Journal: Molecular Cancer

Article Title: Integrated single-cell and spatial transcriptomic profiling decodes lineage plasticity and immune microenvironment remodeling in prostate cancer progression

doi: 10.1186/s12943-026-02617-6

Figure Lengend Snippet: Functional characterization of FOSL1 as a key regulator in double-negative prostate cancer subtype. A Transcription factor activity in malignant epithelial subtypes. Heatmap displaying the top transcription factors with the highest inferred activity (based on SCENIC analysis) in each of the four malignant epithelial subtypes. Color scale (blue to red) represents low to high regulon activity. B Spatial activity of FOSL1 across subtypes. UMAP of malignant epithelial cells colored by the SCENIC-derived regulon activity score of FOSL1, showing its specific enrichment in Subtype 4. Gradient from white to blue indicates low to high activity. C Effect of FOSL1 knockdown on cell proliferation (CCK-8 assay). Growth curves of PC-3 cells transfected with control siRNA or three independent siRNAs targeting FOSL1. Data are presented as mean ± SD; *** P < 0.001, ** P < 0.01 (two-way ANOVA). D Effect of FOSL1 knockdown on clonogenic survival. Representative images of colony formation assays for PC-3 cells treated as in (C). E Effect of FOSL1 knockdown on apoptosis. (Left) Representative flow cytometry plots of Annexin V/PI staining. Quadrants: viable cells (Annexin V⁻/PI⁻), early apoptotic (Annexin V⁺/PI⁻), late apoptotic (Annexin V⁺/PI⁺), and necrotic (Annexin V⁻/PI⁺). (Right) Quantification of total apoptotic cells (early + late apoptosis). Data are mean ± SD; *** P < 0.001, ** P < 0.01 (one-way ANOVA). F Identification of FOSL1-regulated DNPC genes. Venn diagram showing the overlap between predicted FOSL1 target genes (from SCENIC) and the DNPC gene signature, yielding 8 candidate genes. G FOSL1 binding to candidate gene promoters (ChIP-qPCR). ChIP-qPCR analysis showing enrichment of FOSL1 at the promoters of the 8 candidate genes in control vs. FOSL1-knockdown PC-3 cells. Results are presented as % input. Data are mean ± SD; *** P < 0.001, ** P < 0.01, ns = not significant (two-tailed Student’s t-test). H Expression of candidate genes upon FOSL1 knockdown (qRT-PCR). mRNA expression levels of four selected candidate genes in PC-3 cells after FOSL1 knockdown. Data are normalized to control and presented as mean ± SD; *** P < 0.001, ** P < 0.01, ns = not significant (two-tailed Student’s t-test). I Correlation between FOSL1 and HMGA1 expression. Scatter plot showing a significant positive correlation between FOSL1 and HMGA1 expression across all malignant epithelial cells (n = 152,872). The red line indicates the linear regression fit (R² = 0.212, P < 0.0001, Pearson correlation). J Spatial co-expression of FOSL1 and HMGA1. (Left three panels) Spatial mapping of FOSL1 and HMGA1 expression and their co-localization in a representative section (Patient 01). Red spots indicate high co-expression, yellow indicates high FOSL1 alone, green indicates high HMGA1 alone, and gray indicates low expression of both. (Right panel) Correlation analysis of FOSL1 and HMGA1 expression specifically within the co-expressing spots (red spots, left panel), showing a strong positive correlation (R = 0.923, P = 1.28e-10, Pearson correlation). K - M Western blot analysis of downstream pathways. Protein levels of (K) stemness markers (CD44, OCT4, SOX2, NANOG), (L) EMT markers (ZO-1, N-cadherin, E-cadherin, Vimentin, SNAIL), and (M) DNPC markers (DSG3, KRT6A, KRT5), along with FOSL1 and HMGA1, in PC-3 cells under four conditions: Control, FOSL1 knockdown (siFOSL1), HMGA1 overexpression (oeHMGA1), and FOSL1 knockdown combined with HMGA1 rescue (siFOSL1 + oeHMGA1). GAPDH served as a loading control. N Rescue of proliferation by HMGA1 overexpression. CCK-8 proliferation assay of PC-3 cells under the four conditions described in (K-M). Data are mean ± SD; *** P < 0.001, ** P < 0.01, ns = not significant (two-way ANOVA). O Rescue of apoptosis by HMGA1 overexpression. Quantification of total apoptosis (Annexin V⁺ cells) by flow cytometry under the four conditions described in (K-M). Data are mean ± SD; *** P < 0.001, ** P < 0.01, ns = not significant (one-way ANOVA). P Pharmacological inhibition of FOSL1 reduces HMGA1. Western blot showing dose-dependent decrease of FOSL1 and its downstream target HMGA1 in PC-3 cells treated with increasing concentrations (0, 0.2, 2, 5, 10 µM) of the FOSL1 degrader T-5224. Q In vivo combination therapy schematic. Workflow of the xenograft study. Nude mice bearing PC-3 subcutaneous tumors were treated starting at day 13 post-inoculation with vehicle, Docetaxel (1 mg/kg), FOSL1 degrader T-5224 (10 mg/kg), or the combination via intraperitoneal injection every 48 h. Treatment continued until day 28. R Representative images of resected tumors. Photographs of excised tumors from each treatment group at the study endpoint. S Tumor growth curves. Tumor volume (mm³) was measured over time for each treatment group. Data are presented as mean ± SEM; *** P < 0.001 (two-way ANOVA). T Tumor weight at endpoint. Final tumor weights (g) for each group. Data are mean ± SEM; *** P < 0.001, ** P < 0.01 (one-way ANOVA). U Assessment of tumor cell proliferation (IHC). Representative immunohistochemistry images showing Ki67 expression in tumor sections from each treatment group. Scale bar, 100 μm

