Additional file 1 of Targeting cathepsin S promotes activation of OLF1-BDNF/TrkB axis to enhance cognitive function
Creators: name: Lee, Hao-Wei
name: Chen, Szu-Jung
name: Tsai, Kuen-Jer
name: Hsu, Kuei-Sen
name: Chen, Yi-Fan
name: Chang, Chih-Hua
name: Lin, Hsiao-Han
name: Hsueh, Wen-Yun
name: Hsieh, Hsing-Pang
name: Lee, Yueh-Feng
name: Chiang, Huai-Chueh
name: Chang, Jang-Yang
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: Biotechnology
subject: Science Policy
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Mental Health

description: Supplementary Material 1: Supplementary Figure 1. Targeting CTSS improved learning memory. (A) The diagram illustrates the target hole and nontarget holes in the Barnes maze.  (B) The timeline illustrating RJW-58 administration and the training phase of the Barnes maze for assessing learning memory. (C)(E) Alternations in the mean latency time during the training phase in RJW-58-treated mice (C, N = 5 mice / group) and Ctss−/− knockout mice (E, N = 5 mice/group). (D)(F) Determination of the number of correct visits in RJW-58-treated mice (D, N = 5 mice / group) and Ctss−/− knockout mice (F, N = 5 mice/group) at probe trial-1 and probe trial-2. Bar charts and plots indicate the mean ± SEM. Asterisks indicate significant differences, Sidak’s post hoc test in E, Mann-Whitney test in F, *p&lt;0.05,**p&lt;0.01,***p &lt; 0.001 versus the mock group in C and D, versus Ctss+/+ in E and F. Supplementary Figure 2. Suppressing CTSS delayed spatial memory loss in the Barnes maze. (A)(B) Intragroup analyses of the number of correct visits in RJW-58-treated mice (a, Fmock(3,32)=13.60,p&lt;0.001; F2.5mg(3,33) = 1.032,p&gt;0.05; F7.5mg(3,30) = 5.72,p=0.0032; F15mg(3,31)= 3.589, p = 0.0246, N = 10 mice/group) and Ctss−/− knockout mice (B, FCtss−/− (3,12) = 3.738, p = 0.0417, N = 10 mice) from probe trial-1 to probe trial-4. (C)(D)(E)(F) The distribution of visits of RJW-58-treated mice in the Barnes maze during probe trial-1 and probe trial-2. Bar charts indicate the mean ± SEM.  Asterisks indicate significant differences,Tukey’s post-hoc test *p&lt;0.05, **p&lt;0.01, ***p&lt; 0.001 versus probe trial-1 in the corresponding groups in A and B. Supplementary Figure 3. Effects of RJW-58 on intracellular calcium homeostasis. (A) Representative [Ca2+]i traces in HT-22 cells in recording buffer containing 1.5 mM Ca2+ after RJW-58 induction.  The black arrow indicates DMSO or different doses of RJW-58 added.  (B) Representative [Ca2+]i traces in HT-22 cells in Ca2+-freerecording buffer after RJW-58 stimulation. The black arrow indicates DMSO or different doses of RJW-58 added. (C) Analyses of dynamic [Ca2+]i after RJW-58 stimulation by measuring the AUC.  Quantitative analyses of western blotting for band relative intensity in CaMK IV (D), CaMK IV (E), PSD95 (F, G) from murine hippocampal tissues (N = 5 mice/group).  Bar charts in C indicate the mean ± SD, in D, E, F, and G indicate the mean ± SEM.  Asterisks indicate significant differences, Dunn’s multiple comparisons post-hoc test in D, E, F, G; *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001 vs the control group in C, vs the Mockgroup in D, E, F, G. Supplementary Figure 4. Supportive data for neurogenesis. (A) The representative images of BrdU-incorporated cells co-express NeuN in the DG of murine hippocampus after 7-day-consecutive RJW-58 injections (BrdU stained green, NeuN stained red). (B)(C) Quantitative analyses of BrdU+NeuN+ co-expression cells (B), and total NeuN+ cells (C) in the DG of different RJW-58 dosing groups (n = 6 sections/mouse, N= 6 mice/group). (D)(G)(I) The representative images of BrdU+Sox2+ cells (D), BrdU+DCX+ cells (G), and BrdU+NeuN+ cells (I) in the DG of hippocampus of Ctss+/+ and Ctss−/− mice. (E)(F)(H)(J) Quantitative analyses of BrdU+ cells (E), BrdU+Sox2+ cells (F), BrdU+DCX+ cells (H), and BrdU+NeuN+ cells (J) in the DG of hippocampus of Ctss+/+ and Ctss−/− mice (n = 6 sections/mouse, N = 3 mice/strain).  