Fig 1: Prognostic significance of FSTL1-DIAPH1 gene pair. (A) Bioluminescence imaging of systemic metastasis by A253 cells expressing luciferase reporter and control shRNA or shRNA against FSTL1 or DIAPH1 individually or in combination (n = 7). (B) Representation of the total number of metastatic foci in lung sections. Error bars denote mean ± SEM. **, P < 0.0001. (C) Representative images of lung metastatic colonies detected by staining for KRT5 on day 26 postinjection. Bars, 100 μm. (D) Scatter plot of FSTL1 and DIAPH1 expression in 262 patients, classified into four distinct segments by 2D-DDg. (E) Kaplan–Meier survival curves of individual segments. (F) Kaplan–Meier survival curves with statistical significance by log-rank test of two groups, in which patients were grouped as low risk (segments a+b+c) and high risk (segment d).
Fig 2: Serum follistatin‐like 1 (FSTL1) levels in the LETO, OLETF‐CON, and OLETF‐AT groups. Representative immunofluorescence image of serum FSTL1 is shown. The relative serum FSTL1 levels are represented as fold changes from the LETO group. A.U.: Arbitrary units. One‐way ANOVA followed by Fisher's post hoc test. n = 8 in each group
Fig 3: FSTL1 overexpression attenuated DOX-induced damage in vitro. (a, b) Nrf2 expression and Nrf2 activity in NRCMs (n = 6). (c) Nrf2 expression after siRNA infection (n = 6). (d) ROS production in NRCMs (n = 6). (e) The mRNA levels of SOD1 in NRCMs (n = 6). (f) The level of 4-HNE in NRCMs (n = 6). (g, h) Caspase 3 activity and LDH release (n = 6). (i) Cell viability (n = 6). ∗P < 0.05. For (c), the data were analyzed using two-tailed Student's t-tests. For the others, the data were analyzed using one-way ANOVA, followed by Tukey's post hoc analysis.
Fig 4: Correlations between serum FSTL1 and soleus FSTL1 levels (a), arterial endothelial nitric oxide synthase (eNOS) phosphorylation levels (b), arterial nitrite/nitrate (NOx) levels (c), or carotid‐femoral pulse wave velocity (cfPWV) (d) in the LETO, OLETF‐CON, and OLETF‐AT groups. n = 5–8 in each group
Fig 5: The defective switch is proinvasive. (A) Immunostaining of DIAPH1 on normal tongue (n = 11) and HNSCC (n = 64) tissue sections. In comparison with normal tongue, a significant increase in DIAPH1 expression is observed in HNSCC tissue sections. Bars, 100 µm. (B) Expression pattern of miR-198 and DIAPH1 across HNSCC tissue samples indicates a clear inverse correlation. (C and D) Boyden chamber invasion assay shows a significant decrease in the number of cells invading the chamber matrix in siDIAPH1 or siFSTL1 compared with control cells. Representative images of migrated cells detected with Giemsa staining (representative of five independent experiments). Error bars represent SD. Bars, 100 μm. (E) Morphology of SCC cells transduced with control shRNA or shRNA against FSTL1 or DIAPH1 (top) visualized using phalloidin-conjugated TRITC. Knockdown of FSTL1 or DIAPH1 showed a significant difference in morphology compared with control cells (top). Cell trajectory displacement over 24 h (bottom; n = 3). Bars, 10 μm. (F) Analysis of cell displacement in control, shFSTL1, and shDIAPH1 cells (representative of three independent experiments). (G) Organotypic invasion assay with shFSTL1 and shDIAPH1 independently or in combination or control SCC12 cells. SCC12 cells were detected by KRT14 staining (in red). Bars, 100 μm. (H) Histogram represents relative percentage of cells that invade the matrix (representative of three independent experiments; *, P < 0.05; **, P < 0.01). Error bars denote mean ± SEM.
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