Article Snippet: HMGA1 overexpression plasmid (Cat: HG11613-NF), and the empty vector plasmid pCMV3-N-FLAG were purchased from SinoBiological.

Techniques: Functional Assay, Activity Assay, Derivative Assay, Knockdown, CCK-8 Assay, Transfection, Control, Flow Cytometry, Staining, Binding Assay, ChIP-qPCR, Two Tailed Test, Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Proliferation Assay, Inhibition, In Vivo, Injection, Immunohistochemistry

Journal: PLoS ONE

Article Title: Spink2 Modulates Apoptotic Susceptibility and Is a Candidate Gene in the Rgcs1 QTL That Affects Retinal Ganglion Cell Death after Optic Nerve Damage

doi: 10.1371/journal.pone.0093564

Figure Lengend Snippet: (A–C) Retinal whole mounts stained for the ganglion cell marker BRN3A (A) and SPINK2 (Santa Cruz goat polyclonal) (B). A majority of BRN3A positive cells are positive for SPINK2 staining. Additionally, nerve fiber bundles emanating from ganglion cells are SPINK2 positive (arrow). SPINK2 also stains BRN3A negative cells (asterisks), which may be displaced amacrine cells or some of the 10–15% of ganglion cells that do not stain for this transcription factor . SPINK2 also stains cells with morphology typical of astrocytes (arrowhead). BV = blood vessel. Size bar = 30 μm. (D,E) Frozen sections of a control (D) and crush (E) mouse retina (DBA/2J.BALB Rgcs1 mouse shown) stained for an antibody against the C-terminus of mouse SPINK2 (ProSci Inc., rabbit polyclonal). This antibody predominantly stains the cells in the ganglion cell layer (GCL), although some light staining is evident in the innermost cells of the inner nuclear layer (INL) and the inner plexiform layer (IPL) and outer plexiform layer (OPL). This antibody may also stain putative Müller cell processes (see ). There is no appreciable change in the staining pattern of SPINK2 before and after crush when viewed on sections. The overall pattern of staining shown here is consistent among 3 different SPINK2 antibodies tested. Size bar = 50 μm. (F–I) Retinal whole mounts from DBA/2J (F,G) and DBA/2J.BALB Rgcs1 (H,I). Ganglion cells positive for BRN3A (yielding pink colored nuclei with the DAPI counterstain – arrowheads) show diffuse SPINK2 staining (green, rabbit polyclonal, see ref 18) in control retinas (F,H). At 7 days after optic nerve crush, some cells in the retinas of both strains exhibit intense SPINK2 immunoreactivity (examples marked by arrows) that surrounded nuclei with condensed and fragmented chromatin (asterisks). At this stage, ganglion cells exhibit only limited staining for BRN3A (see text). Size bar = 5 μm.

