Erratum to: Protease-activated-receptor-2 affects protease-activated-receptor-1-driven breast cancer

Erratum to: Cell. Mol. Life Sci. (2014) 71:2517–2533 DOI 10.1007/s00018-013-1498-7

In the published article, duplicated or inverted gel sections had been used in the Figs. 1Ai, Aii, D, 2Aii, 4B and 6C. In agreement with the Editor-in-Chief the experiments were repeated and the same results obtained. The primary data from the repeated experiments can be found below.

Fig. 1.

Silencing of hPar2 inhibits association of key signaling partners with the PAR₁ C-tail. A i MCF7 cells stably over-expressing HA-hPar1 and T7-Etk/Bmx were infected with a lentiviral vector driving either shRNA- hPar2 expression or a scrambled shRNA-hPar2 and cultured with or without thrombin activation (1 U/ml) (A i) or the PAR₁ selective ligand, TFLLRN (100 μM) (A ii). Cell lysates were collected at the indicated times and immunoprecipitated using anti-HA antibodies. Immunoprecipitates were separated on SDS-PAGE and Western blotted with anti-T7 antibody to detect T-7 tagged Etk/Bmx. Maximal association of Etk/Bmx is seen after 10 min of thrombin treatment in cells without hPar2 silencing (scrambled shRNA-hPar2); but is not detectable in cells infected with shRNA-Par2. A specific, Etk/Bmx is seen associated after 5′, 10′, 15′, and 20 min following TFLLRN activation in cells expressing both HA-hPar1 and endogenous hPar2 (scrambled shRNA-hPar2). In-contrast, the Etk/Bmx association is not detected in the presence of shRNA-hPar2. B Silencing hPar2 inhibits thrombin-induced association between PAR₁ and Akt. MCF7 clones overexpressing HA-hPar1, with shRNA hPar2 silencing (right), or scrambled shRNA hPar2 (left), were treated with thrombin for the indicated times. Cell lysates were immunoprecipitated with anti-HA to precipitate HA-PAR₁ and analyzed by Western blot with anti-Akt antibodies, followed by antibodies to phosphorylated Akt (pAkt). HA-PAR₁ serves as a loading control. C PAR₂ expression in the MCF7 clone (expressing hPar1, hPar2, and etk/bmx) with and without shRNA silencing as compared with a housekeeping gene GAPDH. D Silencing hPar2 inhibits TFLLRN PAR₁ activation in MDA-MB-231 cells. MDA-MB-231 cells expressing high endogenous levels of both PAR₁ and PAR₂ show a similar pattern (to MCF7 cells) of selective PAR₁ inhibition in the presence of sh-RNA hPar2. TFLLRN activation induces the co-IP between PAR₁ and Akt immediately after 2′, 5′, 10′, and 20′ activation (scrambled sh-RNA hPar2). In the presence of sh-RNA hPar2, this association is markedly inhibited. β-actin serves as a loading control

Fig. 2.

A i Silencing hPar2 in a breast cancer cell line inhibits thrombin- or TFLLRNPNDK-induced colony formation in soft agar. 5 × 10³ MDA-MB-231 cells, either uninfected (a, b) or infected with virus expressing shRNA-GFP-hPar2 (e, f), were plated in soft agar and activated by thrombin (e) or TFLLRNPNDK (f). Uninfected cells were treated with thrombin (c) or TFLLRN (d) as also with SLIGKV (i, j). Controls of either scrambled shRNA hPar2 with thrombin activation (g) and shRNA hPar1 with thrombin activation (h) were shown as well. After 12 days, live images were collected using a Zeiss microscope at ×20 magnification. In uninfected cells, large colonies formed following treatment with either thrombin (c) and somewhat smaller with TFLLRNPNDK (d). MDA-MB-231 cells infected with sh-GFP-hPar2 formed only very small colonies after thrombin (e) or TFLLRNPNDK (f) treatment. Cells containing sh-hPar2 are visualized by GFP fluorescence (e). The colonies formed under different conditions were compared with control non-activated, non-treated cells (control; a, b). The colonies formed in the presence of shRNA-hPar1 and after thrombin activation is significantly smaller (panel h) than colonies formed after thrombin activation (c). Scrambled shRNA-hPar2 had no effect on the colonies formed (panel g). Comparable colony size is obtained by SLIGKV PAR₂ activation (i, j) as found by TFLLRNPNDK (d). Images shown are representative of three independent experiments. A ii RT-PCR analysis of hPar1 and hPar2 mRNA expression before and after shRNA silencing. GAPDH levels were analyzed as a control. A iii Histogram shows mean ± SE of triplicate values from three independent experiments. Post hoc evaluation of multiple comparison (ANOVA Tukey HSD) showed a p value of 0.004 within groups. The mean difference is significant at the 0.05 level. For ANOVA evaluation we used IBM SPSS 20.0 software. B i Silencing hPar2 in MDA-MB-231 cells inhibits TFLLRN PAR₁ invasion. Matrigel invasion in the presence of shRNA-hPar2-infected cells as compared with scrambled shRNA. While TFLLRN specific activation of PAR₁ induces Matrigel invasion in MDA-MB-231 cells, this was attenuated in the presence of shRNA-hPar2-infected cells. In-contrast, no effect was observed when a scrambled shRNA was utilized to infect the cells, demonstrating a markedly induced Matrigel invasion similar to non-treated activated parental MDA-MB-231 cells. B ii Histograms represents quantification of the cells/HPF invaded the Matrigel layer. Unpaired Student’s t test was used. This experiment is a representative of three independent experiments performed in triplicates. B iii Silencing hPar1 in MDA-MB-231 cells did mot inhibit PAR₂ function. In MDA-MB-231 cells silenced for hPar1, thrombin activation resulted with a low level of Matrigel invasion. In-contrast, SLIGKV PAR₂ activation under conditions of sh-hPar1 silencing, resulted with potent Matrigel invasion. B iv Histograms represents quantification of the cells/HPF invaded the Matrigel layer in shRNA hPar1 silenced cells. Unpaired Student’s t test was used. This experiment is a representative of three independent experiments performed in triplicates

