Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Serotonin functions as a bidirectional guidance molecule regulating growth cone motility

doi: 10.1007/s00018-020-03628-2

Figure Lengend Snippet: 5-HT1b and 5-HT2a receptors are expressed in growth cones and filopodia. a–j Sensory neuron growth cones immunostained for serotonin receptors, 5-HT1b (green in a, red in g), 5-HT2a (green, d and h) as well as filamentous actin to highlight the extent of the growth cone (Phalloidin, red; b and e). The receptors 5-HT1b and 5HT2a were observed as a widespread punctate distribution throughout the growth cone (g–h). Punctate expression was evident in filopodia (j) with isolated 5-HT1b (green, *), 5-HT2a (red, #) as well as colocalised (arrowheads) puncta on filopodial shafts. There was prominent colocalization of 5-HT2a and 5-HT1b receptors in peripheral areas (i, arrow). Scale bars are 5 μm (a–i) and 1 μm (j). k Turning responses to serotonin were sensitive to chlorpromazine. Attractive turning of growth cones to 5HT-Lo (n = 7, p = <0.0001) and repulsion to 5HT-Hi (n = 8, p = 0.003) were significantly different when cultures were treated with chlorpromazine. l Axon extension was not significantly different in any experimental treatments (ns, p > 0.05). (Mann–Whitney u-test)

Article Snippet: DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Expressing, Isolation, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Serotonin functions as a bidirectional guidance molecule regulating growth cone motility

doi: 10.1007/s00018-020-03628-2

Figure Lengend Snippet: 5HT-Lo elicits growth cone attraction through the 5-HT2a receptor. a Growth cone attraction was measured in the presence of 5HT-Lo gradients (p = <0.0001) and TCB-2 gradients (p = 0.0002). Growth cone attraction to 5HT-Lo was abolished by pharmacological application of the selective 5-HT2a receptor antagonist, ritanserin (n = 17, p = 0.0001) and the PLC inhibitor, U-73122 (n = 9, p = 0.0018). b Growth cone attraction to 5HT-Lo was not perturbed when growth cones treated with a specific 5-HT1b antagonist, GR55562 (n = 8, p = <0.0001), or the 5-HT3 antagonist ondansetron (n = 8, p = 0.0082). Depletion of ER calcium with thapsigargin (n = 19, p = 0.0003) abolished attractive turning to 5HT-Lo, while inhibition of VGCCs with nifedipine (n = 15, p = 0.0330) had no significant effect. Turning angles were compared to vehicle and 5HT-Lo and only significant differences shown in (a) and (b). Kruskal–Wallis, Dunn’s multiple comparison test. c, d Axon extension was not significantly different in any experimental treatments (ns, p > 0.05). Kruskal–Wallis, Dunn’s multiple comparison test

Article Snippet: DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Inhibition, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Serotonin functions as a bidirectional guidance molecule regulating growth cone motility

doi: 10.1007/s00018-020-03628-2

Figure Lengend Snippet: 5HT-Hi promotes growth cone repulsion through the 5-HT1b receptor. a Growth cone repulsion was measured in the presence of 5HT-Hi gradients (p = <0.0001) and CP-94253 gradients (p = 0.0018). Growth cone repulsion to 5HT-Hi was altered to levels indistinguishable from random growth (vehicle) with application of the selective antagonist to 5-HT1b, GR55562 (n = 16, p = <0.0001), by activating adenylate cyclase with forskolin (n = 8, p = 0.0003) and restoration of cAMP signals with Sp-cAMPs (n = 8, p = 0.0001). b Repulsion to 5HT-Hi was not perturbed when growth cones were treated with the 5-HT2a receptor antagonist ritanserin (n = 8, p = 0.002) or the 5-HT3 antagonist ondansetron (n = 10, p = 0.0003). Inhibition of ER calcium release with thapsigargin (n = 10, p = <0.0001) or calcium influx with nifedipine (n = 8, p = <0.0001) did not alter growth cone repulsion from 5HT-Hi. Turning angles were compared to vehicle and 5HT-Hi. c, d Axon extension was not perturbed by any pharmacological application (ns, p > 0.05). Turning angles were compared to vehicle and 5HT-Hi. (Kruskal–Wallis, Dunn’s multiple comparison test)

