Fig. 5: Autophagy increases in Gal-CC. | Nature Communications

Fig. 5: Autophagy increases in Gal-CC.

From: Exploiting oxidative phosphorylation to promote the stem and immunoevasive properties of pancreatic cancer stem cells

Fig. 5: Autophagy increases in Gal-CC.The alternative text for this image may have been generated using AI.

a Representative brightfield images of 14-day PANC185 Gluc-CC or Gal-CC, taken by optical microscopy. Scale bar = 50 µM. b Representative transmission electron micrographs of 14-day PANC185 Gluc-CC or Gal-CC. Autophagic structures (red arrow heads). c Mean number of autophagic vesicles ± sd in the TEM images of 14-day PANC185 and PANCA6L Gluc-CC and Gal-CC, quantified by ImageJ (n = 6 independent fields; Holm–Sidak t test statistical analysis). **p = 0.0078; ***p = 0.00011. d Representative immunofluorescence images of PANC185 Gluc-CC and Gal-CC stained with anti-LC3BI/II (green), anti-LAMP-1 (red), and DAPI (blue). e Mean percentage of fluorescent area ± sd for LC3BI/II comparing Gluc-CC and Gal-CC with ImageJ (n = 4 independent fields; Holm–Sidak t test statistical analysis). *p = 0.024; **p = 0.0027. f Mean percentage of fluorescent area ± sd for LAMP-1 in PANC185 and PANCA6L Gluc-CC and Gal-CC, using ImageJ (n = 4 independent fields; Holm–Sidak t test statistical analysis). **p = 0.00107; ***p = 0.00038. g WB analysis of LC3BI/II in Gluc-CC and Gal-CC PDAC cultures. GAPDH was used as loading control. h Densitometric quantification of LC3BII expression in g, normalized to GAPDH levels and expressed as relative values for each tumor or pooled samples from the same immunoblot (n = 4 biological replicates in Pool; Holm–Sidak t test statistical analysis). *p = 0.041. i RT-qPCR analysis of relative mRNA expression levels of ATG5 in Gluc-CC and Gal-CC PDAC cultures. mRNA expression levels for each target gene are normalized to β-actin levels (n = 3 biological replicates per tumor, n = 12 for pool; Holm–Sidak t test statistical analysis). Data are presented as mean fold change ± sd. Gluc was set as 1.0. ***p = 0.00002; **p = 0.0016; **p = 0.00105; ***p = 0.000088; ***p = 0.000029. j WB analysis of LC3BI/II in Gluc-CC and Gal-CC untreated (Gluc, Gal) or treated with the autophagy inhibitor (Baf = Bafilomycin). GAPDH was used as loading control. k Mean fold change ± sd of densitometric quantification of autophagic flux (LC3B-II accumulation after autophagy inhibitor addition, subtracting their respective control expression levels), (n = 4 independent immunoblots for each culture; n = 8 pooled values, Holm–Sidak t test statistical analysis). Gluc was set as 1.0. **p = 0.0077; **p = 0.0012; ***p = 0.000022.

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