Fig. 2
Expression and regulation of Sema4c mRNA and Sema4C protein in basal and fear memory conditions. a–c Analysis of β-galactosidase expression in mice expressing the lacZ gene in the Sema4c locus (Sema4C-LacZ mice) across the CA1, CA3, and dentate gyrus (DG) areas of hippocampus (a), anterior cingulate cortex (rACC; b) and basolateral amygdala (BLA) and central amygdaloid nucleus (CeA; c). Typical examples and quantitative summary of densitometric magnitude of LacZ staining in mock-treated mice and at day 3 following fear conditioning with foot shock. In panel b, bar graphs represent mean signal density over unit area over the whole ROI (demarcated by black lines in the examples shown in panel b) as well as a separate analysis of LacZ staining signal intensity in the high and low expression level areas (medial and lateral areas, respectively; indicated by arrow and arrowhead, respectively), normalized on the number of cells present in the ROI n = 3 mice per group; at least 3 sections were analyzed per mouse. d Analysis of Sema4c mRNA expression across the CA1 area of hippocampus (upper panels), rACC (middle panels) and in the central amygdala (lower panels) using RNAScope ISH technique. Typical examples (left), and quantitative summary (bar graphs) of Sema4c probe signal in mock-treated mice and on day 3 following fear conditioning upon foot shock; bar graphs on the left represent the integral signal intensity normalized on the number of cell nuclei, whereas bar graphs on the right represent the mean number of signal dots per nucleus; n = 3 mice/group; at least 3 sections were analyzed per mouse. e Examples and densitometric quantification of Sema4C protein via western blot analysis in lysates of CA1 area of hippocampus (upper panels), rACC (middle panels) and amygdalae-enriched tissue (lower panels) in mock-treated mice and on day 3 following fear conditioning via foot shock; data are represented as fold changes of the ratio of Sema4C over loading control signal (β-Tubulin III); n = 4 mice per group. Student’s t-test was performed; P < 0.05 indicated by * as compared to the corresponding control (mock-treated) groups. Error bars represent SEM. Scale bars represent 100 µm (a, c) and 5 µm (d)