Article Snippet: DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Inhibition, Hi-C, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Serotonin functions as a bidirectional guidance molecule regulating growth cone motility

doi: 10.1007/s00018-020-03628-2

Figure Lengend Snippet: 5HT-Lo exposure causes asymmetric distribution of 5-HT receptors to the turning or motile side of growth cones. Schematic a showing growth cone divided into “near” and “far” regions with respect to micropipet position (located on the upper left side). b–d Growth cones immunostained after turning in response to 5HT-Lo and stained for 5-HT2a (green, b), 5-HT1b (red, c) and merged in (d). Dotted line separates the near and far regions of growth cone (near vs far). (e) Representative image of filopodia with 5-HT2a (green), 5-HT1b (red) receptor puncta. (f) There was no significant near/far bias in the number of filopodia following exposure to 5HT-Lo (n = 8, p = 0.9004), 5HT-Hi (n = 13, p = 0.5450) or BDNF (n = 10, p = 0.7907) compared to vehicle. (Data not normally distributed: Kruskal–Wallis, Dunn’s multiple comparison test). g There were significantly more (n = 36, p = 0.0033) 5-HT2a puncta in filopodia on the near side of growth cones exposed to 5HT-Lo compared to all other treatments. h There was no bias in 5-HT1b puncta in filopodia of growth cones. (Mann–Whitney U-test). (i-j) When entire growth cones were analysed, i 5HT-2a (n = 9, p = 0.0006) and j 5HT-1b (n = 9, p = 0.0028) receptor translocation was significantly biased in growth cones exposed to 5HT-Lo. All ratios were compared to vehicle (Data normally distributed: Shapiro–Wilk. One-way ANOVA, Tukey’s multiple comparison test). Scale bars are 5 μm (Fig. 7b–d) and 1 μm (Fig. 7e)

Article Snippet: DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Staining, Comparison, MANN-WHITNEY, Translocation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Serotonin functions as a bidirectional guidance molecule regulating growth cone motility

doi: 10.1007/s00018-020-03628-2

Figure Lengend Snippet: 5HT-Lo exposure causes asymmetric distribution of membrane 5-HT2a receptors to the motile side of growth cones. a–h Representative growth cones exposed to vehicle and 5HT-Lo gradients (micropipette on upper left side) and stained for membrane localization of receptors 5-HT1b (green, a, e), 5-HT2a (green, c, g) and merged with actin, (red, b, d, f, h). Dotted line separates the near and far regions of the growth cone. i Increase magnification of representative image h to show the presence of 5-HT2a receptor membrane on most filopodia (*) of the growth cone (yellow *, insets). Scale bars are 5 μm. (j-k)There was no significant near/far bias in the amount of f-actin (/area) (j) and the number of filopodia (k) in growth cones exposed to vehicle and 5HT-Lo. Kruskal–Wallis, Dunn’s multiple comparison post hoc test. (l) There was no significant bias in the total percentage of filopodia containing 5HT2a and 5HT1b puncta (60%). One-way ANOVA followed by Tukey’s multiple comparison post hoc test. (m) There was no significant near/far bias in the amount of 5-HT2a and 5-HT1b puncta per filopodia. (n) Analysis of 5-HT receptor membrane localization showed translocation of 5-HT2a (n = 18, p = <0.0001) to the near side of growth cones exposed to 5HT-Lo while no significant translocation of 5-HT1b (n = 15, p = 0.346) was observed. (Mann–Whitney U-test)

Article Snippet: DRG sensory neurons were fixed with paraformaldehyde (PFA) (4%, Sigma-Aldrich) in phosphate buffered saline (PBS) overnight at 4 °C, washed in PBS (3 × 10 min), for some experiments, cells were permeabilised for 1 h with blocking solution (PBS containing 0.4% Triton X-100; 5% goat serum, Sigma-Aldrich & Gibco) and immunostained with a rabbit antibody to serotonin receptors 5-HT1b (1:500; Abcam) or 5-HT2a (1:500; Alomone Labs) in blocking solution overnight at 4 °C.