Fig. 4.

Co-localization of PAR₁ and PAR₂. A Confocal immunofluorescence analysis. HEK-293T cells were transfected with both HA-hPar1 and YFP-hPar2. After 24 h, the cells were serum deprived for an additional 24 h then activated with thrombin for 5 min followed by fixation with cold methanol. PAR₂ was visualized by direct fluorescence (a) and PAR₁ was visualized by immunostaining with anti HA-antibodies followed by Cy3-conjugated IgG secondary antibodies (b). Merge staining for both PAR₁ and PAR₂ revealed co-localization confined to the cell membrane (d). For reference, staining of cell nuclei with Draq5 is shown (c). The bottom panel highlights staining in one cell exhibiting the expression of YFP-hPar2 and HA-hPar1 on the cell membrane prior to activation (i, ii). Significant co-localization recapitulated by merge fluorescence is observed following five minutes thrombin activation (iv). This is detected as compared with cell nuclei staining (iii) and phase-contract analysis (v). The percent of positive cells for HA-hPar1 was 58 % ± 1.2 and for YFP-hPar2 were 55 % ± 1.86; PAR₁-PAR₂ merge was observed in 45 % ± 2.1. B PAR₁ and PAR₂ co-immunoprecipitate. HEK 293T cells expressing either HA-hPar1, YFP-hPar2 or GFP-CXCR4 were treated with thrombin for 10 min and lysed. Cell lysates were then immunoprecipitated before (−) and after (+) thrombin activation by anti HA or IgG, resolved by SDS-PAGE and immunoblotted as indicated (by anti-GFP). While no specific complex is formed when CXCR4 and PAR₁ were co-IP (following 10-min thrombin activation), a specific complex is observed between PAR₁ and PAR₂ following activation. No specific band is seen when IgG were applied in the immunoprecipitation assay. Expression of CXCR4, PAR₂ (as indicated by anti-GFP, first lane), and PAR₁ (as shown by anti-HA) were shown as controls for transfection efficiency. The figure shown is a representative of three independent experiments

Fig. 6.

Impaired tumor development in the presence of PAR₁ and truncated PAR₂. Stable clones of HU cells expressing either hPar2 or hPar1 and hPar2, or hPar1 and truncated hPar2 were injected subcutaneously into nude mice (2 × 10⁶ cell/mouse). Parental non-transfected HU cells were used as a control. A Tumor morphological appearance. Mice injected either with hPar1 and hPar2 or hPar2 developed tumors at the sites of injection. In-contrast, mice inoculated with wt hPar1 and a truncated form of hPar2 did not develop or developed very little tumors (a). By the end of the experiment (e.g., 35 days) the mice were killed and the tumors were excised, measured, and weighed (n = 4/per group). B Mouse tumor growth curve in nude mice. Tumors were excised, weighed and measured at the indicated times and tumor volume (mm³) was calculated. Error bars show SD; * indicates p < 0.006. Data shown are representative of three experiments performed. C i, ii RT-PCR analyses of the indicated clones (a and b; two different clones) show the expression of PAR₁ and PAR₂, truncated PAR₂ and the prime signal protein Etk/Bmx