Fig 2: Effect of Imatinib and Dasatinib treatment on HERC1 and HERC2 expression in primary CML and K562 (Ph+) cells. The HERC1 and HERC2 gene expressions following Imatinib (1 μM) and Dasatinib (0.25 µM) treatment were determined at mRNA and protein level by RT-qPCR (A,C,E,F), immunofluorescence (B,D), and Western Blotting (G). Imatinib (1 µM) and Dasatinib (0.25 µM) treated K-562 cells (24 h) showed an increase in HERC1 gene expression both at mRNA and protein levels compared to non-treated cells, in contrast, an increase in HERC2 mRNA was observed without any change in its protein level. TKIs impaired the phosphorylation (enzymatic actively) of BCR-ABL, which indicates the HERC1 protein and mRNA levels of both HERCs are modulated by the activity of Bcr-Abl1. Vinculin was used as a loading control in Western blot. p ≤ 0.05 = *; p ≤ 0.01 = **.

Fig 3: Gene expression correlation analyses among HERC1, HERC2, and BCR-ABL from CML patients treated with two different TKIs. The scatter plots and Spearman coefficient (rho) showed the correlations occurring among the BCR-ABL, HERC1, and HERC2 transcripts in CML patients’ specimens collected at different time points starting from the beginning of the Imatinib (A) and Dasatinib (B) treatments up to 12 months (0, 3, 6, and 12). Values are expressed either as 2−∆∆Ct for both HERC1 and HERC2 and as I.S. for BCR-ABL, respectively. Each blue and red solid circle refers to a patient who has been treated either with Imatinib or with Dasatinib, respectively.

Fig 4: Leukemic cells’ differentiation towards the erythroid and megakaryocyte-like lineages antagonistically affect the HERC1 levels. Differentiation was performed by incubating Ph+ K562 myeloid leukemia cell lines with 250 nM phorbol 12 myristate 13-acetate (PMA) and 20 U/mL Erythropoietin (EPO). (A) Representative pictures of the morphology of uninduced control and differentiated cells; (B) K562 cells were harvested after 48 h with or without EPO induction and stained with benzidine. Benzidine-positive cells turned blue, indicated with red arrows. Untreated K562 cells served as a control. (C,D) Representative cell morphology images and flow cytometry analysis of PMA treated K562 cells. Prior flow cytometry cells were stained for megakaryocyte-specific cell surface marker CD61. (E,F) Expression changes in HERC1 gene at mRNA and protein level in non-treated, EPO, and PMA induced K562 cells. An increase in HERC1 protein and mRNA level was seen in EPO-treated K562 cells, while a decrease in HERC1 gene expression was observed in PMA treated cells. (G) Interestingly, there is not any change in HERC2 protein level in K562 differentiated cells. p ≤ 0.05 = *; p ≤ 0.01 = **.

Fig 5: Identification of candidate quality control factors for unassembled CCT subunits, related to Figure 1(A) CCT subunits with a C-terminal TST were translated in RRL, affinity-purified under native conditions via the TST, and analyzed by label-free quantitative mass spectrometry. The volcano plots illustrate proteins enriched in CCT subunit pull-downs compared with control pull-downs (see method details for details). The log2 fold difference (x axis) is plotted against the -log10 adjusted p value (y axis). The statistical analysis was done using a two-sided Student’s t test with Benjamini-Hochberg correction for multiple comparisons. The bait and non-bait CCT subunits are shown as magenta and yellow dots, respectively. E3 ligases, including components of E3 ligase complexes, are in blue. ZNRD2 (also called SSSCA1) is in green.(B) CCT subunits with a C-terminal TST were translated in RRL, affinity-purified under native conditions, and analyzed by immunoblotting for HECTD3, HERC1, HERC2, and ZNRD2. The CCT subunit was detected via the TST. Two amounts of total RRL were analyzed in parallel.