Article Snippet: The rabbit polyclonal antibody from ProSci (see Methods) also revealed staining in putative Müller cell processes , while a rabbit polyclonal used in previous studies , exhibited strong immunoreactivity in the nerve fibers of the ganglion cell layer.

Techniques: Staining, Marker, Control

Journal: Cells

Article Title: Phosphorylation in the Charged Linker Modulates Interactions and Secretion of Hsp90β

doi: 10.3390/cells10071701

Figure Lengend Snippet: Determination of occupancy of CL phosphosites in human Hsp90β (HSP90AB1). ( A ) Amino acid sequence of Hsp90β’s CL with S226, S255 and S261 indicated by red arrows. ( B ) Typical peptides spanning S226 or S255 resulting from a tryptic digestion of the CL. The peptides marked in red were selected for targeted MS. ( C ) The complete list of peptides selected for the targeted MS method and their charge state (z number). Normalization peptides are required to determine the H/L ratio for the whole protein. Both phosphopeptides and non-phosphopeptides from the CL were measured. ( D ) Typical chromatogram for a normalization peptide (here SIYYITGESK, left pane), a non-phosphopeptide from the CL (IEDVGSDEEDDSGK, middle pane), and its corresponding phosphopeptide (IEDVGpSDEEDDSGK, right pane). The chromatogram for the heavy (H) peptide is the blue curve, and the light (L) chromatogram the red curve. ( E ) Average and standard deviation of phosphorylation occupancy for 5 human cell lines ( n = 3).

Article Snippet: Plasmids were generated using standard procedures and verified by sequencing. pCMV3-HA-Hsp90β WT (“WT plasmid”) was ordered from SinoBiological Inc. (Beijing, China; HG11381-NY), which served as template to produce pCMV3-Hsp90β S226A/S255A (“AA plasmid”) and no tag control plasmids.

Techniques: Sequencing, Standard Deviation

Journal: Cells

Article Title: Phosphorylation in the Charged Linker Modulates Interactions and Secretion of Hsp90β

doi: 10.3390/cells10071701

Figure Lengend Snippet: Quantitative exploration of the interactome of WT vs. AA. ( A ) Volcano plot of the inverse log 10 of the adjusted p -value as a function of the normalized H/L ratio in log 2 scale for all 83 quantified interactors (SILAC experiment). Points represent individual proteins. Point color code: co-chaperones in green, Hsp40 proteins in pink, Hsp70 in orange, Hsp90 in red, other already known interactors in blue, and putative interactors in grey. Classification of co-chaperones and known interactors is based on the “Hsp90 interactors” table by Didier Picard ( www.picard.ch , accessed on 17 May 2021). Horizontal dashed line marks the 0.05 threshold for the adjusted p -value, vertical dashed lines mark absolute fold-changes greater than 0.4, and vertical red line marks Hsp90β. ( B ) Gene Ontology (GO) term analysis of the proteins enriched in the AA co-IP. Vertical axis: enriched GO terms. Horizontal axis: the GeneRatio is the ratio between the number of proteins enriched with the corresponding GO term and the total number of proteins identified in the input assigned to the corresponding GO term. Spot size is proportional to the count of proteins enriched (legend on the right of the plot). Spot color corresponds to the adjusted p -value as described in the color scale on the right of the plot.

Article Snippet: Plasmids were generated using standard procedures and verified by sequencing. pCMV3-HA-Hsp90β WT (“WT plasmid”) was ordered from SinoBiological Inc. (Beijing, China; HG11381-NY), which served as template to produce pCMV3-Hsp90β S226A/S255A (“AA plasmid”) and no tag control plasmids.