Techniques: Membrane, Staining, Comparison, Translocation Assay, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of coronaviral exoribonuclease activity by TRIM-mediated SUMOylation

doi: 10.1073/pnas.2528398123

Figure Lengend Snippet: TRIM32 restricts SARS-CoV-2 replication in a RING-dependent but IFN-independent manner. ( A ) Validation of CRISPR TRIM32- KO Caco-2 cells. TRIM32 protein abundance in TRIM32 -KO Caco-2 single cell clones (cl-1 and cl-2) and WT control cells, determined in the whole cell lysates (WCLs) by immunoblot (IB) analysis with anti-TRIM32 and anti-Actin (loading control). The asterisk indicates nonspecific band. ( B ) Validation of CRISPR TRIM32- KO Caco-2 cells (cl-1 and cl-2) by assessing TRIM32 transcripts by RT-qPCR. Values indicate relative mRNA (fold), normalized to cellular HPRT1 . ( C ) Virus replication kinetics over a 72-h period in TRIM32 -KO and WT (control) Caco-2 cells that were infected with EGFP-rSARS-CoV-2 (strain K49; MOI 0.02) or mock-treated, determined by measuring RFU for EGFP. Values were autoscaled to the average values for mock-infected cells at 0 h, set to 200. ( D ) SARS-CoV-2 titers in the supernatant of TRIM32 -KO and WT (control) Caco-2 cells that were infected with SARS-CoV-2 (strain WA1; MOI 0.02) for 48 h and 60 h, determined by plaque assay. ( E ) SARS-CoV-2 spike (S) and nucleocapsid (N) protein abundances in TRIM32 -KO and WT Caco-2 cells that were mock-treated (−) or infected for 48 h as in ( D ), determined in the WCLs by IB with anti-S and anti-N. ( F ) TRIM32 protein expression in stably reconstituted TRIM32 -KO Caco-2 cells that inducibly [via 2 μg/mL doxycycline (DOX) treatment for 24 h] expressed either empty vector or FLAG-tagged TRIM32 WT or ΔRING, determined in the WCLs by IB analysis. Stable WT Caco-2 cells expressing empty vector (termed “WT control”) served as control. The asterisk indicates nonspecific band. ( G ) Virus replication kinetics over a 72-h period in TRIM32 -KO Caco-2 cells reconstituted as indicated and either mock-treated or infected with EGFP-rSARS-CoV-2 (strain K49; MOI 0.02), determined by measuring RFU for EGFP. Values were autoscaled as in ( C ). WT control cells were included for comparison. ( H ) SARS-CoV-2 g. Nsp3 transcripts in TRIM32 -KO Caco-2 cells reconstituted as indicated and either mock-treated (−) or infected with SARS-CoV-2 (strain WA1; MOI 0.02) for 48 h, determined by RT-qPCR. ( I and J ) SARS-CoV-2 g. Nsp3 and sg. N&Orf9b transcripts in stable Caco-2 cells that inducibly (by 2 μg/mL DOX treatment) expressed either empty vector (Control Caco-2) or TRIM32 WT (Caco-2 TRIM32) and were infected with SARS-CoV-2 (strain WA1; MOI 0.05) for 48 h in the absence or presence of anti-IFNAR2 antibody (2 µg/mL), determined by RT-qPCR. Data are representative of at least two independent experiments [mean ± SD of n = 3 biological replicates ( B – D and G – J )]. * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t test). ns, not significant.

Article Snippet: Anti-TRIM32 antibody (10326-1-AP, Proteintech or GTX113937, GeneTex) was used at a dilution of 1:1,000.

Techniques: Biomarker Discovery, CRISPR, Quantitative Proteomics, Single Cell, Clone Assay, Control, Western Blot, Quantitative RT-PCR, Virus, Infection, Plaque Assay, Expressing, Stable Transfection, Plasmid Preparation, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of coronaviral exoribonuclease activity by TRIM-mediated SUMOylation

