Fig 2: p38-MAPK levels are greater in the frontal cortex of persons with AD dementia with APOE3/E4 compared with APOE3/E3. The frozen inferior frontal lobe tissue from persons with NCI or AD dementia with different APOE genotypes was homogenized to test phosphorylated p38 (p-p38) and total p38-MAPK protein levels using WB. A p-p38 and p38 protein levels in the cortex from persons with NCI (n = 6 for E3/E3; n = 6 for E3/4). B p-p38 and p38 protein levels in the cortex from persons with AD dementia (n = 6 for E3/E3; n = 8 for E3/E4). NCI, no cognitive impairment; AD, Alzheimer’s disease. WB: Western Blot. Data are represented as mean ± SEM and analyzed by Student’s t-test (two-tailed). *p < 0.05

Fig 3: Effect of Dio on synaptic loss in young AppNL-G-F mice. (A–C) Representative Golgi-staining and quantification showing spines of 3- and 6-month-old male and female WT and AppNL-G-F mice. Scale bar: 0.1 mm. Data are expressed as mean ± SEM (n = 8). * p < 0.05 compared with WT; ** p < 0.01 compared with WT. # p < 0.05 compared with AppNL-G-F; (F,K) TEM image from male and female WT and AppNL-G-F mice showing synapses. Scale bar: 0.5 μm; (F) 3-month-old male and female mice; (K) 6-month-old male and female mice. (D,E,I,J) Immunofluorescence staining of PSD95 and SYP in the cortex and hippocampus in 3- and 6-month-old male and female WT and AppNL-G-F mice. Scale bar: 0.1 mm; (D) 3-month-old male mice; (E) 3-month-old female mice; (I) 6-month-old male mice; (J) 6-month-old female mice. (G,H,L,M) Western blot and quantification of PSD95 and SYP protein expression in the cortex and hippocampus in 3- and 6-month-old male and female WT and AppNL-G-F mice. Data are expressed as mean ± SEM. n = 6 mice/group. * p < 0.05 compared with WT; ** p < 0.01 compared with WT; *** p < 0.001 compared with WT. # p < 0.05 compared with AppNL-G-F; ## p < 0.01 compared with AppNL-G-F.

Fig 4: Expression and activation of 57 signaling molecules in matched SYN+ and SYN− clones isolated from the same tissue section(A) Unsupervised hierarchical clustering analysis using the Ward’s method of matched SYN+ and SYN− clones. Intensity values are displayed on a scale ranging from burgundy to blue, where burgundy indicates high levels and dark blue indicates low levels of expression/activation. On the x axis are listed proteins measured by RPPA; on the y axis are displayed SYN+ (red) and SYN− (blue) clones. Matched samples are identified by the same color on the left side of the panel.(B) List of proteins that increased in four or more samples in matched SYN+ and SYN− cancer cells. Proteins that were increased in four, five, or six samples are color coded in red, blue, and green, respectively. Proteins that were statistically different between SYN+ and SYN− cancer cells are indicated in bold. Samples where significance was retained in pairwise comparisons are indicated in italic.(C) Box plot displaying mean and 95% confidence intervals of fold change in SYN+ cancer cells compared with the SYN− clone. Asterisks highlight changes that were statistically significant; proteins were color coded based on their function.(D) Unsupervised hierarchical clustering analysis using the Ward’s method for one case where SYN+ cancer cells (red) were isolated along with proximal (blue) and distal (green) SYN− cells. Proteins are grouped based on their expression/activation across the three matched samples; in the red box are grouped proteins that increased in SYN+ cells, in yellow the one increased in SYN+ and proximal SYN− cells, and in blue the one increased in distal SYN− cells.

Fig 5: Study workflow and clinical-pathological and molecular characteristics of SYN+ NSCLCs(A) Diagram capturing the steps of the immuno-LCM workflow developed for this study. SYN+ clones were first identified using standard IHC. SYN+ and SYN− clones were isolated under direct visualization using an infrared (IR)-based LCM system. Expression/activation of 57 signal transduction molecules were measured on a continuous scale using the RPPA and compared across different clones to identify signaling events that were uniquely attributable to SYN+ cells.(B) Examples of SYN+ cells identified by IHC in a primary tumor (left site) and a lymph node metastasis (right panel) in patients with NSCLC; SYN+ cells differ in degrees of distribution: from a few to 70% of metastatic cells.(C) Higher rates of distant metastases (p = 0.03), recurrence (p = 0.03), and poorer prognosis (p = 0.05) were detected in patients whose tumor contained SYN+ cells. Bar graph illustrating frequency of distant metastases and outcome of patients with NSCLC with and without SYN+ neuroendocrine-like cells.(D) Bar graphs displaying changes in SYN expression in microdissected matched SYN+ and SYN− cancer cells. IHC intensity staining for each sample is reported along with the RPPA-based fold change values.(E) Selected examples of IHC staining matching the samples displayed in the bar graph in (D).(F) Boxplot illustrating significant changes in SYN expression between matched SYN+ and SYN− microdissected clones (p = 0.02).