EpCAM-positive cells from DDB1 mutant liver proliferate <i>in vitro</i> and repopulate the liver <i>in vivo</i>.

<p>(A) Analysis by flow cytometry of EpCAM⁺ F4/80⁻ cells in non-parenchymal fractions prepared from 4-week old <i>DDB1^F/F</i> and <i>DDB1^F/F;Alb-Cre^+/+</i> mice. The ratio of EpCAM⁺ F4/80⁻ cells from a representative experiment is shown in each panel. (B) <i>In vitro</i> culture of EpCAM⁺ cells sorted by FACS from DDB1 mutant liver and seeded on Matrigel. Cells form colonies after 1, 3, and 9 days of culture (upper panels) and exhibit EpCAM positivity (lower panels). (C) Co-IF staining for A6 and AFP on colonies from cultured EpCAM⁺ cells. (D) Experimental scheme for <i>in vivo</i> differentiation of EpCAM⁺ OCs. Adult <i>DDB1^F/F;Mx1-Cre^+/−;Rosa26-lacZ</i> mice were generated and injected with poly(I:C). EpCAM⁺ cells were isolated by FACS 6 weeks later, and injected intrasplenically to nude mice that had been fed with DDC diet. The recipient nude mice received poly(I:C) injection 2 weeks later and liver collected for analysis on the following day. (E) Isolation of EpCAM⁺ F4/80⁻ cells from donor or control mouse liver. The ratio of EpCAM⁺ F4/80⁻ cells is shown in each panel. (F) IHC staining for ß-gal on liver sections from recipient nude mice. Arrows indicate positively stained hepatocytes.</p

DDB1 mutant mice exhibit minor liver damage compared with DDC-treated mice.

<p>(<b>A</b>): Co-IF staining for A6 and EpCAM on liver sections from DDC-treated mice. (<b>B</b>): Gross appearance of liver from mice fed with DDC diet for 4 weeks. (<b>C</b>): H&E staining of the liver in (<b>B</b>). (<b>D</b>): IHC staining for CD45 (upper panels) and F4/80 (lower panels) on liver sections from <i>DDB1^F/F</i>, <i>DDB1^F/F;Alb-Cre^+/+</i> and DDC-treated mice. (<b>E</b>): Percentage of EpCAM⁺ cells from <i>DDB1^F/F;Alb-Cre^+/+</i> and DDC-diet liver, determined by FACS analysis. Data are representative of 4 independent experiments with 3 mice per group in each experiment. Values are expressed as the means±SEM; n = 3. (<b>F</b>): Serum alanine aminotransferase (ALT) levels. Data are representative of 4 independent experiments with 3 mice per group in each experiment. Values are expressed as means±SEM; n = 3 **P<0.01.</p

Deletion of DDB1 in hepatocytes results in hepatic oval cells activation.

<p>(A) H&E staining of liver sections from 4-week old <i>DDB1^F/F</i> and <i>DDB1^F/F;Alb-Cre^+/+</i> mice. PV, portal vein. (B) IHC staining for DDB1 on liver sections from 4-week old <i>DDB1^F/F</i> and <i>DDB1^F/F;Alb-Cre^+/+</i> mice. (C) Co-IF staining for DDB1 and A6 on 4-week old <i>DDB1^F/F</i> and <i>DDB1^F/F;Alb-Cre^+/+</i> liver sections.</p

Characterization of OCs from DDB1 mutant mice and DDC-treated mice.

<p>(<b>A–C</b>): Quantitative real-time PCR analysis for selected genes expressed in hepatocytes isolated from wild type mice, and EpCAM⁺ cells isolated from DDC-treated and <i>DDB1^F/F; Alb-Cre^+/+</i> mice. All data are normalized to <i>18s</i> rRNA level. Data are representative of 4 independent experiments with 3–4 mice per group. *P<0.05 (<b>A</b>): Expression levels of hematopoietic markers <i>Sca1</i>, <i>Thy1</i> and <i>Cd44</i>, and OC markers <i>CD133</i>, <i>Connexin43</i> and <i>Ncam1</i>. (<b>B</b>): Expression levels of cholangiocyte marker <i>Ck19</i> and <i>Spp1</i>, and hepatocyte marker <i>Alb</i>. (<b>C</b>): Expression levels of candidate OC markers, <i>Reelin</i>, <i>EdnrB</i> and <i>Cd206.</i> (<b>D</b>): IHC staining for reelin on liver sections from <i>DDB1^F/F</i>, <i>DDB1^F/F;Alb-Cre^+/+</i> and DDC-treated mice. Arrows in DDC-treated liver indicate non-specific brown deposits.</p

Deletion of p21 partially restores proliferation of DDB1-deficient hepatocytes and alleviates OC activation.

<p>(A) Western blot for some substrates of DDB1-Cul4A ubiquitin ligase using lysates of hepatocytes isolated from <i>DDB1^F/F</i> and <i>DDB1^F/F;Alb-Cre^+/+</i> mice. (B) Western blot for DDB1 and p21 using lysates of cytoplasmic fraction (C) and nuclear fraction (N) prepared from MEFs. (C) IHC staining for DDB1 and Ki67 on liver sections from <i>DDB1^F/F;Alb-Cre^+/−, DDB1^F/F;p21^−/−</i> and <i>DDB1^F/F;Alb-Cre^+/−;p21^−/−</i> mice. (D) Co-IF staining for A6 and DDB1 on <i>DDB1^F/F;p21^−/−</i> and <i>DDB1^F/F;Alb-Cre^+/−;p21^−/−</i> liver sections. Arrows in <i>DDB1^F/F;Alb-Cre^+/−;p21^−/−</i> mice indicate A6 positive oval cells.</p

Expression of oval cell markers in DDB1 mutant mouse liver.