doi: 10.1073/pnas.2528398123

Figure Lengend Snippet: TRIM32 binds to and SUMOylates SARS-CoV-2 Nsp14. ( A ) Endogenous TRIM32 binding to Nsp14 in Caco-2 cells that were either mock-treated (−) or infected with SARS-CoV-2 (strain WA1; MOI 1) for 48 h, determined by IP with anti-TRIM32 and IB with anti-Nsp14. ( B ) Schematic representation of TRIM32’s domain organization as well as the truncation constructs used in the interaction-mapping experiments. RING, really interesting new gene; BB, B-box; CC, coiled-coil; NHL, NCL-1, HT2A, Lin-41 domain. mutSIM #1 and #2 indicate TRIM32 mutants in which the two identified SIMs are mutated individually. Mutated residues are shown in red. Numbers indicate amino acids. ( C ) Binding of FLAG-tagged Nsp14 (SARS-CoV-2) to V5-tagged TRIM32 WT or truncation proteins in transiently transfected HEK293T cells, assessed by PD with anti-V5 (PD: V5), followed by IB with anti-FLAG. WCLs were probed with anti-FLAG and anti-Actin (loading control). ( D ) SUMOylation of FLAG-tagged Nsp14 (SARS-CoV-2) in HEK293T cells that were cotransfected for 40 h with V5-tagged TRIM32 WT or ΔRING, determined by PD: FLAG and IB with anti-pan-SUMO. ( E ) SUMOylation of FLAG-tagged Nsp14 (SARS-CoV-2) in transiently transfected HEK293T cells that coexpressed either empty vector or V5-tagged TRIM32 WT or ΔRING, along with UBC9-HA and Myc-tagged SUMO1, SUMO2, or SUMO3, determined by PD: FLAG and IB with anti-Myc. ( F ) SUMOylation of Nsp14 in stable Caco-2 cells inducibly expressing FLAG-TRIM32 that were infected for 32 h with SARS-CoV-2 (strain WA1; MOI 2) and then treated for 12 h with DOX (2 µg/mL) to induce TRIM32 expression, assessed by denaturing IP with anti-SUMO2/3 and IB with anti-Nsp14. Hc, antibody heavy chain. The asterisk indicates nonspecific band. ( G ) SUMOylation of FLAG-tagged Nsp14 (SARS-CoV-2) in HEK293T cells that were cotransfected for 40 h with either empty vector or V5-tagged TRIM32 WT or mutants (ΔRING or mutSIM #1 or #2) together with Myc-tagged SUMO2 and HA-tagged UBC9, determined by PD: FLAG and IB with anti-Myc. Data ( A and C – G ) are representative of at least two independent experiments.

Article Snippet: Anti-TRIM32 antibody (10326-1-AP, Proteintech or GTX113937, GeneTex) was used at a dilution of 1:1,000.

Techniques: Binding Assay, Infection, Construct, Transfection, Control, Plasmid Preparation, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of coronaviral exoribonuclease activity by TRIM-mediated SUMOylation

doi: 10.1073/pnas.2528398123

Figure Lengend Snippet: The Nsp14 ExoN domain is SUMOylated at K9 and K200. ( A ) Schematic representation of the affinity purification-mass spectrometry (AP-MS) approach to identify the SUMOylation sites in SARS-CoV-2 Nsp14 as well as Coomassie blue-stained gel of affinity-purified Nsp14. HEK293T cells were transiently transfected with FLAG-tagged Nsp14, V5-tagged TRIM32, HA-tagged UBC9, and Myc-tagged SUMO2 T91R mutant. 40 h later, Nsp14 was affinity-purified by PD: FLAG, followed by SDS-PAGE and Coomassie blue staining ( Center ). Unmodified Nsp14 (arrowhead) and modified Nsp14 versions are indicated. Nsp14 protein was subjected to digestion using trypsin, and peptides modified by SUMO2 T91R Lys-Gly-Gly (KGG) signatures were analyzed using LC–MS/MS. The asterisk indicates antibody heavy chain. Parts of this figure were created using Biorender.com. ( B ) Top : Schematic representation of Nsp14 domain organization with the SUMOylated lysines (Ks) identified by MS analysis indicated. ExoN, exoribonuclease; N7-MTase, N7-methyltransferase. Bottom : Mass spectra of the tryptic peptides of affinity-purified FLAG-tagged Nsp14 (SARS-CoV-2) that identified SUMOylation at K9 and K200, with designated b- and y-ions. Ace denotes acetylated N terminus. ( C ) SUMOylation of FLAG-tagged Nsp14 (SARS-CoV-2) WT or indicated mutants in transiently transfected HEK293T cells that were cotransfected for 40 h with either empty vector or TRIM32-V5 together with UBC9-HA and Myc-SUMO2, determined by PD: FLAG and IB with anti-Myc. ( D ) Binding of FLAG-tagged Nsp14 (SARS-CoV-2) WT or mutants in transiently transfected HEK293T cells that were cotransfected for 40 h with TRIM32-V5, determined by PD: FLAG and IB with anti-V5. (E) Representation of the cryo-EM structure of the SARS-CoV-2 Nsp10–Nsp14–RNA complex (PDB: 7N0B) with Nsp14 (cyan), RNA (orange), and Nsp10 (green). K9 and K200 are depicted in red using spheres. Bound zinc ions are depicted as gray spheres. Data are representative of at least two independent experiments ( C and D ) or are from one unbiased AP-MS analysis ( A and B ).

Article Snippet: Anti-TRIM32 antibody (10326-1-AP, Proteintech or GTX113937, GeneTex) was used at a dilution of 1:1,000.