Techniques: Co-Immunoprecipitation Assay

Journal: Cells

Article Title: Phosphorylation in the Charged Linker Modulates Interactions and Secretion of Hsp90β

doi: 10.3390/cells10071701

Figure Lengend Snippet: Average occupancy and standard deviation (s.d.) determined for Hsp90β immunopurified from conditioned medium of K562 suspension cultures ( n = 3). p -values from a t -test comparing the present occupancy values with K562 intracellular Hsp90β occupancy values.

Article Snippet: Plasmids were generated using standard procedures and verified by sequencing. pCMV3-HA-Hsp90β WT (“WT plasmid”) was ordered from SinoBiological Inc. (Beijing, China; HG11381-NY), which served as template to produce pCMV3-Hsp90β S226A/S255A (“AA plasmid”) and no tag control plasmids.

Techniques: Standard Deviation

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15 associates with Keap1. A Network graph representation of interaction from the BioGRID for TRIM15. Users can select the ‘Network’ tab from the ‘Switch View’ menu to view interactions data when available. B Immunoblot detection of the indicated proteins in a Co-IP assay performed in HEK293 cells. C Immunofluorescence colocalization of TRIM15 with Keap1 in H1299 and H1975 was assessed by rabbit anti-TRIM15 detected with anti-rabbit IgG-Alexa Fluor 488 (green fluorescence), and detection of Keap1 with mouse anti-Keap1 detected with anti-mouse IgG Alexa 594 (red fluorescence). The colocalization of TRIM15 and Keap1 is illustrated by overlay of the images, illustrated by yellow fluorescence. D Immunoprecipitation assay revealing the interaction between TRIM15 with Keap1 in H1299 and H1975 cells. E Co-localization of TRIM15 (green) and Keap1 (red) in 2 NSCLC tissues from two patients by immunofluorescent confocal microscopy

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Immunofluorescence, Fluorescence, Immunoprecipitation, Confocal Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15 promotes Keap1 ubiquitination and degradation. A Immunoblot analysis of Keap1, Flag-TRIM15, and β-actin in HEK293 cells transfected with expression vector for Flag-TRIM15 or with empty vector, with or without treatment of 10 μM MG132. Mutations of Flag-TRIM15-ΔRING impaired the ability of TRIM15 to degrade Keap1 protein in HEK293 cells. B HEK293 cells were transfected with the indicated plasmids. The cells were treated with 50 μg/ml CHX for indicated time periods. Densitometry analysis performed on corresponding immunoblots to assess Keap1 half-life in the indicated conditions. C H1299 or H1650 cells were transfected with the indicated plasmids, treated with 50 μg/ml CHX, harvested at different time points, and then immunoblotted with using the indicated antibodies. Densitometry analysis performed on corresponding immunoblots to assess Keap1 half-life in the indicated conditions. D HEK293 cells were transfected with Flag-TRIM15, Flag-TRIM15-ΔRING, HA-Keap1, and His-ubiquitin plasmids, and the cell lysates were subjected to immunoprecipitation using anti-HA antibodies (left panel) or Ni–NTA pull-down (right panel) under denaturing conditions, followed by immunoblotting with the indicated antibodies. Overexpression of wild-type but not the mutant TRIM15-ΔRING promoted ubiquitination of Keap1. E Knockdown of endogenous TRIM15 decreased the ubiquitination of HA-Keap1 in HEK293 cells analyzed by in vitro ubiquitination assays. F Cell lysates prepared in D were immunoprecipitated with anti-HA. knockdown of TRIM15 inhibits K48-linked but not K63-linked ubiquitination of HA-Keap1

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Over Expression, Mutagenesis, Knockdown, In Vitro

