Journal: bioRxiv

Article Title: Exploration of the proxiOME of large subunit ribosomal proteins reveals Acl1 and Bcl1 as cooperating dedicated chaperones of Rpl1

doi: 10.1101/2025.09.18.677003

Figure Lengend Snippet: (A) Proximity labelling assay with C-terminally miniTurbo-tagged RPL10A (HsRPL10A-miniTurbo), the orthologous human protein of yeast Rpl1, in HeLa cells. The assay was carried out in three experimental replicates, with miniTurbo-EGFP and EGFP-miniTurbo-NLS serving as cytoplasmic and nuclear background controls. The RPL10A bait r-protein and selected enriched proteins are written in bold, the red dot highlights the highly enriched WDR89. (B) AlphaFold3 model of the Bcl1-Rpl1 complex in two different orientations. The seven-bladed β-propeller domain of Bcl1 is coloured in green and the C-terminal extension in light green; the position of residue Asn322 (N322) is indicated to better visualize from where the C-terminal extension emanates. (C) AlphaFold3 model of the WDR89-RPL10A complex (left) and its structural superposition with the AlphaFold3 model of the Bcl1-Rpl1 complex (right). (D) Predicted electrostatic surface potential of Bcl1 (left) and close-up view of two of the three Rpl1 sites, indicating residues predicted to form H-bonds with Bcl1, that are in contact with the negatively charged top surface of the β-propeller (right). (E-G) Y2H interaction assays between the full-length Rpl1 and Bcl1 proteins (E) , between full-length Rpl1 and the C-terminally truncated Bcl1.N366 and Bcl1.N325 variants or the C-terminal extension of Bcl1 (323C) (F) , and between the indicated Rpl1 mutant variants and either Bcl1 or Acl1 (G) . Single-letter abbreviations for the amino acid residues are as follows: A, Ala; E, Glu; K, Lys; R, Arg. (H) In vitro binding assay between Bcl1.N366 and Rpl1. Bcl1.N366-(His)6 or Bcl1.N366 and Rpl1b were co-expressed in E. coli and purified by Ni-NTA affinity purification. Proteins were revealed by SDS-PAGE and Coomassie staining (top) or by Western blotting using anti-Rpl1 and anti- His antibodies (bottom). Bands corresponding to Bcl1.N366-(His)6 and Bcl1.N366 or to Rpl1b are indicated by blue or black arrowheads. M: molecular weight standard, T: total extract, P: pellet fraction, S: soluble extract, E: imidazole eluate.

Article Snippet: The DNA sequence coding for the Homo sapiens RPL10A protein was PCR-amplified from plasmid pADH111-HsRPL10A (pDK10427), generated by cloning the PCR-amplified RPL10A coding sequence (template pNTI194 (Addgene plasmid #84266)) into the Nde I/ Bam HI-restricted plasmid pADH111-LTV1 (pDK3331), and cloned by Gibson assembly (NEB, M5510A) between the Nhe I and Pst I restriction sites of the lentiviral donor vector pSKP-32, a pCW57.1-derived plasmid bearing the MND-Blasticidin resistance cassette instead of the hPGK-Puromycin resistance cassette , to generate plasmid pDS79 containing the RPL10A gene under the transcriptional control of a doxycycline-inducible promoter and fused at its 3’ end to sequences encoding the V5 tag, the miniTurbo (MT) biotin ligase, and the HA tag.

Techniques: Residue, Mutagenesis, In Vitro, Binding Assay, Purification, Affinity Purification, SDS Page, Staining, Western Blot, Molecular Weight

Journal: Nature Communications

Article Title: A stem cell roadmap of ribosome heterogeneity reveals a function for RPL10A in mesoderm production