Techniques: Affinity Purification, Mass Spectrometry, Protein-Protein interactions, Staining, Transfection, Mutagenesis, SDS Page, Modification, Ubiquitin Proteomics, Liquid Chromatography with Mass Spectroscopy, Plasmid Preparation, Binding Assay, Cryo-EM Sample Prep

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of coronaviral exoribonuclease activity by TRIM-mediated SUMOylation

doi: 10.1073/pnas.2528398123

Figure Lengend Snippet: ExoN SUMOylation by TRIM32 inhibits RNA and Nsp10 binding, suppressing ExoN activity. ( A ) Schematic diagram showing the approach to assess the effect of TRIM32-mediated SUMOylation of Nsp14 on its ExoN activity and RNA–substrate binding. FLAG-tagged Nsp14 (SARS-CoV-2) or vector control was transfected into HEK293T cells together with either empty vector or V5-tagged TRIM32 (WT, ΔRING, or mutSIM) and Myc-SUMO2 and UBC9-HA, followed by affinity purification of Nsp14-FLAG by PD: FLAG and subsequent elution using 3X FLAG peptide. One portion of purified Nsp14-FLAG was incubated in vitro with recombinant His-tagged Nsp10 and FAM-labeled RNA substrate (CoV-RNA1-A) to analyze ExoN activity ( Lower Center ). Furthermore, purified Nsp14-FLAG was used for in vitro RNA binding assay with biotinylated CoV-RNA1-A (Biotin-CoV-RNA1-A) and subjected to PD: Strep ( Lower Right ). Purified Nsp14-FLAG was also analyzed by IB for SUMOylation ( Lower Left ). Parts of this figure were created using Biorender.com. ( B ) In vitro ExoN activity of SARS-CoV-2 Nsp14 purified from cells that coexpressed vector or TRIM32 (WT or mutants) and the indicated SUMOylating machinery components and then incubated in vitro with 5′-FAM-labeled CoV-RNA1-A, determined as described in ( A ). ( C ) Structural modeling of SARS-CoV-2 Nsp14 with K9 and K200 SUMOylated. Nsp10 (light green) and RNA (orange) from the Nsp10–Nsp14–RNA cryo-EM structure (PDB: 7N0B) are depicted using surface representation showing steric clashes with SUMO molecules (K9-SUMO and K200-SUMO in Nsp14 (cyan)). ( D ) RNA binding of SARS-CoV-2 Nsp14 purified from cells coexpressing vector or TRIM32 (WT or mutants) and the indicated SUMOylating machinery components and incubated in vitro with biotinylated CoV-RNA1-A (or control RNA), determined by PD: Strep and IB with anti-FLAG. ( E ) SARS-CoV-2 Nsp14 binding to Nsp10 in HEK293T cells that were transfected for 40 h with Nsp14-FLAG, Nsp10-Strep II, UBC9-HA, Myc-SUMO2, and either empty vector or V5-tagged TRIM32 WT or mutants, determined by PD: FLAG and IB with anti-Strep II. Nsp14 SUMOylation was assessed in parallel by PD: FLAG and IB with anti-Myc. ( F ) SARS-CoV-2 replication in stable Caco-2 cells that inducibly (by 2 µg/mL DOX treatment) expressed empty vector (Control Caco-2) or WT TRIM32 (Caco-2 TRIM32) and were transfected for 40 h with si.C, si.UBC9, or si.SUMO2, followed by infection with SARS-CoV-2 (strain WA1; MOI 0.02). At 48 h postinfection, SARS-CoV-2 g. Nsp3 and sg. N&Orf9b transcripts were determined by RT-qPCR. ( G ) SARS-CoV-2 replication in stable Caco-2 TRIM32 cells that were infected with SARS-CoV-2 (strain WA1; MOI 0.02) for 48 h in the presence of 2.5 µM TAK-981 or DMSO (−), determined by RT-qPCR of the indicated viral g/sgRNAs. Control Caco-2 cells were included for comparison. Data ( B and D – G ) are representative of at least two independent experiments [mean ± SD of n = 3 biological replicates ( F and G )]. ** P < 0.01, *** P < 0.001 (Student’s t test). ns, not significant.

Article Snippet: Anti-TRIM32 antibody (10326-1-AP, Proteintech or GTX113937, GeneTex) was used at a dilution of 1:1,000.

