Fig 2: miR-218-5p-sensitized GBC cells to gemcitabine was mediated by PRKCE. (a–d) Efficiency testing of overexpression or interference of PRKCE in GBC cells by using Q-PCR and western blot. (e-h) MTS assay and calculating the corresponding IC50 of gemcitabine in GBC cells transfected with PRKCE construct or PRKCE siRNAs. (i, j) Flow cytometric analysis of Annexin V/PI staining used to quantify apoptosis rate of GBC cells transfected with PRKCE construct or PRKCE siRNAs, followed by exposure to gemcitabine (NOZ, 10 μM; GBC-SD, 1 μM) for 72 h. (k, l) IC50 of gemcitabine in GBC cells co-transfected with miR-218-5p mimic and PRKCE construct. GAPDH was used to normalize the Q-PCR results, and β-actin was the loading control in western blot assay. All n=3; bar, S.E.M. *P<0.05; **P<0.01; ***P<0.001; Student’s t-test

Fig 3: miR-218-5p directly targets the 3′-UTR of PRKCE and downregulated its expression. (a) PRKCE and SFMBT1 were potential targets of miR-218-5p in all six miRNA target prediction algorithms. (b, c) Q-PCR to detect PRKCE and SFMBT1 mRNA level in GBC cells when transfected with miR-218-5p mimic or antagomir. n=3; bar, S.E.M. (d) Western blot to analyze PRKCE and SFMBT1 protein levels in GBC cell lines when transfected with miR-218-5p mimic or antagomir. (e) A schematic diagram showing the predicted miR-218-5p binding sites and the designed mutant sequence in the 3′-UTR of PRKCE (up), and the luciferase reporter constructs (down). (f, g) Firefly luciferase activity analysis of PRKCE 3′-UTR performed after co-transfection with PRKCE-wild type or PRKCE-mutant pGL3 constructs and miR-218-5p mimic GBC cell lines. n=3; bar, S.E.M. (h) Q-PCR analysis of PRKCE mRNA levels in 36 pairs of GBC and CNG tissues. (i) Semi-quantitative analysis and the representative images (× 400) of IHC staining for PRKCE protein in 82 paired GBC and CNG FFPE tissues. (j) The correlation between miR-218-5p and PRKCE expression in 36 GBC tissues measured by Q-PCR. (k) POS analysis based on PRKCE protein expression levels in 82 GBC patients. GAPDH was used to normalize the Q-PCR results, and β-actin was the loading control in western blot assay. NS, not significant, *P<0.05; **P<0.01; ***P<0.001; Student’s t-test

Fig 4: miR-218-5p negatively regulates resistance to gemcitabine through PRKCE/MDR1 axis in GBC cells. (a–d) Q-PCR and western blot to analyze MDR1, MRP1, and BCRP mRNA and protein expression, respectively, in GBC cell lines when transfected with miR-218-5p mimic or antagomir. (e, f) Q-PCR and western blot to analyse MDR1 mRNA and P-gp expression, respectively, in GBC cell lines when transfected with PRKCE construct or PRKCE siRNAs. (g–i) Q-PCR and western blot to detect MDR1 mRNA and P-gp expression, respectively, in GBC cell lines when co-transfected with miR-218-5p mimic and PRKCE construct. (j) IC50 of gemcitabine in GBC cells when co-transfected with miR-218-5p mimic and MDR1 construct. GAPDH was used to normalize the Q-PCR results, and β-actin was the loading control in western blot assay. All n=3; bar, S.E.M. *P<0.05; **P<0.01; Student’s t-test

Fig 5: PKCε mRNA (PRKCE) is downregulated in motor cortex in a subset of sporadic ALS patients. (a). Transcriptomic data extrapolated from E-MTAB-2325 dataset showing a statistically significant downregulation of PRKCE mRNA expression level in motor cortex in a subset of sporadic ALS patients (SALS2) compared to CTRL (control = 10, SALS1 = 18, SALS2 = 13 patients, respectively). (b). Transcriptomic data derived from GSE124439 dataset showing a statistically significant downregulation of PRKCE mRNA expression level in motor cortex in a subset of ALS patients (ALS-Glia) compared to non-neurological controls (NA) (NA = 15, ALS-TE = 8, ALS-OX = 51, ALS-Glia = 21 patients, respectively). Data were extrapolated from ArrayExpress (http://www.ebi.ac.uk/arrayexpress/ accessed on 1 June 2021) and Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/ accessed on 1 January 2023), respectively, and analyzed as described in the Materials and Methods section. Tukey–Kramer post hoc test: ** p < 0.01 vs. CTRL, *** p < 0.001 vs. NA. Circles, squares or triangles indicate the PRKCE expression level of single patients for each experimental group from the two datasets.