Journal: bioRxiv

Article Title: The architecture of protein synthesis in the developing neocortex at near-atomic resolution reveals Ebp1-mediated neuronal proteostasis at the 60S tunnel exit

doi: 10.1101/2020.02.08.939488

Figure Lengend Snippet: (A) Schematic of the experimental system to measure the architecture of active protein synthesis (polysomal ribosomes) from the ex vivo neocortex across embryonic (E12.5, E14, E15.5, E17) and early postnatal (P0) neurogenesis. See text for details. (B) MS analysis of neocortical polysomal complexes across development. Scatter plots compare early neurogenesis E12.5 vs. each subsequent stage, demonstrating the enrichment of Ebp1 (red arrow) among ribosomal proteins (RPs) of the large (Rpl, blue) and small (Rps, yellow) subunits, in contrast to other translation-associated proteins (black). (C) Stoichiometry cluster heat maps quantifying the differential enrichment of each RP (Rpl, blue; Rps, yellow), translation-associated protein (black), and Ebp1 (red arrow) per developmental stage. Expression of adjacent proteins on the x-axis is shown as higher (orange), lower (purple), or similar (black) relative to each protein on the y-axis. Legend and histogram at top left for each stage. See also - .

Article Snippet: Pooled 80S and polysomal ribosomes were purified ex vivo by preparative 10-50% sucrose density gradient ultracentrifugation from dissected frozen P0 mouse neocortex tissue as described above, but with the following adaptations optimizing for cryo-electron microscopy (cryo-EM).

Techniques: Ex Vivo, Expressing

Journal: bioRxiv

Article Title: The architecture of protein synthesis in the developing neocortex at near-atomic resolution reveals Ebp1-mediated neuronal proteostasis at the 60S tunnel exit

doi: 10.1101/2020.02.08.939488

Figure Lengend Snippet: Mouse neocortical lysates were subjected to preparative sucrose density gradient ultracentrifugation fractionation in biological triplicate to purify 80S and polysomal ribosome complexes at E12.5, E14, E15.5, E17, and P0. Input lysate, pooled 80S fractions, and pooled polysome fractions were analyzed by LC-MS/MS.

Article Snippet: Pooled 80S and polysomal ribosomes were purified ex vivo by preparative 10-50% sucrose density gradient ultracentrifugation from dissected frozen P0 mouse neocortex tissue as described above, but with the following adaptations optimizing for cryo-electron microscopy (cryo-EM).

Techniques: Fractionation, Liquid Chromatography with Mass Spectroscopy

Journal:

Article Title: Surprising features of plastid ndhD transcripts: addition of non-encoded nucleotides and polysome association of mRNAs with an unedited start codon

doi: 10.1093/nar/gkh217

Figure Lengend Snippet: Mapped ndhD mRNA termini and editing status of ndhD mRNAs. RNAs from fraction 10 of the EDTA-containing control gradient (C10; see Fig. ​Fig.2)2) and fractions 1 and 6 of the EDTA-free gradient were circularized with RNA ligase and analyzed as displayed in Figure ​Figure1.1. Arrows indicate mapped termini, with the number of clones given for each terminus. Asterisks indicate ambiguity with respect to the terminal nucleotide (due to the presence of identical nucleotides at the putative 5′ and 3′ ends). The stem–loop indicates a potential secondary structure within the 3′ UTR which is typical of plastid mRNAs. The number of unedited versus edited clones is given in parentheses above the initiation codon.

Article Snippet: A 2 µl aliquot of RNA from fractions P1 and P6 (EDTA-free polysome gradients) and 0.5 µl of RNA from fraction C10 (EDTA-containing polysome gradient) were ligated at 37°C for 1 h with 20 U of T4 RNA ligase (New England Biolabs) in a final volume of 100 µl. cDNA synthesis was performed as follows.