Techniques: Binding Assay, Activity Assay, Plasmid Preparation, Control, Transfection, Affinity Purification, Purification, Incubation, In Vitro, Recombinant, Labeling, RNA Binding Assay, Cryo-EM Sample Prep, Infection, Quantitative RT-PCR, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inhibition of coronaviral exoribonuclease activity by TRIM-mediated SUMOylation

doi: 10.1073/pnas.2528398123

Figure Lengend Snippet: Nsp14 SUMOylation and virus restriction by TRIM32 are broadly conserved for coronaviruses. ( A ) Sequence alignment of the Nsp14 protein regions containing K9 and K200 from the indicated coronaviruses, determined by ClustalW. Asterisks indicate positions which have a single, fully conserved residue; colons illustrate conservation between groups of strongly similar properties; periods indicate conservation between groups of weakly similar properties. ( B ) SUMOylation of Nsp14 from the indicated coronaviruses in HEK293T cells that were transiently transfected with plasmids expressing FLAG-tagged coronaviral Nsp14s together with UBC9-HA, Myc-SUMO2 and either vector or V5-tagged TRIM32 (WT or ΔRING), assessed by PD: FLAG and IB with anti-Myc. ( C ) TRIM32 binding of the indicated coronaviral Nsp14 proteins in HEK293T cells that were transiently transfected with plasmids expressing FLAG-tagged coronaviral Nsp14s (or with empty vector) together with V5-tagged TRIM32, determined by PD: FLAG and IB with anti-V5. FLAG-tagged Nsp10 (SARS-CoV-2) served as control. ( D–F ) MERS-CoV ( D ), HCoV-OC43 ( E ), and MHV ( F ) replication in TRIM32 -KO Caco-2 single cell clones (cl-1 and cl-2) or WT control cells that were either mock-treated (−) or infected with virus as indicated (MOI 0.02 for each) for 48 h, determined by RT-qPCR of sg. M or s g.N transcripts. ( G–I ) MERS-CoV ( G ), HCoV-OC43 ( H ), and MHV ( I ) replication in stable Caco-2 cells that inducibly (by 2 µg/mL DOX) expressed TRIM32 (Caco-2 TRIM32) and were transfected for 40 h with si.C, si.UBC9 or si.SUMO2, followed by virus infection (MOI 0.02 for each) for 48 h, determined by RT-qPCR of sg. M or s g.N transcripts. Control Caco-2 cells were included for comparison. ( J ) Representative knockdown efficiency of endogenous UBC9 and SUMO2 for the experiments in ( G – I ), determined by RT-qPCR. Data ( B – J ) are representative of at least two independent experiments (mean ± SD of n = 3 biological replicates ( D – J ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Student’s t test).

Article Snippet: Anti-TRIM32 antibody (10326-1-AP, Proteintech or GTX113937, GeneTex) was used at a dilution of 1:1,000.

Techniques: Virus, Sequencing, Residue, Transfection, Expressing, Plasmid Preparation, Binding Assay, Control, Single Cell, Clone Assay, Infection, Quantitative RT-PCR, Comparison, Knockdown

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 1 TRIM32 expression is decreased in failing human hearts and hypertrophic mouse hearts (A and B) Representative Western blots (A) and quantitative results (B) of ANP, β-MHC and TRIM32 protein expression in normal donor hearts and in human hearts with DCM (n = 8 hearts per experimental group; *P < 0.05 compared with donor hearts). (C and D) Representative Western blots (C) and quantitative results (D) of ANP, β-MHC and TRIM32 protein expression in a hypertrophic mouse heart induced by AB for the times indicated (W, weeks) (n = 6 mice per experimental group; *P < 0.05 compared with sham). (E and F) Representative Western blots (E) and quantitative results (F) of ANP, β-MHC and TRIM32 protein expression in cultured NRCMs treated with AngII for 24 or 48 h (n = 4 samples per experimental group; four independent experiments; *P < 0.05 compared with PBS). Results are means +−S.D., and statistical analyses were determined using an unpaired Student’s t test. Molecular masses are indicated in kDa in the Western blot panels.

