Journal: eLife

Article Title: Differential translation of mRNA isoforms underlies oncogenic activation of cell cycle kinase Aurora A

doi: 10.7554/elife.87253

Figure Lengend Snippet: Figure 2. Aurora Kinase A (AURKA) shows 3′UTR isoform-dependent protein expression. (A) Top: UTR-dependent protein expression reporters. Venus coding sequence (CDS) is flanked by AURKA 5′UTR and 3′UTR, WT or polyadenylation signal (PAS)-mutated. Bottom: representative snapshots of transfected U2OS cells. Scale bar 50 µm. (B) Mean and SEM of median Venus/mCherry mean fluorescence intensity (MFI) ratios from transfected U2OS cells from three biological replicates. n ≥ 129 cells per condition. Ordinary one-way ANOVA with Tukeys multiple-comparisons test. (C) 3′RACE of endogenous AURKA APA isoforms. (D) RT-qPCR of endogenous AURKA short/long ratio (SLR) in U2OS cells. Long isoform abundance plotted as fold change over total AURKA mRNA. 18S rRNA used as reference target. (E) Same as (B) but in MCF10A (left) and RPE1 (right) cells. n ≥ 55 cells per condition. Unpaired t-test. ns, not significant; *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: For TRIPS reporters, the DHFRsfGFP ORF was PCR amplified from pHR- DHFRY100I- sfGFP- NLS- P2A- NLS- mCherry- P2A_Emi1 5' and 3'UTR plasmid, a gift from Ron Vale (Addgene plasmid #67930), and inserted into pBI- CMV1- mCherry (TRIPS-Δ) or assembled with AURKA 5′UTR and AURKA wt 3′UTR (TRIPS- WT), short 3′UTR (TRIPS- S), or long 3′UTR (TRIPS- L) before insertion.

Techniques: Expressing, Sequencing, Transfection, Fluorescence, Quantitative RT-PCR

Journal: eLife

Article Title: Differential translation of mRNA isoforms underlies oncogenic activation of cell cycle kinase Aurora A

doi: 10.7554/elife.87253

Figure Lengend Snippet: Figure 3. Aurora Kinase A (AURKA) alternative polyadenylation (APA) isoforms are translated with different efficiency. (A, B) RT-qPCR of reporter mRNAs abundance (A) and decay rate (B) from transfected U2OS cells. mCherry mRNA used as reference target. Ordinary one-way ANOVA with Tukey’s multiple-comparisons test; ns, not significant. (C) Decay rate of endogenous AURKA mRNA as in (B). 18S rRNA used as reference target. Abundance of long isoform plotted as fold change over total AURKA mRNA. (D) Design of the nascent chain immunoprecipitation (NC IP) reporters and assay. (E) Mean and SEM of median FlagVenus/mCherry mean fluorescence intensity (MFI) ratios from transfected U2OS cells from two biological replicates. n ≥ 160 cells per condition. Unpaired t-test; **p<0.005. (F), (G) Immunoblots of NC IP fractions using Δ (F) or Flag-Δ (G) reporter. mCherry used as negative control. (H) RT-qPCR of eluted reporter mRNAs. Results representative of three biological replicates.

Article Snippet: For TRIPS reporters, the DHFRsfGFP ORF was PCR amplified from pHR- DHFRY100I- sfGFP- NLS- P2A- NLS- mCherry- P2A_Emi1 5' and 3'UTR plasmid, a gift from Ron Vale (Addgene plasmid #67930), and inserted into pBI- CMV1- mCherry (TRIPS-Δ) or assembled with AURKA 5′UTR and AURKA wt 3′UTR (TRIPS- WT), short 3′UTR (TRIPS- S), or long 3′UTR (TRIPS- L) before insertion.

Techniques: Quantitative RT-PCR, Transfection, Immunoprecipitation, Fluorescence, Western Blot, Negative Control

Journal: Molecular Metabolism

Article Title: Conditional hepatocyte ablation of PDIA1 uncovers indispensable roles in both APOB and MTTP folding to support VLDL secretion

doi: 10.1016/j.molmet.2024.101874

Figure Lengend Snippet: Hepatocyte-specific Pdia1 deletion induces severe hypolipidemia and hepatic steatosis with a blockade of hepatic TG export. A. Schematic shows floxed exons 1 and 2 of the Pdia1 allele ( Pdia1 f/f ). Generation of hepatocyte-specific PDIA1-ablated mice ( Pdia1 -LKO) was produced using Ad-CMV-Cre or AAV8-TBG-Cre. B. Transduction of Pdia1 f/f with AAV8-TBG-Cre eliminated Pida1 mRNA in the liver but did not affect the mRNA levels of other PDI protein family members. C. PDIA1 was absent in the livers of Pdia1 -LKO accompanied with upregulation of PDIA4. D. Plasma levels of total cholesterol and TG were both greatly reduced in Pdia1 -LKO mice (n = 5). E and F. APOB100, APOB48 and APOE were nearly absent in plasma of Pdia1 -LKO mice, with a reduced level of ApoA1 but plasma albumin levels were not changed. Each lane represents a sample from individual mouse. G. Liver images and H/E-stained liver sections demonstrate severe liver fat accumulation in Pdia1 -LKO mice. Scale bar: 200 μm. H. Hepatic TG content was markedly increased in Pdia1 -LKO mice (n = 5). I. Dramatic decrease in hepatic VLDL-TG secretion in Pdia1 -LKO mice (n = 3). ∗∗P < 0.01.