Bar charts indicate the mean ± SEM. NS: non significantly,p&gt;0.05, versus mock group in B and C, versus Ctss+/+ strain in E, F, H, and J. Supplementary Figure 5. Validation of BDNF protein expression level. (A) Quantitative analysis of relative intensity of BDNF in HT-22 cells treated with different doses of RJW-58 ranging from 0 (DMSO) to 5 μM (N = 5).  (B) Quantitative analysis of relative intensity of BDNF in murine hippocampal tissues treated with 7-day consecutive RJW-58 injection ranging from 0 (Mock) to 15 mg/kg (N = 7 mice/group). (C) Western blot of proBDNF and BDNF proteins in HT-22 cells treated with 2.5 µM RJW-58 for 0 h and 24 h.  (D) Quantitative analysis of relative intensity of BDNF in HT-22 cells treated with 2.5 µM RJW-58 for 0 h and 24 h (N = 4).  (E) Characterizations of primary cortical neurons on the tenth day. MAP2 was stained green, and NeuN was stained red.  (F) Western blot indicated that BDNF expression increased in cortex-derived primary cortical neurons from Ctss−/− mice than in those derived from Ctss+/+ mice.  Bar charts indicate the mean ± SEM, Dunnett’s multiple post hoc test in A, Dunn’s post hoc test in B, unpaired two-tailedt-test in d, NS: non significantly, *p&lt;0.05,**p&lt;0.01,***p &lt; 0.001 vs the control group in C, vs the Mockgroup in D, E, F, G. Supplementary Figure 6. Supportive data of promoter assay and IP experiments. (A) The colored frame highlighted sequences are the predicted OLF-1 binding sites of designated primers in the promoter assay.  (B)(C) Quantitative analyses of relative fold change of OLF-1 (B) and pCREB (C) in the time courses of nuclear/cytosol separation experiments (N = 3). (D)(E)(F) Quantitative analyses of relative fold change of CBP (D), CREB (E), and OLF-1 (F) in IP experiments (N=3).  Bar charts indicate the mean ± SEM, Dunnett’s post hoc test in B and C, unpaired two-tailed t-test in D, E, and F, NS: non significantly, *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001 vs the 0 min group in B and C, vs the DMSOgroup in D, E, F. Supplementary Figure 7.  Western blot quantitative data for BDNF/TrkB signaling. Quantitative analyses of pERK (A), and pCREB (B) in cultured HT-22 cells treated with different dosages of RJW-58 ranging from 0 to 5 μM (N = 3).  Band intensity analyses of pERK (C) and pCREB (D) along the time axis after RJW-58 treatment (N = 3).  Band intensity analyses of BDNF/TrkB signaling including pTrkB (E), pCaMK II (F), pERK (G), and pCREB (H) in cultured primary cortical neurons (N = 3). Band intensity analyses of pTrkB (I), pERK (J), and pCREB (K) in single dose of RJW-58 administered murine hippocampus (N = 4 / group). Analyses of pTrkB (L) in 7-day consecutive RJW-58 injected hippocampus at the eighth day (N = 7 / group), and pERK (M) and pCREB (N) at the seventh week (N = 6 / group).  Bar charts indicate the mean ± SEM, Dunnett’s post hoc test in A-H, Dunn’s post hoc test in I-N, NS: non significantly, *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001 vs the control or DMSO group in A-H, versus the Mockgroup in I-N. Supplementary Figure 8. Supportive data for BDNF/TrkB signaling. (A) Screening of the phosphorylation site of TrkB in response to RJW-58 treatment in the hippocampal tissue.  (B) Western blot analysis of pTrkB and TrkB in murine hippocampal tissues isolated in the seventh week after different dosing treatments of RJW-58.  (C)(D) Western blotting of HT-22 (C) and murine hippocampal tissue (D) for probing pAkt and Akt after designated RJW-58 stimulation for 1 h in HT-22 cells and RJW-58 treatment for 7 consecutive days in murine hippocampal tissues.