<p>(A) Co-IF staining for Cytokeratin-19 (CK19) and Albumin (Alb) (upper panels), E-cadherin and DDB1 (middle panels), α-fetoprotein (AFP) and CD133 (lower panels). (B–D) Co-IF staining for EpCAM and A6 on liver sections from 4-week old <i>DDB1^F/F;Alb-Cre^+/+</i> (B), 6-week old <i>DDB1^F/F;Alb-Cre^+/−</i> (C), and adult <i>DDB1^F/F;Mx1-Cre^+/−</i> mice at 6 weeks after receiving poly(I:C) injection (D).</p

PPI-Miner: A Structure and Sequence Motif Co-Driven Protein–Protein Interaction Mining and Modeling Computational Method

Protein–protein interactions (PPIs) play important roles in biological processes of life, and predicting PPIs becomes a critical scientific issue of concern. Most PPIs occur through small domains or motifs (fragments), which are challenging and laborious to map by standard biochemical approaches because they generally require the cloning of several truncation mutants. Here, we present a computational method, named as PPI-Miner, to fish potential protein interacting partners utilizing protein motifs as queries. In brief, this work first developed a motif-matching algorithm designed to identify the proteins that contain sequential or structural similar motifs with the given query motif. Being aligned to the query motif, the binding mode of the discovered motif and its receptor protein will be initially determined to be used to build PPI complexes accordingly. Eventually, a PPI complex structure could be built and optimized with a designed automatic protocol. Besides discovering PPIs, PPI-Miner can also be applied to other areas, i.e., the rational design of molecular glues and protein vaccines. In this work, PPI-Miner was employed to mine the potential cereblon (CRBN) substrates from human proteome. As a result, 1,739 candidates were predicted, and 16 of them have been experimentally validated in previous studies. The source code of PPI-Miner can be obtained from the GitHub repository (https://github.com/Wang-Lin-boop/PPI-Miner), the webserver is freely available for users (https://bailab.siais.shanghaitech.edu.cn/services/ppi-miner), and the database of predicted CRBN substrates is accessible at https://bailab.siais.shanghaitech.edu.cn/services/crbn-subslib

DDB1 deletion in mouse brain causes learning and memory defects.

<p>All behavior analysis data are shown as mean ± SEM. (A) Novel object recognition to test texture-associated short-term memory in mice. Contacts of mice towards the familiar (A) or novel (C) object were recorded after training. <i>Ddb1</i>^<i>F/F</i><i>;Camk2a-Cre</i> mice could not differentiate object C from A while <i>Ddb1</i>^<i>F/F</i> mice could (<i>Ddb1</i>^<i>F/F</i> <i>n</i> = 11, <i>Ddb1</i>^<i>F/F</i><i>;Camk2a-Cre n</i> = 10, <i>P</i> = 0.026). (B) Barnes maze. The time for <i>Ddb1</i>^<i>F/F</i><i>; Camk2a-Cre</i> mice (<i>n</i> = 10) or <i>Ddb1</i>^<i>F/F</i> <i>mice</i> (<i>n</i> = 11) to enter the escape tunnel were recorded during the training days (<i>P</i> < 0.05 for each block). Two-way repeated-measures <i>ANOVA</i> (genotype versus day) revealed a genotype effect (<i>F</i>_{<i>(1</i>,<i>19)</i>} = 6.518, <i>P</i> = 0.0194) and day effect (<i>F</i>_{<i>(2</i>,<i>38)</i>} = 6.318, <i>P</i> = 0.0043) for both groups. (C) Barnes maze. The errors (numbers of entrance into wrong tunnel) made by mice were recorded during the training days (<i>P</i> = 0.014 for block day 7–9). Two-way repeated-measures <i>ANOVA</i> revealed a genotype effect (<i>F</i>_{<i>(1</i>,<i>17)</i>} = 13.68, <i>P</i> = 0.0018) for both groups. (D) Barnes maze. Incidence distribution of strategy for mice to search the escape tunnel were recorded during the training days. Two-way repeated-measures <i>ANOVA</i> revealed a significant differences in strategy use (<i>F</i>_{<i>(3</i>,<i>16)</i>} = 29.00, <i>P</i> < 0.0001) and a genotype × strategy effect (<i>F</i>_{<i>(3</i>,<i>16)</i>} = 13.16, <i>P</i> = 0.0001) for both groups. (E) Barnes maze. The time for mice spent in the target quadrant (where the removed escape tunnel was located) were recorded on the probe day (<i>P</i> = 0.0027).</p

Currents of BK channel are decreased in CRL4^CRBN deficient glioma cells.

<p>(A) The whole cell currents of U87mG cells were detected by voltage clamp. BK currents were presented as the difference between the currents under basal condition and after treatment of paxilline (10 μM, BK channel blocker) for at least 10 mins. The I-V curve of BK currents in WT cells, <i>CRBN</i> KO cells and <i>CRBN</i> KO cells treated with MLN4924 (0.1 μM) for 24 hrs were shown on the bottom right (<i>n</i> = 5 for each group, <i>P</i> < 0.01). (B) The whole cell currents in <i>CRBN</i> KO cell lines stably expressing vehicle, WT CRBN and R419X mutation-bearing CRBN were detected. The I-V curve of BK currents (paxalline-subtracted currents) in these cell lines were shown on the right panel (<i>n</i> = 5 for each group, <i>P</i> < 0.01). (C) BK single-channel currents were recorded with a pipette voltage from 20 mV to 80mV using the excised inside-out configuration and were then identified by its pharmacological sensitivity. Mean single channel current and mean NPo versus voltage relationships were derived from data sets exemplified in the current traces (<i>n</i> = 5 for each group).</p

sgRNA sequences for gene editing.

<p>sgRNA sequences for gene editing.</p