Fig 2: HSP70/ATF3/APP expression reflecting neuronal stress. (A,F) The highlighted area refers to the area represented in each row. (B–E) HSP70 (red) and APP (green) double staining in the dorsal cortex. While APP expression remains elevated through 7 d, HSP70 expression is comparatively decreased at 7 d. Few neurons are HSP70+/APP+. (G–J) ATF3 (red) and APP (green) double staining in the habenular nuclei. ATF3 follows the same expression pattern as HSP70, with decreased marker uptake at 7 d. In all cases, sham animals do not express any of the tested markers, with only the baseline marker uptake visible. Scale bar = 150 µm (Scale bar = 100 µm in image (F)).

Fig 3: Knockdown of GRP78 or CD44 alters cell morphology, reduces cell attachment, and impedes cell spreading in MCF7-LR cells.(A) Left: Western blot analysis of WCLs prepared from MCF7-LR cells transfected with control siRNA (sictrl) or siRNAs targeting GRP78 coding sequence, si78(1), or 3' untranslated region (3'-UTR), si78(2), using antibodies against GRP78 (MAb159) or HSP70 (C92F3A-5). GAPDH served as a loading control. Right: Quantification of relative levels of GRP78 and HSP70. Data represents mean ± SEM from three biological repeats. (B) Sequence comparison of GRP78 siRNA target sites on human GRP78 and HSP70 genes. (C) Bright-field micrographs showing the morphology of MCF7-LR cells transfected with sictrl or siRNA targeting GRP78 or CD44. siCD44(1) targets 3'-UTR and siCD44(2) targets the common coding sequence. Scale bar, 100 µm. (D) Epifluorescent micrographs showing the F-actin organization of MCF7-LR cells transfected with sictrl or siRNA targeting GRP78 or CD44. The arrows indicate the F-actin bundles. Red, F-actin labeled with rhodamine phalloidin; blue, nuclei stained by DAPI. Scale bar, 50 µm. (E) Cell attachment assay. MCF7-LR cells were transfected with sictrl or siRNA targeting GRP78 or CD44 for 60 h before re-seeding onto collagen I–coated culture plates for 1 h. Attached cells were visualized by crystal violet staining. Scale bar, 50 µm. (F) Quantification of relative levels of cell attachment described in panel (E). Crystal violet staining of adherent cells was dissolved in 100% methanol, and the OD was measured at 595 nm. Data represent mean ± SEM from three biological repeats. *P < 0.05, **P < 0.01 (t test). (G) Kinetic measurement of cell spreading area. MCF7-LR cells were transfected with sictrl or siRNA targeting GRP78 or CD44 for 60 h before re-seeding onto collagen I–coated culture plates for the indicated times. Cells were stained with rhodamine phalloidin and visualized by epifluorescent microscopy. Cell areas were quantified by the FIJI-Image J software, and the results were represented by the box and whisker plot. n, number of analyzed cells; h, hour. **P < 0.01, ***P < 0.001 (t test). (H) Data obtained from panel (G) were represented by mean ± SEM for each condition.

Fig 4: Effect of heat shock treatment on HSP70 and inflammatory cytokines expression in H5N1-infected mice. Mice in the heat-shocked group were exposed to 39°C for 4 h before being infected with 3 LD50 H5N1 HPAIV intranasally. Mice in the non-shocked group were maintained at room temperature (25°C). After infection, all the mice were maintained at room temperature. Mice were sacrificed at Days 0, 1, 3, and 6 post-infection for lung tissues harvest. HSP70 expression was determined by qPCR (A) and western blot (B). IL-6, TNF-a, IFN-ß, and IFN-? expressions were determined by qPCR (C) and ELISA (D). Gene expression was normalized with ß-actin as an internal standard (n = 3–5, *p < 0.05, **p < 0.01, ****p < 0.0001 by two-way ANOVA with Sidak's multiple comparisons test).

Fig 5: Tx-01 recognized membrane-bound HSP70 (mHSP70) on the circulating tumor cells (CTCs) in ascites and led to favorable prognosis(A) Representative fluorescence microscopy images showing Tx-01 and mHSP70 colocalization in the EpCAM(+) CTCs from serous OvCa patient ascites. Scale bar, 100 µm. Right upper graph shows the summary of Tx-01 and HSP70 colocalization percentage in all 16 ascites samples. (B) Tx-01 expression in the ascitic CTCs was significantly correlated with mHSP70 (p < 0.0001; R2 = 0.8141). (C) High Tx-01 binding in the ascitic CTCs was significantly associated with higher PFS (p = 0.03). (D) High mHSP70 expression in the ascitic CTCs was associated with borderline favorable PFS compared with low mHSP70 expression (p = 0.09). PFS, progression-free survival.