Fig 2: cAMP signaling enhances the DNA damage-induced LC3-II/LC3-I ratio(A and B) REH cells (0.6×106 cells/ml) were incubated in the presence or absence of forskolin (Forsk, 60 µM) for 45 min prior to irradiation (IR, 10Gy), and total lysates were subjected to immunoblot analyses with antibodies against LC3B or calnexin (CANX). The numbers indicated below the LC3 images represent the LC3-II/LC3-I signal ratios relative to the CANX signals, normalized to the ratio in untreated (Ctrl) cells. (A) When indicated, BafA1 (2 nM) was added from the start of the cultures. The cells were harvested at the indicated time points after IR, and one representative Western blot of three is shown. (B) Left panel: The cells were harvested 24 hours after IR, and BafA1 (2 nM) was added for the last 4 hours, as indicated. One representative Western blot of 8 is shown. Right panel: Ratios of the LC3-II/LC3-I signal intensities relative to the CANX signals, normalized to ratio in untreated (Ctrl) cells. The data represent the mean +/- SEM, n=8. *p< 0.05 (paired t test).

Fig 3: IFIT3 is detected in sEVs isolated via iodixanol density gradients from PrCa cells devoid of the ß6 integrin subunit.(A) IB analysis of sEV fractions (F1-F10) isolated via iodixanol density gradients and derived from PC3 wild-type (PC3-WT) cells containing the ß6 integrin subunit, (PC3-WT). PC3-WT TCL (20 µg), PC3-WT (PC3-WT 100K) sEV lysate (10 µg) and PC3-WT sEV fraction lysates were separated using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have densities of 1.103, 1.117,1.134,1.151, 1.169, 1.186, 1.193, 1.193, 1.200, and 1.207 g/ml, respectively. Expression of the ß6 integrin subunit, CD63, CD81 (left panel, non-reducing SDS–PAGE) as well as STAT1, IFIT3 and CD9 (right panel, reducing SDS–PAGE) was analyzed in sEV fractions 1 to 10. The STAT1 membrane (right panel, reducing SDS–PAGE) was stripped to visualize calnexin (CNX). CNX (right panel, reducing SDS–PAGE) in PC3-ß6KO C7, PC3-WT TCL, PC3-WT 100K as well as 10 consecutive PC3-WT derived sEV fractions was analyzed. PC3-WT 100K lysate was the input for the density gradient. (B) IB analysis of PC3-ß6KO C7 derived sEV fractions isolated via density gradients. Total lysates (TCL 20 µg) from PC3-WT cells containing the ß6 integrin subunit, as well as the PC3-CRISPR clone devoid of the ß6 integrin subunit, PC3-ß6KO C7, and PC3-ß6KO C7 sEV fraction lysates were separated using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have densities of 1.075, 1.099, 1.123, 1.134, 1.151, 1.165, 1.169, 1.182, 1.186, and 1.193 g/ml, respectively. Expression of the ß6 integrin, CD63, CD81 (left panel, non-reducing SDS–PAGE), STAT1, IFIT3 and CD9 (right panel, reducing SDS–PAGE) was analyzed in sEV fractions 1 to 10. The STAT1 membrane (right panel, reducing SDS–PAGE) was stripped to visualize calnexin (CNX). CNX (right panel, reducing SDS–PAGE) in the PC3-CRISPR clone lacking the ß6 integrin subunit, PC3-ß6KO C7 TCL and PC3-ß6KO C7 derived sEV fractions was analyzed. A and B, TCL = total cell lysates.

Fig 4: STAT1 down-regulation affects IFIT3 protein expression in PrCa cells and their sEVs.(A) IB analysis of PC3-CRISPR control cells and PC3-CRISPR clones devoid of STAT1, PC3-STAT1KO C21 as well as PC3-STAT1KO C1C5 TCL (total cell lysates) (30 µg). TCL were separated using 12.5% SDS–PAGE. TCL were examined for the expression of STAT1, IFIT3, and Actin (reducing SDS–PAGE). The IFIT3 membrane (reducing SDS–PAGE) was stripped to visualize Actin. Actin was used as a protein loading control. (B) NTA measurement of size distribution and concentration of PC3-STAT1KO C21 derived sEV fractions 2-5. (C) IB analysis of sEV fractions derived from a PC3-CRISPR clone lacking STAT1, designated as PC3-STAT1KO C21, and isolated via density gradients. PC3-ß6KO C7, PC3-STAT1KO C21 TCL (20 µg), PC3-STAT1KO C21 (STAT1KO C21 100K) sEV lysate (10 µg) and PC3-STAT1KO C21 sEV fraction lysates were separated by using 12.5% SDS–PAGE. Comparable volumes of each sEV fraction were loaded (30 µl). The 10 consecutive sEV fractions have a density of 1.106, 1.123,1.127,1.144 1.165, 1.169,1.186, 1.193, 1.200, and 1.207 g/ml, respectively. Expression of STAT1, IFIT3, TSG101, CD9 and CD81 (reducing SDS–PAGE) was analyzed in sEV fractions 1 to 10. The IFIT3, STAT1 and CD9 membranes (reducing SDS–PAGE) were stripped to visualize TSG101, calnexin (CNX), and CD81 respectively. CNX expression (reducing SDS–PAGE) was analyzed in PC3-CRISPR clone devoid of the ß6 integrin subunit, PC3-ß6KO C7 as well as PC3-CRISPR clone lacking STAT1, PC3-STAT1KO C21 TCL, STAT1KO C21 100K and 10 consecutive PC3-STAT1KO C21 derived sEV fractions. PC3-STAT1KO C21 100K sEV lysate was used as input for the density gradient.

Fig 5: p53 is involved in IR-, but not in cAMP-induced autophagy(A-D) REH cells (4×106 cells) were transfected with siRNA against p53 (or with scrambled siRNA as control) as described in Materials and Methods, and after 12 hours the cells were treated with or without forskolin and IR as described in Figure 1. (A) Knock-down of p53 by siRNA demonstrated by Western blot analyses of untreated (Ctrl) and irradiated (IR) cells. Signal intensities of p53 relative to GAPDH, normalized to Ctrl, are indicated as numbers below the p53 images. (B-D) BafA1 (2 nM) was added for the last 4 hours of the 24 hours incubations. (B) 24 hours after IR, the cells were harvested for Western blot analyses of LC3-II/I ratios, and one representative of 4 independent experiments is shown. The numbers indicated below the LC3 images represent the LC3-II/LC3-I signal ratios relative to the CANX signals, normalized to the ratio in untreated (Ctrl) cells. (C) Quantifications of the Western blots in panel B, presented as the ratios of LC3-II/LC3-I signal intensities relative to the CANX signals, normalized to the ratio in untreated (Ctrl) cells. The data represent the mean +/- SEM, n=4. *p<0.05 (paired t test). (D) The same cells as in panel A were subjected to CYTO-ID staining, and the fluorescence intensity was analysed by flow cytometry 24 hours after IR. The data represent the mean CYTO-ID fluorescence intensity +/- SEM, n=4. *p<0.05 (paired t test).