,9705 0,852 0,4355 Maximum 0 0,933 0,995 0,876 0,656 Mean 0 0,7428 0,8578 0,7928 0,2692 Std. Deviation 0 0,1843 0,126 0,103 0,2472 Std. Error of Mean 0 0,06143 0,04201 0,03434 0,1009 Lower 95% CI of mean 0 0,6011 0,7609 0,7136 0,00976 Upper 95% CI of mean 0 0,8844 0,9547 0,872 0,5286 Sum 0 6,685 7,72 7,135 1,615 (B) ANOVA summary F 57,59 P value < 0,0001 P value summary **** Are differences among means statistically significant? (P < 0.05) Yes R square 0,8616 Brown-Forsythe test F (DFn, DFd) 4,110 (4, 37) P value 0,0074 P value summary ** Significantly different standard deviations? (P < 0.05) Yes Bartlett's test Bartlett's statistic (corrected) P value < 0,0001 P value summary **** 19 Significantly different standard deviations? (P < 0.05) Yes ANOVA table SS DF MS F (DFn, DFd) P value Treatment (between columns) 4,913 4 1,228 F (4, 37) = 57,59 P < 0,0001 Residual (within columns) 0,7891 37 0,02133 Total 5,702 41 Data summary Number of treatments (columns) 5 Number of values (total) 42 (C) Anova Analysis : Multiple Comparison Number of families 1 Number of comparisons per family 10 Alpha 0,05 Tukey's multiple comparisons test Mean Diff, 95% CI of diff, Significant? Summary control vs. 5 nm -0,7428 -0,9401 to - 0,5454 Yes **** control vs. 20 nm -0,8578 -1,055 to - 0,6604 Yes **** control vs. 80 nm -0,7928 -0,9901 to - 0,5954 Yes **** control vs. BTTN -0,2692 -0,4898 to - 0,04850 No ns 5 nm vs. 20 nm -0,115 -0,3124 to 0,08237 No ns 5 nm vs. 80 nm -0,05 -0,2474 to 0,1474 No ns 5 nm vs. BTTN 0,4736 0,2529 to 0,6943 Yes **** 20 nm vs. 80 nm 0,065 -0,1324 to 0,2624 No ns 20 nm vs. BTTN 0,5886 0,3679 to 0,8093 Yes **** 80 nm vs. BTTN 0,5236 0,3029 to 0,7443 Yes **** 20 Test details Mean 1 Mean 2 Mean Diff, SE of diff, n1 n2 q DF control vs. 5 nm 0 0,7428 -0,7428 0,06884 9 9 15,26 37 control vs. 20 nm 0 0,8578 -0,8578 0,06884 9 9 17,62 37 control vs. 80 nm 0 0,7928 -0,7928 0,06884 9 9 16,29 37 control vs. BTTN 0 0,2692 -0,2692 0,07697 9 6 4,945 37 5 nm vs. 20 nm 0,7428 0,8578 -0,115 0,06884 9 9 2,362 37 5 nm vs. 80 nm 0,7428 0,7928 -0,05 0,06884 9 9 1,027 37 5 nm vs. BTTN 0,7428 0,2692 0,4736 0,07697 9 6 8,702 37 20 nm vs. 80 nm 0,8578 0,7928 0,065 0,06884 9 9 1,335 37 20 nm vs. BTTN 0,8578 0,2692 0,5886 0,07697 9 6 10,81 37 80 nm vs. BTTN 0,7928 0,2692 0,5236 0,07697 9 6 9,62 37 Number of families 1 Number of comparisons per family 10 Alpha 0,05 Tukey's multiple comparisons test Mean Diff, 95% CI of diff, Significant? Summary control vs. 5 nm -0,7428 -0,9401 to - 0,5454 Yes **** control vs. 20 nm -0,8578 -1,055 to - 0,6604 Yes **** control vs. 80 nm -0,7928 -0,9901 to - 0,5954 Yes **** control vs. BTTN -0,2692 -0,4898 to - 0,04850 Yes * 5 nm vs. 20 nm -0,115 -0,3124 to 0,08237 No ns 5 nm vs. 80 nm -0,05 -0,2474 to 0,1474 No ns 5 nm vs. BTTN 0,4736 0,2529 to 0,6943 Yes **** 20 nm vs. 80 nm 0,065 -0,1324 to 0,2624 No ns 20 nm vs. BTTN 0,5886 0,3679 to 0,8093 Yes **** 80 nm vs. BTTN 0,5236 0,3029 to 0,7443 Yes **** Test details Mean 1 Mean 2 Mean Diff, SE of diff, n1 n2 q DF 21 Figure SS1 (A) Raw data concentration ( µg/ml) 0 10 20 50 Number of values 39 36 33 39 Minimum 0 15,7 25,85 18 25% Percentile 0 22,19 46,15 45 Median 0 32,18 73,91 65 75% Percentile 10 48,97 91,24 94,4 Maximum 21 90,81 138,9 181,7 Mean 5,462 36,66 72,23 73,54 Std. Deviation 6,966 19,09 30,06 42,2 Std. Error of Mean 1,115 3,181 5,233 6,757 Lower 95% CI of mean 3,204 30,2 61,57 59,86 Upper 95% CI of mean 7,719 43,12 82,89 87,22 Sum 213 1320 2384 2868 (B) Statistic Analysis Table Analyzed AVERAGE BRANCH LENGTH ANOVA summary F 51,24 P value < 0,0001 P value summary **** control vs. 5 nm 0 0,7428 -0,7428 0,06884 9 9 15,26 37 control vs. 20 nm 0 0,8578 -0,8578 0,06884 9 9 17,62 37 control vs. 80 nm 0 0,7928 -0,7928 0,06884 9 9 16,29 37 control vs. BTTN 0 0,2692 -0,2692 0,07697 9 6 4,945 37 5 nm vs. 20 nm 0,7428 0,8578 -0,115 0,06884 9 9 2,362 37 5 nm vs. 80 nm 0,7428 0,7928 -0,05 0,06884 9 9 1,027 37 5 nm vs. BTTN 0,7428 0,2692 0,4736 0,07697 9 6 8,702 37 20 nm vs. 80 nm 0,8578 0,7928 0,065 0,06884 9 9 1,335 37 20 nm vs. BTTN 0,8578 0,2692 0,5886 0,07697 9 6 10,81 37 80 nm vs. BTTN 0,7928 0,2692 0,5236 0,07697 9 6 9,62 37 22 Are differences among means statistically significant? (P < 0.05) Yes R square 0,5181 Brown-Forsythe test F (DFn, DFd) 15,31 (3, 143) P value < 0,0001 P value summary **** Significantly different standard deviations? (P < 0.05) Yes Bartlett's test Bartlett's statistic (corrected) 94,54 P value < 0,0001 P value summary **** Significantly different standard deviations? (P < 0.05) Yes ANOVA table SS DF MS F (DFn, DFd) P value Treatment (between columns) 119511 3 39837 F (3, 143) = 51,24 P < 0,0001 Residual (within columns) 111175 143 777,4 Total 230686 146 (C) Multiple Comparison test Number of families 1 Number of comparisons per family 3 Alpha 0,05 Dunnett's multiple comparisons test Mean Diff, 95% CI of diff, Significant? Summary 0 vs. 10 -31,2 -46,50 to - 15,90 Yes **** 0 vs. 20 -66,77 -82,43 to - 51,11 Yes **** 0 vs. 50 -68,08 -83,07 to - 53,08 Yes **** Test details Mean 1 Mean 2 Mean Diff, SE of diff, n1 n2 q DF 0 vs. 10 5,462 36,66 -31,2 6,444 39 36 4,841 143 23 0 vs. 20 5,462 72,23 -66,77 6,595 39 33 10,12 143 0 vs. 50 5,462 73,54 -68,08 6,314 39 39 10,78 143