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15 stabilizes Nrf2 through binding with Keap1. A HEK293 cells transfected with Flag-TRIM15, HA-Keap1, and Myc-Nrf2 were subjected to immunoprecipitation with HA antibody. Lysates were analyzed by western blotting. B TRIM15 reduced the interaction between Nrf2 and Keap1. Cell lysates were immunoprecipitated with an anti-Keap1 antibody and blotted with an anti-Nrf2 antibody. C – F Subcellular fractionation was used to isolate cytoplasmic and nuclear proteins, and immunoblotting was performed to examine the localization of Nrf2 following the downregulation or overexpression of TRIM15. Nuclear and cytoplasmic levels of Nrf2 are quantified. G Effect of TRIM15 knockdown (H1299 cells) or overexpression (H1650 cells) on the mRNA expression of the Nrf2-regulated genes. NAD(P)H quinone dehydrogenase1(NOQ1), thioredoxin (TXN), peroxiredoxin 1(PRDX1), hemoxygenase 1(HMOX1), glutamate-cysteine ligase catalytic subunit (GCLC), glutathione S-transferase μ1(GSTM1), glutathione S-transferase μ3(GSTM3), ferritin light chain (FTL). H Representative IHC staining images of Nrf2 in the same set of NSCLC tissue slices. Correlation analysis of TRIM15 and Nrf2 expression in NSCLC samples. Spearman correlation coefficients are shown. Scale bars, 100 μm. Statistical analyses were performed by two-tailed unpaired Student’s t -test. ** P < 0.01

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot, Fractionation, Over Expression, Knockdown, Expressing, Immunohistochemistry, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15-mediated Nrf2 signaling regulates growth and invasion in NSCLC cells in vitro. A Western blot analyses of TRIM15, Nrf2, Keap1, and Nrf2 target NQO1 in H1299 cells with TRIM15 knockdown with or without subsequent Nrf2 overexpression and H1650 cells overexpressing TRIM15 with or without subsequent knockdown of Nrf2. B ARE Luc reporter activity assessed in H1299 cells expressing shTRIM15, sh TRIM15 + Nrf2 or H1650 cells overexpressing TRIM15 with or without subsequent knockdown of Nrf2. Up-regulating of Nrf2 expression in H1299 cells or down-regulating of Nrf2 expression in H1650 cells was set as a control. C – F Cell proliferation ( C , D ), invasion ( E ) and ROS formation ( F ) in H1299 cells with or without shTRIM15 and Nrf2 rescue or in H1650 cells with or without TRIM15 overexpression and shNrf2 rescue. Statistical analyses were performed by two-tailed unpaired Student’s t -test. ** P < 0.01

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: In Vitro, Western Blot, Knockdown, Over Expression, Activity Assay, Expressing, Control, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15 mediated increase in Nrf2 regulates growth and invasion in vivo. A Nude mice were randomized into three groups and subcutaneously injected with H1650 cells that had been transfected with control (empty vector), TRIM15, or TRIM15 + shNrf2 plasmids. Tumors formed in nude mice were collected 30 days after grafting, and the tumor weight were measured. B Measurement of tumor volume in experimental groups over time. C Western blotting analysis was performed to evaluate the levels of TRIM15, Nrf2, Keap1, and NQO1 in harvested tumors. D , E Up-regulation of TRIM15 significantly promoted lung metastasis in H1650 xenograft nude mice models, whereas the suppression of Nrf2 prevented the tumor metastasis of TRIM15 overexpressing cells. Representative pictures of the lung metastases in nude mice by H&E staining. Quantification of lung metastases in all groups. Scale bar: 200 μm. F , G A representative image of tumor growth in nude mice subcutaneously inoculated with H1299 cells tranfected with shCtrl, shTRIM15 or shTRIM15 + Nrf2 plasmids. Tumor volumes were measured on the indicated days. H Western blotting analysis was performed to evaluate the levels of TRIM15, Nrf2, Keap1, and NQO1 in xenograft tumors. I Representative pictures of the lung metastases in nude mice by H&E staining. Quantification of lung metastases in all groups. Scale bar: 200 μm. J TRIM15 was significantly upregulated in NSCLC and that increased TRIM15 was associated with poor survival. TRIM15 promoted tumor proliferation and metastasis by activating Nrf2 signaling. Furthermore, TRIM15 regulated Nrf2 activity by modulating Keap1 and inducing its ubiquitination and degradation in NSCLC cells. Activation of Nrf2 facilitated tumor cell proliferation and invasion. N.S. represents no significant. Statistical analyses were performed by two-tailed unpaired Student’s t -test. ** P < 0.01

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: In Vivo, Injection, Transfection, Control, Plasmid Preparation, Western Blot, Staining, Activity Assay, Activation Assay, Two Tailed Test