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Expressing, Western Blot, Cell Culture

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 2 TRIM32 attenuates AngII-induced cardiomyocyte hypertrophy in vitro (A) The protein expression level of TRIM32 in NRCMs after infection with AdshRNA, AdshTRIM32, AdGFP or AdTRIM32 was determined by Western blotting (n = 4 samples per experimental group). Left: representative blots. Right: quantitative results. (B) Representative images of NRCMs infected with AdshRNA, AdshTRIM32, AdGFP or AdTRIM32 and treated with AngII (1 μmol/l) or PBS for 48 h (n = 4 samples per experimental group; blue, nucleus; green, α-actinin; scale bar, 20 μm). (C) Quantitative results of the cell surface area of NRCMs infected with AdshTRIM32 or AdshRNA subjected to AngII (1 μmol/l) or PBS for 48 h (n⩾50 cells per experimental group; *P < 0.05 compared with AdshRNA/PBS; #P < 0.05 compared with AdshRNA/AngII). (D) Quantitative results of the cell surface area of NRCMs infected with AdTRIM32 or AdGFP subjected to AngII (1 μmol/l) or PBS for 48 h (n⩾50 cells per experimental group; *P < 0.05 compared with AdGFP/PBS; #P < 0.05 compared with AdGFP/AngII). (E) Relative mRNA levels of ANP, BNP and β-MHC in AdshTRIM32-infected NRCMs

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: In Vitro, Expressing, Infection, Western Blot

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 3 TRIM32 overexpression attenuates pressure overload-induced cardiac hypertrophy and heart failure (A) Schematic diagram of the construction of TG mice with a full-length murine Trim32 cDNA under the control of the α-MHC promoter. (B and C) Representative Western blots (B) and quantitative results (C) of TRIM32 protein expression in heart tissue from four TG lines and MCT mice (n = 3 mice per experimental group; three independent experiments).

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Over Expression, Control, Western Blot, Expressing

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 4 Generation of global TRIM32-deficient mice (A) One sgRNA targeted a region downstream of the 5′ end of exon 2 in the Trim32 mouse gene. (B) Representative results of the T7E1 assay from pups subjected to microinjection. Sizes are indicated in bp. (C) Representative results of the DNA sequencing from mice with frameshift mutations. (D) Agarose gel photograph illustrating genotyping results of PCR products from WT (+/+), heterozygous (+/−) and TRIM32-KO (−/−) mice. Sizes are indicated in bp. (E) Representative Western blots of TRIM32 expression in heart tissues from global TRIM32-KO mice and their littermate controls (n = 4 mice per experimental group). Molecular masses are indicated in kDa.

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Microinjection, DNA Sequencing, Agarose Gel Electrophoresis, Western Blot, Expressing

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 5 TRIM32 deficiency aggravates pressure overload-induced cardiac hypertrophy and heart failure (A) Statistical results for HW/BW and HW/TL in WT mice and TRIM32-KO mice at 4 weeks after sham or AB (n = 8–10 mice per experimental group). (B) Representative images of gross morphology, H&E staining, WGA staining and PSR staining of hearts from the indicated groups (n = 5 mice per experimental group; scale bar, 20 μm for lower H&E staining,

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Staining

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 6 TRIM32 inhibits activation of Akt-dependent signalling pathways upon hypertrophic stresses

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Activation Assay

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 7 Blockage of Akt-dependent signalling pathways rescues pressure overload-induced cardiac abnormalities in TRIM32-KO mice (A and B) Representative Western blots (A) and quantitative results (B) of total (T-) and phospho- (P-) Akt-dependent signalling pathway protein levels in the hearts from LY294002- or DMSO-treated TRIM32-KO mice 4 weeks after AB surgery (n = 4 mice per experiment group; GAPDH was used as a loading control). Molecular masses are indicated in kDa in (A). (C) Statistical results for HW/BW and HW/TL in the indicated groups (n = 8–10 mice per experimental group). (D) Representative images of gross morphology, H&E staining and PSR staining from hearts in the indicated groups at 4 weeks after AB (n = 5 mice per experimental group; scale bar, 20 μm for lower H&E staining and PSR staining). (E) Statistical results for the cardiomyocyte cross-sectional area in the indicated groups (n⩾100 cells per experimental group). (F) Quantification of left ventricular collagen volume in the indicated groups (n⩾25 fields per experimental group). (G) Measurements of LVESD, LVEDD and FS in the indicated groups (n = 5 mice per experimental group). *P < 0.05 compared with KO/AB/DMSO; n.s. indicates no significant difference. Results are means +−S.D., and statistical analyses were performed using an unpaired Student’s t test or one-way ANOVA.