doi: 10.1038/s41467-022-33263-3

Figure Lengend Snippet: a Schematic of hESC differentiation down the endoderm lineage to the mid/hindgut and down the mesoderm lineage to the sclerotome. b Schematic of polysome isolation by sucrose-gradient fractionation and labeling with tandem mass tags (TMT) to quantify each RP in a differentiated cell sample relative to the hESC starting population. c Heatmap of relative polysome abundance for RPs that change significantly by at least 10% in at least two differentiated cell types relative to hESCs. Several show progressive changes in abundance during mesoderm differentiation (red dashed boxes). One RP (RPS5/uS7) that does not change significantly is also included as an illustrative example. Heatmap values are median ratios of polysome abundance in each differentiated cell relative to hESCs in log2 scale, n = 6 for each cell type except for anterior primitive streak and mid/hindgut ( n = 7 each), and P values for each RP were calculated by ANOVA. d Western blot of hESC ( n = 2) and sclerotome ( n = 4) polysome samples, each loaded as a serial dilution (1, 0.5, 0.25 µg). RPL10A/uL1 and RPS25/eS25 expression were normalized to the non-heterogeneous RPS5/uS7 and the middle dilution (0.5 µg) was used for quantification (shown as mean +/− SEM). Student’s t test P value = 0.03 for RPL10A/uL1, 0.008 for RPS25/eS25. e Location of the 31 heterogeneous RPs in the polysome mass spectrometry on the human 80 S ribosome (PDB: 4v6x). Small and large subunit heterogeneous RPs are blue and red, respectively. Non-heterogeneous RPs are dark gray and rRNA light gray. RPL10A/uL1 (red arrow) is located near the mRNA exit tunnel. f Fraction of the surface area that is solvent-exposed for each RP on the human ribosome. The 31 RPs that change significantly in polysomal abundance are significantly more solvent-accessible (Mann–Whitney test P = 0.0055), indicating they are enriched at the surface of the ribosome. Box is the interquartile range (IQR), center line is the median, whiskers represent 1.5*IQR from the box boundaries, and each point represents a single RP. * P value < 0.05; ** P value < 0.01. Source data are provided as a Source Data file.

Article Snippet: This phenotype was also present at double the frequency in Vangl2 Lp/+ ; Rpl10a LOF/+ double heterozygotes compared to Vangl2 Lp/+ embryos, though it did not achieve statistical significance due to its overall rarity (Supplementary Fig. ).

Techniques: Isolation, Fractionation, Labeling, Western Blot, Serial Dilution, Expressing, Mass Spectrometry, MANN-WHITNEY

Journal: Nature Communications

Article Title: A stem cell roadmap of ribosome heterogeneity reveals a function for RPL10A in mesoderm production

doi: 10.1038/s41467-022-33263-3

Figure Lengend Snippet: a Schematic of possible mechanisms regulating changes in RP abundance in the polysomes across cell types. b Scatterplots of median polysomal and cytoplasmic protein relative abundance in each differentiated cell relative to hESCs for RPS10/eS10, RPL10A/uL1, and RPS16/uS9 with Spearman’s correlation coefficients (ρ) in upper right corners. Error bars are standard error and points are color-coded by cell type. N = 6 for all cell types and sample preparations except for the following: n = 7 for anterior primitive streak and mid/hindgut polysome samples and n = 8 for mid/hindgut cytoplasmic samples. Dashed arrows indicate the locations of these RPs in the histogram in ( c ). c Histogram of Spearman’s correlation coefficients for each RP comparing polysomal and cytoplasmic protein abundance (same n as in ( b )). d Histogram of Spearman’s correlation coefficients for each RP comparing polysomal (same n as in ( b )) and whole-cell protein abundance ( n = 5 for early somite, paraxial mesoderm, and mid/hindgut whole-cell samples; n = 6 for anterior primitive streak, sclerotome, anterior-most primitive streak, and definitive endoderm whole-cell samples). e Histogram of Spearman’s correlation coefficients for each RP comparing polysomal (same n as in ( b )) and mRNA abundance ( n = 3 for mesoderm lineage, n = 1 for endoderm lineage). MHG mid/hindgut, DE definitive endoderm, AmPS anterior-most primitive streak, APS anterior primitive streak, PXM paraxial mesoderm, ESom early somite, Scler sclerotome.

Article Snippet: This phenotype was also present at double the frequency in Vangl2 Lp/+ ; Rpl10a LOF/+ double heterozygotes compared to Vangl2 Lp/+ embryos, though it did not achieve statistical significance due to its overall rarity (Supplementary Fig. ).