Techniques: Clone Assay

Journal: iScience

Article Title: Manganese suppresses tumor growth through hyper-activating IRE1α

doi: 10.1016/j.isci.2025.113121

Figure Lengend Snippet: Mn 2+ promotes the terminal UPR downstream of IRE1α but spares the adaptive UPR unaffected (A) The oligomerization of IRE1α detected by immunoblotting assay. MDA-MB-231 cells were treated with Tg (0.2 μM) and MnCl 2 (100 μM) alone or in combination for 24 h. Cell lysates were separated by sucrose density gradient centrifugation (20%–40%). (B) Representative immunofluorescence image (Left) and the quantitative result (Right) of hIRE1α-sfGFP puncta in MDA -MB-231 IRE1α KO cells that stably express hIRE1α-sfGFP cells in a Dox-inducible way. Magnified views of the red-boxed regions are shown on the right of each original image. Cells were pretreated with Dox (5 μg/mL) for 20 h, followed by MnCl 2 (100 μM) treatment for 4 h. Hoechst was used to identify the nucleus. Scale bar: 10 μm. (C) Phosphorylation of IRE1α and activation of its downstream signaling pathway in MDA-MB-231 WT and IRE1α KO cells measured by immunoblotting assay. Cells were treated with Tg (0.2 μM), MnCl 2 (100 μM), and KIRA8 (1 μM) alone or in combination for 24 h. Diamond denotes non-specific band. (D) Quantitative gray value analysis of p-IRE1α/t-IRE1α (by Phos-tag), p-JNK/JNK, XBP1s/GAPDH, and cleaved caspase-3/caspase-3 in (C) ( n = 3). Relative ratio was shown, with ratio in lane 3 (Tg treatment for IRE1α WT) set as 1. (E) Agarose gel of XBP1 cDNA amplicons from MDA-MB-231 WT and IRE1α KO cells. Cells were treated as in ( C ). U, unspliced XBP1 ; S, spliced XBP1 . (F) Quantitative gray value analysis of S/(S + U) in ( E ) ( n = 4). (G) Immunoblotting assay of IRE1α phosphorylation and its downstream signaling pathways in MDA-MB-231 IRE1α KO cells that stably express exogenous IRE1α in a Dox-inducible way. Cells were pretreated with Dox (200 ng/mL) for 24 h, and then incubated with MnCl 2 (200 μM) for 48 h. Relative ratio was shown, with ratio in lane 3 (OE IRE1α WT) set as 1. (H) Quantitative gray value analysis of p-IRE1α/t-IRE1α (by Phos-tag), p-JNK/JNK, and XBP1s/GAPDH in (G) ( n = 4). (I) The mRNA expression level of BLOS1 in MDA-MB-231 cells detected by RT-qPCR. Cells were pretreated with ActD (2 μg/mL) for 1 h, then treated with Tg (0.2 μM), MnCl 2 (100 μM), and KIRA8 (1 μM) alone or in combination for 24 h ( n = 3). (J) The mRNA expression level of BLOS1 in MDA-MB-231 cells detected by RT-qPCR. Cells were pretreated with ActD (2 μg/mL) for 1 h, then treated with Tm (1 μg/ml), MnCl 2 (100 μM), and KIRA8 (1 μM) alone or in combination for 48 h ( n = 3). (K) The mRNA expression level of BLOS1 in BT-474 and MDA-MB-453 cells detected by RT-qPCR. BT-474 and MDA-MB-453 cells. BT-474 and MDA-MB-453 cells were pretreated with ActD (2 μg/mL) for 1 h, then treated with Tg (0.2 μM), MnCl 2 (100 μM), and KIRA8 (1 μM) alone or in combination for 12 h and 24 h, respectively ( n = 3). (L) The interaction of IRE1α and TRAF2 in MDA-MB-231 cells detected by co-immunoprecipitation. Cells were treated with Tg (0.2 μM) and MnCl 2 (100 μM) alone or in combination for 24 h. (M) Cell death percentage in MDA-MB-231 cells measured by Annexin V staining. Cells were pretreated with or without CC401 (5 μM) for 1 h, then incubated with Tg (0.2 μM) and MnCl 2 (100 μM) alone or in combination for 24 h ( n = 3). (N) Cell death percentage in MDA-MB-231 cells measured by Annexin V staining. Cells were pretreated with z-VAD-FMK (z-VAD, 20 μM), ferrostatin-1 (Fer, 1 μM), necrostatin-1 (Nec, 80 μM), bafilomycin A1 (Baf, 10 nM), disulfiram (Dis, 10 μM) for 24 h or cycloheximide (CHX, 2 μM) for 6 h, and further treated with Tg (0.2 μM) and MnCl 2 (100 μM) alone or in combination for 24 h ( n = 3). Data are presented as mean ± SEM. Statistical analysis was performed by unpaired Student’s t test. ns, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also and .

Article Snippet: Input samples and polysome fraction samples were performed reverse transcription (First-strand cDNA synthesis kit, TIANGEN, Cat# KR116-03) and qPCR according to manufacturer’s protocols.

Techniques: Western Blot, Gradient Centrifugation, Immunofluorescence, Stable Transfection, Phospho-proteomics, Activation Assay, Agarose Gel Electrophoresis, Protein-Protein interactions, Incubation, Expressing, Quantitative RT-PCR, Immunoprecipitation, Staining