Figure 3.

(A) Representative confocal image of HeLa cells after transfection with two tsRNAs (5′ tsRNA-Ala^AGC labeled with FAM, 5′ tsRNA-Gly^GCC labeled with Atto590, each 100 nM) in separately produced liposomes. Left image shows merge of both signals (5′ tsRNA-Ala^AGC in cyan, 5′ tsRNA-Gly^GCC in magenta) with inset showing separation of both 5′ tsRNA-derived signals. To monitor the induction of SG formation, indirect immunofluorescence with an antibody against G3BP1 (magenta) was used. (B) Representative confocal image of HeLa cells after transfection with two tsRNAs (5′ tsRNA-Ala^AGC labeled with FAM, 5′ tsRNA-Gly^GCC labeled with Atto590, each 100 nM) in the liposomes (pooled). Left image shows merge of both signals (5′ tsRNA-Ala^AGC in cyan, 5′ tsRNA-Gly^GCC in magenta) with inset showing co-localization of both 5′ tsRNA-derived signals. To monitor the induction of SG formation, indirect immunofluorescence with an antibody against G3BP1 (magenta) was used. (C) Representative confocal image of HeLa cells after transfection with 5′ tsRNA-Gly^GCC labeled with Atto590, but containing a 3′ CycP, 100 nM). Left image shows 5′ tsRNA-Gly^GCC in magenta, while SG formation was monitored by indirect immunofluorescence using an antibody against G3BP1 (cyan). Dashed region was digitally magnified to show the presence of G3BP1-positive SG. Inset: DNA (black). (D) Representative confocal image of HeLa cells after transfection with 5′ tsRNA-Ala^AGC labeled with FAM, 100 nM). Left image shows 5′ tsRNA-Ala^AGC in cyan, while SG formation was monitored by indirect immunofluorescence using an antibody against G3BP1 (magenta). Dashed region was digitally magnified to show the presence of G3BP1-positive SG. Inset: DNA (black). (E) Quantification of SG formed by transfection of HeLa cells with dsRNA (poly-IC,155 pM) or after time-limited exposure (1 h) to As[III] (0.3 mM). SG formation was quantified by counting cells with exactly one (1 SG) and more than one SG per cell (>1 SG) using ImageJ. Number of analyzed cells in parentheses. (F) Representative confocal image of HeLa cells after transfection with dsRNA (poly-IC,155 pM). SG formation was monitored by indirect immunofluorescence using an antibody against G3BP1 (magenta). Arrows point at necroptotic cells. DNA is false-colored in cyan. (G) Quantification of SG formed by transfection of HeLa cells with various 5′ tsRNAs or a small RNA control (related to Supplementary Figure S3A) at the highest molarity tested (100 nM) SG formation was quantified as described in (E). Number of analyzed cells in parentheses. (H) Quantification of SG formed by transfection of HeLa cells with two molarities (A: 10 nM, B: 100 nM) of tsRNAs (5′ tsRNA-Ala^AGC and 5′ tsRNA-Gly^GCC) in separately produced liposomes or the same liposomes. Transf. (ctrl) designates SG counts in cells that were treated with Lipofectamine only. Number of analyzed cells in parentheses. (I) Representative confocal image of HeLa cells after transfection with transfection reagents only (+P3000). SG formation was monitored by indirect immunofluorescence using an antibody against G3BP1 (magenta). Arrows point at cells with SG. DNA is false-colored in cyan.