Techniques:

Journal: Nature Communications

Article Title: A stem cell roadmap of ribosome heterogeneity reveals a function for RPL10A in mesoderm production

doi: 10.1038/s41467-022-33263-3

Figure Lengend Snippet: a Median relative abundance of RPL10A/uL1 in the polysomes of the mesoderm lineage relative to hESCs. N = 6 for all cell types, except n = 7 for primitive streak and error bars are standard error. Student’s t test P values: anterior primitive streak to early somites 0.006, anterior primitive streak to sclerotome 0.03. b Schematic of the genetic complementation test to identify loss-of-function mutations. c Observed frequencies of Rpl10a null/null homozygotes, Rpl10a deletion/deletion homozygotes, Rpl10a extended/null double heterozygotes, and Rpl10a extended/deletion double heterozygotes. d Lateral views of E8.5, E9.5, E10.5, and E12.5 Rpl10a LOF/LOF and control embryos. The tail bud is traced in white and indicates the posterior trunk truncation; the hindlimb is also traced in red on the E12.5 images. e Quantification of posterior trunk length at E8.5, E9.5, E10.5, and E12.5. Graph shows average length relative to wild-type with SEM error bars. No significant differences were observed between wild-type and Rpl10a LOF/+ embryos; Rpl10a LOF/LOF embryos were significantly different from both wild-type and Rpl10a LOF/+ embryos at all stages except E8.5. For E8.5 wild-type n = 5, Rpl10a LOF/+ n = 7, Rpl10a LOF/LOF n = 8; for E9.5 wild-type n = 13, Rpl10a LOF/+ n = 11, Rpl10a LOF/LOF n = 25; for E10.5 wild-type n = 10, Rpl10a LOF/+ n = 8, Rpl10a LOF/LOF n = 15; for E12.5 wild-type n = 8, Rpl10a LOF/+ n = 13, Rpl10a LOF/LOF n = 19. Wild-type vs. Rpl10a LOF/LOF Student’s t test P values: 0.65 (E8.5), 2.55 × 10 −7 (E9.5), 2.45 × 10 −5 (E10.5), 1.13 × 10 −5 (E12.5); Rpl10a LOF/+ vs. Rpl10a LOF/LOF P values: 0.10 (E8.5), 5.31 × 10 −6 (E9.5), 8.89 × 10 −7 (E10.5), 1.20 × 10 −10 (E12.5). f Schematic of gastrulation at the tail bud to produce the paraxial mesoderm lineage, with the embryonic tissues represented in the in vitro hESC differentiation indicated. APS anterior primitive streak, PXM paraxial mesoderm, ESom early somite, Scler sclerotome, * P value < 0.05; ** P value < 0.01, *** P value < 0.001. Source data are provided as a Source Data file.

Article Snippet: This phenotype was also present at double the frequency in Vangl2 Lp/+ ; Rpl10a LOF/+ double heterozygotes compared to Vangl2 Lp/+ embryos, though it did not achieve statistical significance due to its overall rarity (Supplementary Fig. ).

Techniques: In Vitro

Journal: Nature Communications

Article Title: A stem cell roadmap of ribosome heterogeneity reveals a function for RPL10A in mesoderm production

doi: 10.1038/s41467-022-33263-3

Figure Lengend Snippet: a Western of whole-cell lysates from Rpl10a LOF/LOF and Rpl10a LOF/+ embryos, unedited hESCs, and hESC clones either homozygous or heterozygous for the Rpl10a LOF insertion. b Cell viability of wild-type, heterozygous, and homozygous Rpl10a LOF/LOF hESCs as measured by Cell Titer Glo ( n = 44). Data are shown as mean +/− SEM and significance was calculated using Student’s t tests. c Cell viability of heterozygous and homozygous Rpl10a LOF/LOF hESCs as measured by Cell Titer Glo during differentiation down the paraxial mesoderm lineage ( n = 12 for each cell line for anterior primitive streak, n = 10 for each cell line for paraxial mesoderm, n = 5 for each cell line for sclerotome, n = 5 for Rpl10a LOF/+ cells and n = 4 for Rpl10a LOF/LOF cells for early somites). Data are shown as mean +/− SEM. Student’s t test P values: 0.77 (anterior primitive streak), 0.04 (paraxial mesoderm), 0.03 (early somite), 0.002 (sclerotome). APS anterior primitive streak, PXM paraxial mesoderm, ESom early somite, Scler sclerotome. * P value <0.05; ** P value <0.01. Source data are provided as a Source Data file.

Article Snippet: This phenotype was also present at double the frequency in Vangl2 Lp/+ ; Rpl10a LOF/+ double heterozygotes compared to Vangl2 Lp/+ embryos, though it did not achieve statistical significance due to its overall rarity (Supplementary Fig. ).

