Fig 2: Effects of geldanamycin on gene silencing involving miRNAs.(A) Stability of AKT. To verify the efficacy of geldanamycin (GM) in HeLa cells, AKT that is a HSP90 client was examined in cells treated with various concentrations (nM) of geldanamycin by means of Western blotting. Alpha-tubulin was examined as an internal control. (B) Activation of HSF1 by GM. The pGL4-HSE (black bars) and pGL4-minP (gray bars; as a control) reporter plasmids were co-transfected with phRL-TK vector into HeLa cells. After 18 h-incubation, culture medium was changed to fresh medium containing 10 µM GM or DMSO (vehicle), and after 30 min a half of each treated cells were subjected to a mild hyperthermia at 40°C for 12 h. The expression levels of luciferase reporter genes were examined by a dual luciferase assay, and the activity of Photinus (target) luciferase was normalized to that of Renilla (control) luciferase, and further normalized to the data of pGL4-minP given as 1. The data are averages of three independent experiments and error bars represent standard deviations. (C) Gene silencing profiles in the presence of GM. The constructed reporter plasmids for assessment of gene silencing involving miRNAs were transfected into HeLa cells, and the cells were subjected to the same treatments as in B. The assessment of gene silencing was performed as in Fig. 1C. The data are averages of three independent experiments and error bars represent standard deviations. The upper and lower panels indicate the data in the presence and absence of GM, respectively.

Fig 3: GKAP Regulates Cancer Cell Invasion through NMDAR Activity and Downstream Effectors FMRP and HSF1(A) Western blot for GKAP and p-GluN2b in control and GKAP-knockdown ßTC-3 cells under unstimulated cell culture conditions. The numbers below indicate levels of p-GluN2b and GKAP normalized to untransfected ßTC-3.(B) Fluorescence reporter assay was performed in ßTC-3 small hairpin RNA (shRNA) control and GKAP-knockdown (KD) cells lines, comparing calcium transients induced by puffing an NMDA solution onto the cells. Clusters of cultured control ßTC-3 cells or GKAP-KD ßTC-3 cells were analyzed under a bright-field microscope after an NMDA solution (1 mM, 1 s) was puffed through perfusion pipette at left (upper panels). The arrows indicate the direction of puffing. ßTC-3 shRNA control KD cells: 78 of 284 cells examined showed a response (orange bars). ßTC-3 shRNA-GKAP KD cells: 5 of 178 cells showed minor NMDA responses (light blue bars). p < 10-11, Wilcoxon rank-sum test. Histogram of transient amplitude, denoting time-resolved fluorescence signals (sampling frequency/frame rate = 12.5 Hz). Light orange bar and blue bar indicate cells with no response.(C) Invasion assay in control and GKAP-KD ßTC-3 cells. Two-way ANOVA, Bonferroni multiple comparisons test: **p < 0.01; n.s., not significant (mean ± SEM, n = 3 invasion assay devices/condition in one experiment; two independent experiments were performed with consistent results).(D) IHC staining of FMRP in B6 PanNETs. Similarly sized invasive versus non-invasive primary tumors on the same section were used for comparison (tumor borders marked by yellow dashed line in the representative images). Rare, multiple metastatic lesions in the liver from one mouse (indicated by the red arrowheads, tumor borders marked by yellow dashed line). Images shown are representative of an analysis of >50 PanNETs from >10 B6 RIP1Tag2 mice, one section per mouse, and all staining was performed in the same experiment. Magnified lesion is representative of >100 metastases from one liver.(E) Western blot shows the efficiency of FMRP knockdown in ßTC-3 cells. The numbers below indicate levels of FMRP normalized to GAPDH. Bar graph: invasion assay. Unpaired t test: *p < 0.05; **p < 0.01. FMRP #1 and #2 indicate two different siRNA constructs used (mean ± SD, n = 3 invasion assay devices per condition in one experiment; two independent experiments).(F) Western blots comparing FMRP and p-HSF1 expression in ßTC-3 cancer cells infected with control shRNA or shRNA-GKAP lentiviral vectors (left), and comparing expression of p-GluN2b, FMRP, and p-HSF1 in ßTC-3 cancer cells treated with either vehicle or MK801 (right). GAPDH was used as a loading control and the numbers below indicate levels of p-GluN2b, FMRP, and p-HSF1 normalized to GAPDH. (n = 3).(G) Left: tissue immunostaining shows expression of total and active GluN2b, FMRP, and total and active HSF1 in tumors from saline- and MK801-treated B6 RIP1Tag2 mice. Data shown are representative of 9–21 random pictures from >15 PanNETs from three mice per group. Scale bar in the blow-up picture represents 25 µm. Right: quantification of FMRP and pHSF1 expression in saline- and MK801-treated tumors. Mean ± SEM. FMRP, n = 9 pictures in saline treated groups, n = 21 in MK801-treated group; p-HSF1, n = 15 pictures in saline treated groups, n = 20 in MK801-treated group. Mann-Whitney test: *p < 0.05; **p < 0.01.See also Figure S2.

Fig 4: Effective targeting of HSF1 activity reduces the levels of molecular chaperone proteins.(A) Western blot analysis showing the depletion of specific molecular chaperone proteins in aptamer or control expressing cells. Hsp90 co-expression rescues specific molecular chaperones observed in HSF1 inhibited aptamer expressing cells. The asterisk indicates PARP degradation product, a marker of apoptosis. The left most three lanes are a serial dilution of parental line extracts that provides a quantification standard curve. (B) Quantification of the results observed in panel A (n = 4, error indicates %SEM).

Fig 5: iaRNAHSF1 expression attenuates transformed growth.HSF1 inhibition by iaRNAHSF1 inhibits transformed growth in soft agar. Soft agar analysis of non-transfected HeLa cells (top left) or control RNA over-expressing HeLa (bottom left), shows that iaRNAHSF1 over-expression (bottom right) inhibits cellular transformation (colony formation) in a similar manner as treatment of HeLa cells with 150 nM 17-AAG (top right) (Day 14).