Figure 4.
Multiomic analysis identifies the Dck as a downstream target of Herc1. (A) Representation of the functional domains of HERC1 protein. (B) Volcano plot showing protein abundance in sgHerc1-MA cells vs sgCtrl MA cells. P values were calculated using t tests and were corrected for multiple hypothesis testing with Benjamin-Hochberg method. The dashed line represents P = .05. (C) Pathway enrichment analysis of 106 proteins that were significantly abundant in sgHerc1-MA cells. (D) Volcano plot showing differential expressed genes in sgHerc1-MA cells vs sgCtrl MA cells. The dashed line represents log2 fold change (FC) of ±1.5. (E) Integration of transcriptomic and proteomic data with the CRISPR/Cas9 screen to identify potential targets of Herc1. Potential candidates that passed the following criteria of (1) greater protein abundance (>1.5-fold) (shown in panel B) and (2) no corresponding change in mRNA transcript abundance (<1.5-fold) (shown in panel D). (F) Assessment of DCK protein levels in Herc1–gene edited cells. Representative western blot of whole cell lysates shows the DCK protein level in MA and HM-sgCtrl and sgHerc1 knockout cells using 3 different sgRNAs. (G) Quantification of DCK western blot as shown in panel F. Proteins are represented in arbitrary units (a.u.), normalized against sgCtrl. Values are means ± SEM of n = 8 (MA) and n = 7 (HM) independent replicates. Significance was determined using Wilcoxon signed-rank test. ∗P ≤ .05; ∗∗P < .01. (H) Quantitative PCR shows the mRNA levels of Dck in MA cells. Values are means ± SEM, (n = 4). Significance was determined using a 1-way ANOVA, followed by Dunnett multiple comparisons test. ∗P ≤.05; ∗∗P < .01. (I) Assessment of DCK protein levels in MA and HM cells treated with the proteasome inhibitor MG132 (10 μM, 8 hours). Representative western blot of whole cell lysates shows DCK protein level in MA and HM cells. (J) Quantification of DCK western blot as shown in panel I. Proteins are presented in a.u., normalized to the vinculin loading control. Values are means ± SEM (n = 4). Significance was determined using an unpaired 2-tailed t test. ∗P ≤ .05; ∗∗P < .01.

Multiomic analysis identifies the Dck as a downstream target of Herc1. (A) Representation of the functional domains of HERC1 protein. (B) Volcano plot showing protein abundance in sgHerc1-MA cells vs sgCtrl MA cells. P values were calculated using t tests and were corrected for multiple hypothesis testing with Benjamin-Hochberg method. The dashed line represents P = .05. (C) Pathway enrichment analysis of 106 proteins that were significantly abundant in sgHerc1-MA cells. (D) Volcano plot showing differential expressed genes in sgHerc1-MA cells vs sgCtrl MA cells. The dashed line represents log2 fold change (FC) of ±1.5. (E) Integration of transcriptomic and proteomic data with the CRISPR/Cas9 screen to identify potential targets of Herc1. Potential candidates that passed the following criteria of (1) greater protein abundance (>1.5-fold) (shown in panel B) and (2) no corresponding change in mRNA transcript abundance (<1.5-fold) (shown in panel D). (F) Assessment of DCK protein levels in Herc1–gene edited cells. Representative western blot of whole cell lysates shows the DCK protein level in MA and HM-sgCtrl and sgHerc1 knockout cells using 3 different sgRNAs. (G) Quantification of DCK western blot as shown in panel F. Proteins are represented in arbitrary units (a.u.), normalized against sgCtrl. Values are means ± SEM of n = 8 (MA) and n = 7 (HM) independent replicates. Significance was determined using Wilcoxon signed-rank test. ∗P ≤ .05; ∗∗P < .01. (H) Quantitative PCR shows the mRNA levels of Dck in MA cells. Values are means ± SEM, (n = 4). Significance was determined using a 1-way ANOVA, followed by Dunnett multiple comparisons test. ∗P ≤.05; ∗∗P < .01. (I) Assessment of DCK protein levels in MA and HM cells treated with the proteasome inhibitor MG132 (10 μM, 8 hours). Representative western blot of whole cell lysates shows DCK protein level in MA and HM cells. (J) Quantification of DCK western blot as shown in panel I. Proteins are presented in a.u., normalized to the vinculin loading control. Values are means ± SEM (n = 4). Significance was determined using an unpaired 2-tailed t test. ∗P ≤ .05; ∗∗P < .01.

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