Techniques: Western Blot, Clone Assay

Journal: Nature Communications

Article Title: A stem cell roadmap of ribosome heterogeneity reveals a function for RPL10A in mesoderm production

doi: 10.1038/s41467-022-33263-3

Figure Lengend Snippet: a Whole-mount in situ hybridizations in control and Rpl10a LOF/LOF embryos at E9.5 and E10.5. The region of expression at the tail bud is demarcated with a solid white line. A dashed white line outlines the tail bud in the Shh in situ. b Quantification of the area of staining relative to total tail bud area. For each probe, stage, and genotype n = 3 with the exception of Fgf8 ( n = 4 for each genotype) and T ( n = 4 for E9.5 Rpl10a LOF/LOF ). Data are shown as mean +/− SEM, Student’s t test P values: 0.04 ( T E9.5), 0.03 ( T E10.5), 0.09 ( Msgn1 E9.5), 0.045 ( Msgn1 E10.5), 0.005 ( Tbx6 E9.5), 0.049 ( Tbx6 E10.5), 0.40 ( Fgf8 E9.5). c Quantification of the distance between the end of the notochord, as demarcated by Shh in situ, and the tip of the tail bud ( n = 3). Distances were normalized to the distance between limb buds and otic placode in each embryo to account for any differences in embryo size. Data are shown as mean +/− SEM, and significance was calculated using Student’s t tests ( P value = 0.04). d Whole-mount in situ hybridizations for Uncx4.1 in control and Rpl10a LOF/LOF embryos at E9.5, lateral view (top), and view of the posterior end of the embryo (bottom). The dashed white line demarcates the region of expression; black arrows indicate abnormalities in somite boundaries and spacing. * P value < 0.05; ** P value < 0.01. Source data are provided as a Source Data file.

Article Snippet: This phenotype was also present at double the frequency in Vangl2 Lp/+ ; Rpl10a LOF/+ double heterozygotes compared to Vangl2 Lp/+ embryos, though it did not achieve statistical significance due to its overall rarity (Supplementary Fig. ).

Techniques: In Situ, Expressing, Staining

Journal: Nature Communications

Article Title: A stem cell roadmap of ribosome heterogeneity reveals a function for RPL10A in mesoderm production

doi: 10.1038/s41467-022-33263-3

Figure Lengend Snippet: a (Left) Polysome traces of wild-type, Rpl10a LOF/+ , and Rpl10a LOF/LOF whole E13.5 embryo extracts show similar distributions of polysomes, monosomes, and free subunits. (Right) Western blots of protein precipitated from each sucrose-gradient fraction show the extended RPL10A/uL1 protein is incorporated into polysomes equivalently to wild-type. b (Left) Fluorescence image of E9.5 Meox1 Cre ; Ai9 embryo illustrating the presomitic/somitic mesoderm tdTomato reporter expression. (Right) OP-Puromycin incorporation rates in Meox1 Cre ; Ai9; Rpl10a LOF/LOF E9.5 presomitic/somitic mesoderm (tdTom+) and in the rest of the embryo (tdTom−) ( n = 3). Values are normalized to OP-Puro incorporation rates of TdTom+ and TdTom– cells of Meox1 Cre ; Ai9; Rpl10a LOF/+ E9.5 control littermates. Data shown are mean +/− SEM, and significance was calculated using Student’s t test. c Comparison of RNA-seq and ribosome profiling for whole E8.5 Rpl10a LOF/LOF and wild-type embryos ( n = 3 each). Genes changing significantly (false discovery rate (FDR) < 0.1) only in mRNA abundance are blue; genes changing significantly (FDR < 0.1) only in ribosome occupancy are red; genes changing significantly in both datasets are purple. d Selected Wnt signaling gene sets that were significantly (FDR < 0.1) enriched in CAMERA gene set enrichment analysis of genes with altered translation efficiency in Rpl10a LOF/LOF embryos compared to wild-type. All gene sets shown are translationally downregulated. Node size = gene set size; edge size = gene set overlap. e Genes from the “Wnt signaling pathway, planar cell polarity pathway” set with statistically significant changes in ribosome-protected footprints (RPF) but not in total RNA-Seq (RNA). f E9.5 wild-type ( n = 2) and Rpl10a LOF /LOF ( n = 4) gradient RT-qPCR for Vangl2 showing the fraction of the total mRNA found in each of the five fractions of the 25–50% sucrose gradient demarcated in the schematic. Data shown are mean +/− SEM. Vangl2 is significantly decreased in Rpl10a LOF /LOF medium and heavy polysomes relative to wild-type (Student’s t test P values = 0.04 and 0.008, respectively). * P value < 0.05; ** P value < 0.01. Source data are provided as a Source Data file.

