Fig 2: Autophagy caused by MTOR inhibition does not require MAPK8/9 in immortalized MEFs. (a) The amount of RPS6KB1, p-Thr389 RPS6KB1, and TUBA (tubulin alpha) in WT and mapk8-/- mapk9-/- immortalized MEFs after incubation without or with 250 nM torin 1 (2 or 4 h) was examined by immunoblot analysis. (b) WT and mapk8-/- mapk9-/- immortalized MEFs were transduced with a lentivirus vector that expresses GFP-LC3B. Puncta formation following incubation of the cells with 250 nM torin 1 (2 and 4 h) was examined by fluorescence microscopy. Scale bar: 30 µm. (c) LC3B and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression by WT and mapk8-/- mapk9-/- immortalized MEFs after incubation (2 h) without or with 250 nM torin 1 in the absence or presence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the mean of WT control condition (first lane). The ‘Change in MAP1LC3B-II’ was calculated by subtracting MAP1LC3B-II:GAPDH (media+ CQ condition) from MAP1LC3B-II:GAPDH (torin 1+ CQ condition). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Two-way ANOVA was used for the analysis of LC3B-II expression and Student’s t-test is used for the flux analysis. (d) The amount of SQSTM1 and TUBA in WT and mapk8-/- mapk9-/- immortalized MEFs after incubation with 250 nM torin 1 (2 or 4 h) was examined by immunoblot analysis. The SQSTM1:TUBA ratio was quantified and normalized to SQSTM1 expression in WT cells treated without torin 1 (mean ± SEM; n = 3 independent experiments; **, p < 0.01 (two-way ANOVA). (e) RPS6KB1, p-Thr389 RPS6KB1, AKT, p-Thr308 AKT, p-Ser473 AKT, and GAPDH expression by WT and mapk8-/- mapk9-/- immortalized MEFs after incubation (2 or 4 h) without or with 200 nM rapamycin was examined by immunoblot analysis. (f) LC3B and TUBA expression by WT and mapk8-/- mapk9-/- immortalized MEFs after incubation (2 h) without or with 200 nM rapamycin in the presence or absence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:TUBA ratios were normalized to the mean of WT control (first lane). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Two-way ANOVA was used for the analysis of LC3B-II expression and Student’s t-test was used for the flux analysis.

Fig 3: Effect of starvation and MTOR inhibition on MAPK8/9 activation is not sufficient to cause autophagy. (a and b) MAPK8/9 activation in immortalized MEFs was examined by immunoblot analysis of p-Thr183/Tyr185 MAPK8/9, MAPK8/9, and GAPDH in cells after incubation (2 or 4 h) with EBSS containing 5 mM glucose (a) or 250 nM torin 1 (b). Lanes 1 and 2 represent positive and negative controls: lysates of WT MEFs exposed to 60 J/m2 UV and mapk8-/- mapk9-/- MEFs, respectively. (c) WT and mapk8-/- mapk9-/- immortalized MEFs were exposed to UV radiation (60 J/m2) and cell extracts were prepared at 45 min post-irradiation. The expression of LC3B, p-Thr183/Tyr185 MAPK8/9, MAPK8/9, and GAPDH was examined by immunoblot analysis.

Fig 4: Molecular consequences of SNRNP200, SRRM1 and SRSF3 silencing in 22Rv1 cell line. a) Basal phospho-AKT, phospho-ERK1/2 and phospho-JNK levels in SNRNP200-, SRRM1- and SRSF3 silenced 22Rv1 cells (si-SNRNP200, si-SRRM1 and si-SRSF3, respectively; 24 h; n = 3). Protein levels were normalized by total AKT, ERK and JNK protein levels. Representative images are shown in right panel. Protein data were represented as percent of scramble cells. b) Expression levels of selected transcripts in response to SNRNP200 (upper panel), SRRM1 (central panel) and SRSF3 (bottom panel) silencing (24 h) in 22Rv1 cells. Ratio between the expression of splicing variants is shown in bars with dotted pattern. c) Expression levels of KHDRBS1, SFPQ and U2AF2 in response to SNRNP200- and SRRM1-silencing in 22Rv1 cells. d) Expression levels of C-MYC, PTEN and TP53 in response to SNRNP200-, SRRM1- and SRSF3-silencing in 22Rv1 cells. mRNA levels were determined by qPCR and adjusted by normalization factor calculated from ACTB and GAPDH expression levels. Data were represented as percent of scramble-treated control cells (mean ± SEM). Asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001) indicate statistically significant differences between groups.

Fig 5: Autophagy caused by starvation does not require MAPK8/9 in immortalized MEFs. (a) RPS6KB1, p-Thr389 RPS6KB1, and TUBA expression by WT and mapk8-/- mapk9-/- immortalized MEFs after incubation with EBSS containing 5mM glucose (2 or 4 h) was examined by immunoblot analysis. (b) WT and mapk8-/- mapk9-/- immortalized MEFs were transduced with a lentivirus vector that expresses GFP-LC3B. Puncta formation following incubation with EBSS containing 5 mM glucose (2 h) was examined by fluorescence microscopy. Scale bar: 30 µm. (c) LC3B and GAPDH in WT and mapk8-/- mapk9-/- immortalized MEFs after incubation (2 h) in medium or with EBSS containing 5 mM glucose in the presence or absence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the average of WT control condition (first lane). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p < 0.05; **, p < 0.01; ***; p < 0.001; ****; p < 0.0001. Two-way ANOVA was used for the analysis of LC3B-II expression and Student’s t-test was used for the flux analysis. (d) The amount of SQSTM1 and GAPDH in WT and mapk8-/- mapk9-/- immortalized MEFs after incubation with EBSS containing 5 mM glucose (2 or 4 h) was examined by immunoblot analysis. The SQSTM1:GAPDH ratio was quantified and normalized to WT cells treated without EBSS (mean ± SEM; n = 3 independent experiments); *, p < 0.05; **, p < 0.01; ***; p < 0.001 (two-way ANOVA).