Fig 2: Loss of one Trp53 allele affects NF-?B pathway via regulation of I?Ba mRNA expression.A Levels of P-I?Ba and I?Ba in untreated and LPS-treated (100 ng/ml) BMDMs from p53 WT, HEM and KO mice at the indicated times. Representative image of a western blot analysis from three independent experiments is shown. B Quantification of I?Ba levels from three independent western blot analysis. A graph for basal levels (left panel) and a graph for LPS treatment (right panel) are shown. Expression values were referred to tubulin levels. C Quantification of P-I?Ba levels from three independent western blot analysis. Expression values were referred to tubulin levels. D Relative expression of Nfkbia mRNA in BMDMs from WT, HEM and KO mice treated with LPS (100 ng/ml) at the indicated times. A graph for basal levels (left panel) and a graph for LPS treatment (right panel) are shown. Values refer to biological replicates in three independent experiments. E p65 ChIP qPCR of Nfkbia promoter. WT, HEM, and KO BMDMs were untreated and treated with LPS (100 ng/ml, 60 min) before ChIP was performed. Values were normalized to input and the results are shown as fold change related to control immunoglobulin (Ig) sample. Values refer to biological replicates of two independent experiments. F p53 ChIP qPCR of Nfkbia promoter. BMDMs were untreated or treated with LPS (100 ng/ml, 60 min) before ChIP was performed. Values are normalized to input and the results are shown as fold change related to Ig sample. Values refer to biological replicates of three independent experiments for WT and KO, and one for HEM BMDMs. In B, D, E and F, bars represent mean values ± SEM. In B, C and D (right panels) mean values ± SEM are represented. AU arbitrary units. Significant differences and p values were derived from an unpaired t-test, two-tailed. wt vs HEM or KO. ****p value < 0.0001, **p value < 0.005, *p value < 0.05. HEM vs KO. ###p value < 0.0005, #p value < 0.05.

Fig 3: Effect of engeletin on PMA-induced IKK phosphorylation, IkBa phosphorylation, and IkBa degradation, in NCI-H292 cells. NCI-H292 cells were incubated with varying concentrations of engeletin for 24 h and treated with 50 ng/mL PMA for 30 min. Cytoplasmic extracts were fractionated and then subjected to western blot analysis using phospho-specific I?Ba (Ser 32/36) antibody or antibody against anti-I?Ba. Whole cell lysates were prepared and then subjected to western blot analysis using phospho-specific IKKa/ß (Ser 176/180) antibody. Equal protein loading was evaluated by ß-actin levels. *Significantly different from control (p<0.05). †Significantly different from PMA alone (p<0.05). Cont: control, En: engeletin, IkBa: inhibitory kappa Ba, IKK: inhibitory kappa B kinase, concentration unit is µM.

Fig 4: Effect of LAMP-2C expression on CMA substrates. (A) Cellular levels of CMA substrates Chk1, I?Ba, and p-I?Ba in DM331 pCMV and DM331 2C myc cells were examined by western blotting. Relative protein levels were calculated by setting the normalized expression to one for DM331 pCMV cells. (B) mRNA levels of CHEK1 and NFKBIA transcripts were analyzed by qPCR and normalized to ACTB expression. mRNA levels in DM331 pCMV cells were normalized and set to one. (C) Proteasome activity was measured using the Proteasome-Glo Chymotrypsin-Like Cell-Based Assay. Cells were incubated with a substrate succinyl-LLVY-aminoluciferin which penetrates into the cytoplasm. This substrate is cleaved by the proteasome to release aminoluciferin which is released from cells. Luciferase is added to these cells, cleaving aminoluciferin to a luminescent product detectable using a luminometer. Data were analyzed by two-way ANOVA or by two-tailed, unpaired Student’s t-test. *p < 0.05, ***p < 0.001, and ****p < 0.0001 (n = 3).

Fig 5: Effect of betulin on PMA-induced IKK phosphorylation, IkBa phosphorylation, and IkBa degradation, in NCI-H292 cells. NCI-H292 cells were incubated with varying concentrations of betulin for 24 h and treated with 50 ng/mL PMA for 30 min. Cytoplasmic extracts were fractionated and then subjected to western blot analysis using phospho-specific I?Ba (Ser 32/36) antibody or antibody against anti-I?Ba. Whole cell lysates were prepared and then subjected to western blot analysis using phospho-specific IKKa/ß (Ser 176/180) antibody. Equal protein loading was evaluated by ß-actin levels. *Significantly different from control (p<0.05). †Significantly different from PMA alone (p<0.05) (cont: control, B: betulin, IkBa: inhibitory kappa Ba, IKK: inhibitory kappa B kinase, concentration unit is µM).