Article Snippet: This phenotype was also present at double the frequency in Vangl2 Lp/+ ; Rpl10a LOF/+ double heterozygotes compared to Vangl2 Lp/+ embryos, though it did not achieve statistical significance due to its overall rarity (Supplementary Fig. ).

Techniques: Western Blot, Fluorescence, Expressing, RNA Sequencing Assay, Quantitative RT-PCR

Journal: Nature Communications

Article Title: A stem cell roadmap of ribosome heterogeneity reveals a function for RPL10A in mesoderm production

doi: 10.1038/s41467-022-33263-3

Figure Lengend Snippet: a Schematic of 3xFLAG-RPL10A/uL1 and RPL22/eL22-3xFLAG ribosome immunoprecipitation from in vitro differentiated sclerotome and measurements of association with target mRNAs by RT-qPCR. b Genes identified as differentially translated in Rpl10a LOF/LOF embryos showed increased association with RPL10A-uL1-containing ribosomes compared to control transcripts that were unchanged in the ribosome profiling. Data shown are mean +/− SEM. Student’s t test P values: 0.05 ( Gapdh ), 0.11 ( Foxc2 ), 0.07 ( Meox1 ), 0.08 ( Smad4 ), 0.06 ( Tgfbr1 ), 0.005 ( Dhcr24 ), 0.002 ( Fgfr1 ), 0.007 ( Vangl2 ), 0.12 ( Fzd3 ), 0.003 ( Sfrp2 ), 0.02 ( Rac1 ). * P value < 0.05; ** P value < 0.01. Source data are provided as a Source Data file.

Article Snippet: This phenotype was also present at double the frequency in Vangl2 Lp/+ ; Rpl10a LOF/+ double heterozygotes compared to Vangl2 Lp/+ embryos, though it did not achieve statistical significance due to its overall rarity (Supplementary Fig. ).

Techniques: Immunoprecipitation, In Vitro, Quantitative RT-PCR

Journal: Nature Communications

Article Title: A stem cell roadmap of ribosome heterogeneity reveals a function for RPL10A in mesoderm production

doi: 10.1038/s41467-022-33263-3

Figure Lengend Snippet: a Whole-embryo X-gal staining of Axin2 LacZ/+ ; Rpl10a +/+ and Axin2 LacZ/+ ; Rpl10a LOF/LOF embryos. Decreased staining in Axin2 LacZ/+ ; Rpl10a LOF/LOF tail buds is indicated with red arrows; decreased staining in Axin2 LacZ/+ ; Rpl10a LOF/LOF neural tube is indicated with an orange arrow. b Quantification of the area of X-gal staining in tail bud relative to overall tail bud area. E8.5 and E9 n = 2 each, E9.5 n = 4, E10.5 n = 3 for Rpl10a LOF/LOF and n = 6 for control. Data are shown as mean +/− SEM. Student’s t test P values: 0.9 (E8.5), 0.03 (E9), 0.006 (E9.5), 0.03 (E10.5). c Quantification of the area of X-gal staining in the neural tube relative to overall neural tube area in E10.5 embryos ( n = 3 for Rpl10a LOF/LOF and n = 6 for control). Data are shown as mean +/− SEM and significance was calculated using Student’s t tests ( P = 0.005). d Abundance of Axin2 mRNA relative to Nupl1 control transcript as measured by RT-qPCR in in vitro differentiated paraxial mesoderm derived from heterozygous and homozygous Rpl10a LOF/LOF hESCs ( n = 3 each). Data are shown as mean +/− SEM, and significance was calculated using Student’s t tests ( P = 0.03). * P value < 0.05; ** P value < 0.01. Source data are provided as a Source Data file.

Article Snippet: This phenotype was also present at double the frequency in Vangl2 Lp/+ ; Rpl10a LOF/+ double heterozygotes compared to Vangl2 Lp/+ embryos, though it did not achieve statistical significance due to its overall rarity (Supplementary Fig. ).