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: Linkage analysis and whole-genome sequencing identify novel disease gene ZCCHC8 in familial pulmonary fibrosis with low telomerase RNA ( TR ). ( A ) Pedigree with pulmonary fibrosis proband (arrow) with affected relatives are indicated by the shaded symbols (key). The clinical history below each of the four shaded pedigree symbols refers to the age of onset of lung disease including idiopathic pulmonary fibrosis (IPF). (?) Asymptomatic individuals who had unknown affected status at the time of clinical assessment; (gray shading) unknown cause of death; (*) individuals with DNA who were included in the linkage analysis. ( B ) TR levels measured by quantitative real time PCR (qRT-PCR) in lymphoblastoid cell lines (LCLs). Arrow refers to proband (red) and pedigree identifiers refer to A . TR level from a DKC1 mutation carrier is a positive control. The data represent a mean of three experiments, each from independent RNA isolations. ( C ) Telogram shows age-adjusted lymphocyte telomere length by flow cytometry and fluorescence in situ hybridization (flowFISH) in the proband (arrow) and family (pedigree designations as in A ). The validated telogram is based on 192 controls. ( D ) Phenotype assignments used in linkage (key) and genotype below each individual refers to ZCCHC8 SNP. Italicized genotypes refer to obligate carriers. ( E ) Log of the odds (LOD) ratio across autosomal chromosomes calculated from SNP data from 14 individuals, with arrow on chromosome 12 pointing to maximum LOD. ( F ) p.P186L conservation across eight vertebrate ZCCHC8 species with darker shading denoting more conserved residues. CCHC refers to Zinc-knuckle domain; PSP refers to proline-rich domain.

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: Sequencing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Mutagenesis, Positive Control, Flow Cytometry, Fluorescence, In Situ Hybridization

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: ZCCHC8 loss of function is sufficient to cause low TR levels. ( A ) Immunoblot of ZCCHC8 in lymphoblastoid cell lines (LCLs) from healthy controls (C1 and C2) and unaffected relatives and mutation carriers labeled with pedigree identifiers from A. Quantification from one blot and result replicated twice from independently harvested protein lysates. ( B ) Immunoblot of ZCCHC8, SKIV2L2, and RBM7 levels in proband's primary skin fibroblasts. ( C ) Immunoblot of transfected Myc-tagged (293FT cells) and endogenous ZCCHC8. ( D ) Mean ZCCHC8 mRNA levels ± SEM from LCLs in unaffected family members ( n = 4) and ZCCHC8 p.P186L mutation carriers ( n = 3). ( E ) Chromatogram showing that ZCCHC8 p.P186L mutation is expressed in LCL mRNA from proband (also verified in two other mutation carriers). ( F ) Immunoblot showing efficiency of shRNA knockdown of Luciferase (Luc), ZCCHC8, and NAF1 in HeLa cells. ( G ) Total TR levels measured by qRT-PCR (mean ± SEM from three independent knockdowns and RNA isolations). ( H ) Northern blot of TR after stable knockdown of ZCCHC8 and NAF1 (replicated twice with independent RNA isolations). (**) P < 0.01; (***) P < 0.001 (Student's t -test, two-sided).

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: Western Blot, Mutagenesis, Labeling, Transfection, shRNA, Luciferase, Quantitative RT-PCR, Northern Blot