Article Snippet: Expression vector encoding human APOB48 was from Addgene (138334; Watertown, MA).

Techniques: Produced, Transduction, Clinical Proteomics, Staining

Journal: Molecular Metabolism

Article Title: Conditional hepatocyte ablation of PDIA1 uncovers indispensable roles in both APOB and MTTP folding to support VLDL secretion

doi: 10.1016/j.molmet.2024.101874

Figure Lengend Snippet: Secretion of APOB48 is completely blocked in Pdia1 -deleted hepatocytes and is rescued by complementary expression of wild type PDIA1 (PDI) or catalytically inactive PDIA1 (PDImt). A . Pulse-chase analysis revealed that Pdia1 -deletion did not affect APOB48 synthesis (lane 5 vs lane 1) but it completely inhibited APOB48 secretion (lanes 12–14 vs lanes 9–11, respectively). B . Complementary expression of PDI or PDImt alone rescued APOB48 secretion. Hepatocytes isolated from Pdia1 f/f and Pdia1 -LKO mice were infected with the indicated adenoviruses at 20 h after plating. At 18 h post-transduction, the hepatocytes were pulse-labeled with 35 S-Met/Cys in the presence of 0.3 mM oleic acid complexed with BSA (OA-BSA) for 3 h. The 35 S-labeled ApoB's and albumin were immunoprecipitated with rabbit polyclonal antibodies against mouse APOB and albumin, respectively. Immunoblotting (IB) demonstrated that no endogenous MTTP was rescued in the Ad-PDI- or Ad-PDImt-infected Pdia1 -LKO hepatocytes. C. Complementary expression of PDI or PDImt alone did not rescue secretion of 3 H-labeled TG by the Pdia1 -LKO hepatocytes, neither did forced expression of MTTP alone. Hepatocytes isolated from Pdia1 f/f and Pdia1 -LKO mice were infected with the indicated adenoviruses. At 18 h p.i., hepatocytes were incubated with DMEM containing 0.3 mM oleic acid-BSA and 3 H-glycerol for 4 h. The 3 H-labeled TG in cells and media were isolated and the 3 H-radioacivity was measured and expressed as DPM/mg cell protein/h. Each bar represents average +/− SD of triplicate wells. ∗, P < 0.05; ∗∗, P < 0.01.

Article Snippet: Expression vector encoding human APOB48 was from Addgene (138334; Watertown, MA).

Techniques: Expressing, Pulse Chase, Isolation, Infection, Transduction, Labeling, Immunoprecipitation, Western Blot, Incubation

Journal: Molecular Metabolism

Article Title: Conditional hepatocyte ablation of PDIA1 uncovers indispensable roles in both APOB and MTTP folding to support VLDL secretion

doi: 10.1016/j.molmet.2024.101874

Figure Lengend Snippet: Wild type PDIA1 (PDI) and catalytically inactive PDIA1 (PDImt) directly interact with the peptide region between APOB27 and APOB48, respectively . A. PDI and PDImt interact with APOB48 in transfected COS-7 cells. COS-7 cells were transfected with human APOB48 (hAPOB48), C-terminal-FLAG-tagged PDI (PDI-f) or PDImt (PDImt-f) expression vectors as indicated. The transfected cells were harvested 30 h post-transfection and subjected to FLAG-immnunoprecipitation (IP) analysis using M2 anti-FLAG magnetic beads. Co-IP of hAPOB48 with PDI-f (lanes 10 & 11) or PDImt-f (lanes 12 & 13) indicate their direct interactions. B. Neither APOB17 nor APOB27 interact with PDI or PDImt. COS-7 cells were transfected PDI-f or or PDImt-f expression vectors in the presence of Ad-hAPOB15 (hAPOB15) or Ad-hAPOB27. FLAG-IP assays were performed on the DNA transfected COS-7 cells at 30 h post-transfection. No hAPOB15 nor hAPOB27 were pulled down with PDI-f or PDImt-f. C. PDIA1 is not required for secretion of APOB17 and APOB27. Hepatocytes isolated from Pdia1 f/f and Pdia1 -LKO mice were transduced with Ad-GFP (GFP), Ad-hAPOB15 (B15), or Ad-hAPOB27 (B27). At 48 h post-transduction, hepatocytes, and conditioned media (20 h incubation time) were harvested. Cellular and secreted human ApoB15 and apoB27 were immunoprecipitated with rabbit anti-human ApoB followed by immunoblot analysis using goat anti-human ApoB.

Article Snippet: Expression vector encoding human APOB48 was from Addgene (138334; Watertown, MA).

Techniques: Transfection, Expressing, Magnetic Beads, Co-Immunoprecipitation Assay, Isolation, Transduction, Incubation, Immunoprecipitation, Western Blot