Techniques: Staining, Quantitative RT-PCR, In Vitro, Derivative Assay

Journal: Nature Communications

Article Title: A stem cell roadmap of ribosome heterogeneity reveals a function for RPL10A in mesoderm production

doi: 10.1038/s41467-022-33263-3

Figure Lengend Snippet: a Lateral views of E12.5 wild-type, Rpl10a LOF /+ , Vangl2 Lp /+ , and Rpl10a LOF /+ ; Vangl2 Lp /+ embryos and graph of looped tail phenotype frequencies for each genotype. The Rpl10a LOF /+ ; Vangl2 Lp /+ embryo shown has the looped tail phenotype (arrow). The increased frequency in Rpl10a LOF /+ ; Vangl2 Lp /+ embryos is statistically significant ( P = 0.02, Fisher’s exact test). b Schematic of basal foot polarization in the multiciliated cells of the trachea epithelium. Basal feet point in the proximal direction (toward the larynx) in wild-type embryos and are less uniformly oriented when PCP signaling is perturbed. c Transmission electron microscopy of wild-type and Rpl10a LOF /LOF E18.5 trachea with basal bodies with visible basal feet demarcated with red arrowheads. Basal feet orientation relative to proximal–distal axis of the trachea, with the oral direction set to 0°, are shown in rose plots with wild-type n = 151 and Rpl10a LOF /LOF n = 127. The circular grid lines represent frequencies, and the difference in distribution between wild-type and Rpl10a LOF /LOF is statistically significant (Watson’s U 2 test P value < 0.001). * P value < 0.05; ** P value < 0.01. Source data are provided as a Source Data file.

Article Snippet: This phenotype was also present at double the frequency in Vangl2 Lp/+ ; Rpl10a LOF/+ double heterozygotes compared to Vangl2 Lp/+ embryos, though it did not achieve statistical significance due to its overall rarity (Supplementary Fig. ).

Techniques: Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: Dynamic Regulation of Cholesterol Metabolism Genes in Dopaminergic Neurons following Methamphetamine Treatment as Revealed by Single-Nucleus RNA Sequencing

doi: 10.1101/2025.07.28.667272

Figure Lengend Snippet: (A) Representative RNAscope HiPlex images showing DAPI-labeled nuclei (blue), dopaminergic neurons identified by Slc6a3 (green), Ldlr (red), and Hmgcr (purple). Scale bar, 1,000 μm. (B) Representative RNAscope HiPlex images on the same brain section as in (A) with DAPI (blue), Th (green) labeling dopaminergic neurons, Insig1 (red), and Msmo1 (purple). Scale bar, 1,000 μm. (C) Representative image of RNAscope co-labeling for Slc6a3 (green) and Hmgcr (red). Scale bar, 200 μm. (D) Co-labeling of Slc6a3 (green) and Hmgcr (red). The region occupied by Slc6a3 -positive cells was defined as the region of interest (ROI), and red dots within the ROI were quantified for Hmgcr expression (Scale bar, 50 μm). (E) Violin plot showing Hmgcr expression in different cell types in the VTA and SNc. (F) Dot plot depicting expression levels of cholesterol metabolism-associated genes across different cell types. Abbreviations: OPC, oligodendrocyte precursor cells; GABA, GABAergic neurons; Glu, glutamatergic neurons; DA, dopaminergic neurons; Oligo, oligodendrocytes; Endo, endothelial cells; Mixed, neurons expressing multiple markers.

Article Snippet: To facilitate accurate dissection of the VTA and SNc, Slc6a3 -EGFP/ Rpl10a (Dat bacTRAP) mice ( ) (Jackson Laboratory, Stock No. 012365) were used for identification of midbrain regions containing dopaminergic neurons.

Techniques: RNAscope, Labeling, Expressing

Journal: bioRxiv

Article Title: Simultaneous measurement of nascent transcriptome and translatome using 4-thiouridine metabolic RNA labeling and translating ribosome affinity purification

doi: 10.1101/2023.02.15.525786

Figure Lengend Snippet: (A) MA plot of cytosolic RNA-seq, P-TRAP-seq, and L10a-TRAP-seq data from TPG-treated versus DMSO-treated HEK293 cells. Differentially expressed genes (adjusted p-value < 0.01 and log2 fold change > 1.5) are highlighted in red (up-regulated) or blue (down-regulated). (B) CPM (counts per million) values of selected ER stress response genes ( HSPA5, DDIT3, PDIA4, DNAJC3 , and HERPUD1 ) and a control gene ( GAPDH ) in the cytosolic RNA-seq (RNA), P-TRAP-seq (P0), and L10a-TRAP-seq (L10a) in response to DMSO-(black) or TPG-(red) treatments. The averages and standard deviations of three replicates are shown. (C) Cumulative distributions of the log2 fold changes of selected gene categories (preferentially translated by polysome upon eIF2α phosphorylation) compared to all genes, together with p-values of Mann-Whitney U test.