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: ZCCHC8 is required for its 3′ end maturation and telomerase function. ( A ) Compound heterozygous frameshift (fs) mutations introduced using CRISPR/Cas9 in HCT116 pseudodiploid cells. ( B ) Immunoblot for ZCCHC8 in HCT116-edited cells. ( C ) Scheme summarizing TR 3′ rapid amplification of cDNA ends sequencing (3′RACE-seq). TR 3′ ends were generally divided into mature (451 bp) and extended (>451 bp) where extensions are denoted by gray N's. ( D ) Summary of TR 3′RACE-seq fractions in isogenic ZCCHC8 +/+ and ZCCHC8 −/− cells. Color-coded key shows four categories of TR forms: mature (451 nt), adenylated (A)n, short genomically extended (g)n (<465 nt), and long genomically extended (>465 nt). Data are mean of three independent 3′RACE-seq analyses from three RNA isolations each from a different aliquot of a single clone. ( E ) qRT-PCR of extended TR forms beyond the 451 mature end (>20, >51, >784 nt). Data are mean of three independent RNA isolations similar to D . ( F ) TR Northern blot of edited ZCCHC8 +/+ and ZCCHC8 −/− cells. ( G ) Total TR levels by Northern bot (six blots from three RNA isolations). ( H ) Telomerase activity measured by telomere repeat amplification protocol (TRAP) assay in ZCCHC8 +/+ , ZCCHC8 −/− , and NAF1 S329/S329 HCT116 cell extracts. Activity was quantified on serially diluted extracts (1, 1/5, 1/25, and 1/125) against a PCR-amplified internal control (IC). RNase-treated wild-type extract and no template PCR reaction are included as negative controls. ( I ) Mean TRAP activity of 1/5× diluted extracts (three independent TRAP assays, each from a different lysate). ( J ) Summary of 3′RACE-seq of TR forms from control and proband's primary skin fibroblasts with speciation as in D . ( K ) qRT-PCR values of extended TR forms in primary skin fibroblasts as in E , mean of three technical replicates). ( L ) Amplified TR from input and Myc-ZCCHC8 immunprecipitated fractions (293FT cells) using primers falling within the mature TR sequence. ( M ) qRT-PCR of extended TR (>51 nt extended beyond the 3′ mature TR end) after transfection of tagged ZCCHC8, DIS3, EXOSC10/RRP6, and PARN into HCT116 ZCCHC8 −/− cells (three to four independent transfections/experiment). Data are expressed as mean ± SEM (*) P < 0.05; (**) P < 0.01 (Student's t -test, two-sided).

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: CRISPR, Western Blot, Rapid Amplification of cDNA Ends, Sequencing, Quantitative RT-PCR, Northern Blot, Activity Assay, Amplification, TRAP Assay, Transfection

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: Zcchc8- null mice have TR insufficiency. ( A – C ) Immunoblot for ZCCHC8, SKIV2L2, and RBM7, respectively, on lysates from mouse ear fibroblasts. ( D , E ) Northern blot for mouse TR and quantification. For E , mean reflects mice Zcchc8 +/+ ( n = 4, 2M/2F), Zcchc8 +/− ( n = 4, 2M/2F), Zcchc8 −/− ( n = 3M), mTR +/− ( n = 2, sex unknown) and mTR −/− ( n = 2, sex unknown). ( F ) TR 3′ extended levels (>20 bp) relative to Hprt as measured by qRT-PCR. Mouse numbers and M/F designations as in E . Data are expressed as mean ± SEM. (*) P < 0.05; (**) P < 0.01 (Student's t -test, two-sided).

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: Western Blot, Northern Blot, Quantitative RT-PCR

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: ZCCHC8 complete loss causes progressive and fatal neurodevelopmental phenotype. ( A , top row) Images showing head profile of Zcchc8 wild-type, heterozygous, and homozygous null mice (41–46 d-old). The labels show the genotype with a male ( left ) and a female ( right ) for each genotype. Zcchc8 −/− mice have abnormal head profiles with domed crania, as outlined by the dashed line. ( Middle row ) CT head mid-sagittal images show Zcchc8 −/− mice have dome-shaped crania. ( Bottom row). Volume-rendered (VR) CT images of mouse calvaria show widened cranial sutures in Zcchc8 −/− mice (seen in three of six imaged). None of Zcchc8 +/+ or Zcchc8 +/− mice (four mice imaged/genotype) had this feature. Each vertical image group is from the same mouse except the last column (two different females). ( B ) Representative H&E coronal sections from 8-wk-old heads (all male) show no differences in Zcchc8 +/− mice (11 examined) compared with Zcchc8 +/+ mice (10 examined). In contrast, Zcchc8 −/− mice had severe ventricular dilation (nine of 11 examined). ( C ) E12.5 brain sections show Zcchc8 −/− have microcephaly but intact brain structures with no ventriculomegaly in utero. ( D ) Image of E12.5 embryos from a single dam showing expected Mendelian ratios but Zcchc8 −/− embryos have small crania. ( E ) Cranial area of newborn (P0) pups measured on VR CT images and corrected to left femur length on the same images ( Zcchc8 +/+ n = 7, 3M/4F; Zcchc8 +/− n = 10, 3M/7F; Zcchc8 −/− n = 4, 3M/1F). ( F ) Mean cranial area ± SEM relative to age for all three genotypes showing that Zcchc8 −/− mice develop macrocephy after birth. Newborn mice include those listed in E and older mice ( Zcchc8 +/+ , n = 4, 3M/1F; Zcchc8 +/− , n = 4, 3M/1F; Zcchc8 −/− , n = 5, 3M/2F). Dashed lines denote 95% confidence intervals. (**) P < 0.01 (Student's t -test, two-sided).

