PMC

Control (A) and SMILE (B) siRNA transfected HeLa cells cultured 24 h with RPMI+vehicle (DMSO). Endoplasmic reticulum vacuolization in HeLa cells treated 24 h with 20 nM Bortezomib and transfected with SMILE siRNA (D) compared to cells transfected with control siRNA (C).

The luminescent signal measured in arbitrary units (AU) is proportional to the amount of proteasome activity and is decreased in SMILE siRNA versus control (C- siRNA) siRNA-transfected Hela cells (*p = 0.0313, Wilcoxon signed rank test).

(A) XBP-1 transcripts are significantly increased in SMILE siRNA-transfected HeLa cells after 6 h treatment with Bortezomib at 20 nM (*p = 0.0156, Wilcoxon signed rank test). (B) XBP-1 transcripts are also significantly increased in SMILE siRNA-transfected keratinocytes after 6 h treatment with Bortezomib at 20 nM (**p = 0.0078, Wilcoxon signed rank test), and are increased in SMILE siRNA-transfected keratinocytes without treatment (p = 0.0547, Wilcoxon signed rank test).

(A) PDIA3 labeling is localized in the endoplasmic reticulum. (B) SMILE labeling is mainly present in vesicles and in some cells in the reticulum area. (C) Merged picture showing the colocalization of PDIA3 and SMILE in the endoplasmic reticulum. (D) A higher magnification of (C) showing yellow dots in the endoplasmic reticulum. Bar in A is 40 µm. Bar in D is 10 µm.

Graphic representations of viable cell cluster numbers in clonogenic assays. (A) Absolute number of clusters of cells transfected with control siRNA (white bars) compared to cells transfected with SMILE siRNA (black bars) (**p = 0.0045, Mann-Whitney test). Absolute number of clusters of cells transfected with control siRNA (white bars) compared to cells transfected with SMILE siRNA (black bars) and treated with 5 nM Bortezomib (B, *p = 0.0317, Mann-Whitney test), 100 nM Thapsigargin (C, p = 0.3939, Mann-Whitney test) or 180 nM Etoposide (D, p = 0.4, Mann-Whitney test).

(A) SMILE mRNA transcripts were increased in the PBMC of operationally tolerant patients (TOL, n = 8) compared to patients with stable graft function under standard immunosuppressive therapy (STA, n = 9, **p<0.01) and deteriorating graft function under standard immunosuppressive therapy with biopsy-proven chronic antibody-mediated rejection (CAMR, n = 14, *p<0.05) (*p = 0.0205, Kruskal-Wallis test,). (B) The ROC curve measuring the ability of SMILE mRNA quantity to correctly classify operationally tolerant patients versus patients with stable function.