Article Snippet: Antibodies against ribosomal proteins uL3 (GTX114725, GeneTex), L10a (A305-061A, BETHYL), uS2 (GTX114734, GeneTex), uS3 (GTX103964, GeneTex), uS15 (GTX101839, GeneTex), and PABP4 (A301-467A, BETHYL), CBP80 (A301-793A, BETHYL) and GAPDH (M171-7, MBL) were purchased commercially.

Techniques: RNA Sequencing, Control, Phospho-proteomics, MANN-WHITNEY

Journal: bioRxiv

Article Title: Simultaneous measurement of nascent transcriptome and translatome using 4-thiouridine metabolic RNA labeling and translating ribosome affinity purification

doi: 10.1101/2023.02.15.525786

Figure Lengend Snippet: (A) MA plot of cytosolic RNA-seq, P-TRAP-seq, and L10a-TRAP-seq data from TPG-treated versus DMSO-treated HEK293 cells. Differentially expressed genes (adjusted p-value < 0.01 and log 2 fold change > 1.5) are highlighted in red (up-regulated) or blue (down-regulated). (B) CPM (counts per million) values of selected ER stress response genes ( HSPA5, DDIT3, PDIA4, DNAJC3 , and HERPUD1 ) and a control gene ( GAPDH ) in the cytosolic RNA-seq (RNA), P-TRAP-seq (P0), and L10a-TRAP-seq (L10a) in response to DMSO-(black) or TPG-(red) treatments. The averages and standard deviations of three replicates are shown. (C) Cumulative distributions of the log 2 fold changes of selected gene categories (preferentially translated by polysome upon eIF2α phosphorylation, localized at endoplasmic reticulum, ATF4/CHOP transcriptional targets, and ATF6/XBP1 transcriptional targets) compared to all genes, together with p-values of Mann-Whitney U test.

Article Snippet: Antibodies against ribosomal proteins uL3 (GTX114725, GeneTex), L10a (A305-061A, BETHYL), uS2 (GTX114734, GeneTex), uS3 (GTX103964, GeneTex), uS15 (GTX101839, GeneTex), and PABP4 (A301-467A, BETHYL), CBP80 (A301-793A, BETHYL) and GAPDH (M171-7, MBL) were purchased commercially.

Techniques: RNA Sequencing, Control, Phospho-proteomics, MANN-WHITNEY

Journal: Cell

Article Title: Late Endosomes Act as mRNA Translation Platforms and Sustain Mitochondria in Axons

doi: 10.1016/j.cell.2018.11.030

Figure Lengend Snippet:

Article Snippet: 1:200 rabbit anti-RPS3A (14123-1-AP, Proteintech), 1:200 mouse anti-RPS3A (ab194670, Abcam), 1:200 mouse anti-RPL19 (ab58328, Abcam), 1:200 rabbit anti-RPL24 (17082-1-AP, Proteintech), 1:400 rabbit anti-Vg1RBP (gift from Dr. Standart), 1:250 rabbit anti-LAMP-1 (ab24170, Abcam), 1:400 mouse anti-FXR (gift from Dr. Khandjian), 1:50 mouse anti-Rab5 (sc-46692, Santa Cruz), 1:300 rabbit anti-Rab7 (ab137029, Abcam), 1:50 mouse anti-GM130 (BD610822, BD Biosciences), rabbit anti-Rpl10A (16681-1-AP, Proteintech), 1:100 rabbit anti-phospho-AKT (Ser473) (9271T, Cell Signaling), 1:200 rabbit anti-phospho-mTOR (Ser2448) (ab109268, Abcam), 1:200 rabbit anti-mTOR (#2983, Cell Signaling), 1:800 rabbit anti-phospho-S6 Ribosomal Protein (Ser235/236) (#2211, Cell Signaling), 1:200 rabbit anti-SFPQ (ab38148, Abcam).

Techniques: Control, Virus, Recombinant, Fluorsave, Plasmid Preparation, SYBR Green Assay, Mutagenesis, In Situ, Affinity Purification, Software