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: In Utero

Journal: Genes & Development

Article Title: ZCCHC8 , the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation

doi: 10.1101/gad.326785.119

Figure Lengend Snippet: The Zcchc8 −/− transcriptome shows up-regulation and misprocessing of low-abundance intronless RNAs other than TR . ( A ) Heat map with dendrogram of gene expression showing unsupervised analysis of 9788 high-quality genes from brain RNA-seq analysis. Colors denote mean-subtracted FPKM expression values on a log 2 scale ( Zcchc8 +/+ , n = 5; Zcchc8 +/− , n = 3; Zcchc8 −/− , n = 6 embryonic brains sequenced). Each column is labeled below by WT, HET, and KO followed by the embryo number (1, 2, 3, etc.), referring to respective Zcchc8 genotypes. The log 2 expression value was subtracted from the mean log 2 expression value of the entire cohort. The dendrogram showing relatedness of the samples is above , and relatedness of the gene transcripts is at the left . The differential change in RNA expression is shown as positive and negative change on color scale in the key above the top right corner. ( B , C ) Volcano plots depicting the log 2 -fold changes ( X -axis) versus −log 10 P -values calculated by two-tailed one-way ANOVA ( Y -axis) for the Zcchc8 +/− and Zcchc8 −/− versus Zcchc8 +/+ comparisons, respectively. Each dot represents a single transcript. ( D ) Histogram of number of genes at each expression value denoted on the x -axis by the mean log 2 FPKM values obtained from Zcchc8 wild-type embryos ( n = 5). RNAs that have more than two SD higher levels in the Zcchc8 +/+ versus Zcchc8 −/− comparison are shown in red ( n = 197) and fall on the low end of the histogram with TR and its mean FPKM in wild-type embryos shown. Down-regulated RNAs, defined as less than two SD ( n = 43), are shown in blue appear uniformly distributed on the distribution. ( E ) Histogram of the most up-regulated (>2SD) transcripts in the Zcchc8 −/− versus Zcchc8 +/+ by exon number shows the largest subset is intronless RNAs (42 of 188 with known gene structure, 22%). The pie chart divides the intronless RNAs by functional category. ( F ) Annotation of 28 up-regulated intronless RNAs ( TR , histones and cilia) shows a majority of the histones represented are replication-dependent histones (RDH) (23 of 24, 96%). The majority have an annotated transcript size in the range of TR between 400 and 560 (22 of 28). Columns referring to 5′ end and 3′ end refer to visualized additional reads beyond annotated gene boundaries with 5′ end reads referring to upstream reads that are not necessarily contiguous (manually identified in the Integrative Genome Viewer [IGV]). ( G , H ) Genome browser read coverage plots from IGV viewer showing extended 3′ ends as labeled above from two histone genes in each of Zcchc8 +/+ , Zcchc8 +/− , Zcchc8 −/− transcriptomes. ( I , J) Coverage plots for two coiled-coil domain containing cilia genes Ccdc89 and Ccdc182 , respectively, by genotype. For Ccdc89 , there is also an increase in discontinuous upstream of gene 5′end reads that resemble so-called promoter upstream transcripts (PROMPTs).

Article Snippet: Myc-tagged mouse ZCCHC8 cDNA (NM_028151) was purchased in a pCMV3 expression vector (MG51487-NM, Sino Biological).

Techniques: Expressing, RNA Sequencing Assay, Labeling, RNA Expression, Two Tailed Test, Functional Assay