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Western Blot, Control, Staining

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 is selectively expressed in inner EGL and displays an uneven cytoplasmic distribution during GNP differentiation. a RT-qPCR analysis of Trim32 in P0, P7, P14, and adult mouse cerebella. Data are expressed as means ± SD (n = 3). An asterisk indicates P < 0.05 and triple asterisks indicate P < 0.001. b Immunoblotting analysis of Trim32, Gli1, and MycN in P0, P7, P14, and adult mouse cerebellums. c Immunofluorescence staining of Trim32 (red) in the EGL of P7 Math1-GFP transgenic mouse cerebellum. Nuclei were counterstained with DAPI (blue). EGL external granule layer, ML molecular layer, PCL Purkinje cells layer, and IGL internal granule layer. The scale bars represent 100 µm in the first panel and 25 µm in the second panel. d Confocal analysis (left) and quantification of immunofluorescence intensities (right) of distribution of Trim32 (green) in the dividing GNPs in different phases of the cell cycle. The dashed line highlights the cells that are in the indicated phase of cell cycle. The cell-cycle phases were identified by PH3 staining (red) for DNA. Nuclei were counterstained with DAPI (blue). The scale bars represent 10 µm. Data are expressed as means ± SD (n = 3). ns indicates P > 0.05 and triple asterisks indicate P < 0.001

Article Snippet: The human Trim32 cDNA and GLi1 cDNA plasmid were ordered from OriGene (#RC201289L2, #RC201110).

Techniques: Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Transgenic Assay

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 knockout enhances GNP proliferation in the postnatal developing cerebellum. a Expression of the Math1-GFP protein (GFP, green) in P7 mouse cerebellar sections from the Math1-GFP/Trim32wt mice and Math1-GFP/Trim32KO mice. Nuclei were counterstained with DAPI (blue). Graph in a representing Math1 positive cells normalized to the length of the EGL edge. The scale bar represents 25 µm. Immunofluorescence staining of NeuN (red, b) and Ki67 (red, c) in P7 mouse cerebellar sections from the Math1-GFP/Trim32wt mice and Math1-GFP/Trim32KO mice. Nuclei were counterstained with DAPI (blue). Graph in b and c respectively representing NeuN or Ki67-positive cells normalized to the length of the EGL edge. The scale bar in b represents 50 µm. The scale bar in c represents 25 µm. oEGL outer external granule layer, iEGL inner external granule layer, ML molecular layer, and IGL internal granule layer. d P7 and P18 cerebellar midsagittal sections were stained for DAPI to show the overall morphology of cerebellum in Trim32wt mice and Trim32ko mice. The scale bar represents 500 µm

Article Snippet: The human Trim32 cDNA and GLi1 cDNA plasmid were ordered from OriGene (#RC201289L2, #RC201110).

Techniques: Knock-Out, Expressing, Immunofluorescence, Staining

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 antagonizes the SHH signaling activity. a RT-qPCR analysis of Trim32, the granule neuronal progenitor marker Maht1, and SHH target genes in P7 cerebellar granule neuronal progenitors (cGNPs) from Trim32wt mice and Trim32KO mice. Data are expressed as means ± SD (n = 3). Double asterisks indicate P < 0.01 and triple asterisks indicate P < 0.001. b Immunoblotting analysis of Trim32, Gli1, Ccnd1, Ccnd2, and MycN in P7 mouse cerebellum from Trim32wt mice and Trim32KO mice. c, d RT-PCR mRNA expression of the SHH target genes Gli1 and MycN in HEK293T cells overexpressing Trim32-GFP or GFP control vector. The cells were cultured with or without SHH for 24 and 48 h. Data are expressed as means ± SD (n = 3). Double asterisks indicate P < 0.01 and triple asterisks indicate P < 0.001. Gli-RE-luciferase activity in human medulloblastoma cell line D283 (e) and HEK293T cells (f), following transfected the indicated vectors. The luciferase activity was evaluated relative to Renilla activity. The means ± SD from three experiments are shown

Article Snippet: The human Trim32 cDNA and GLi1 cDNA plasmid were ordered from OriGene (#RC201289L2, #RC201110).

Techniques: Activity Assay, Quantitative RT-PCR, Marker, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Plasmid Preparation, Cell Culture, Luciferase, Transfection

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 interacts with Gli1 though the NHL domain. a In vivo assay for binding between Trim32 and Gli1. Expression vectors encoding TRIM32-GFP and Flag-tagged Gli1 were transfected into HEK293T cells. Whole cell lysates were immunoprecipitated with anti-Trim32 or anti-FLAG antibody and immunoblotted with anti-Trim32 and anti-Gli1 antibodies. b Co-immunoprecipitation of exogenously expressed Trim32-HA with the endogenous Gli1 in HEK293T cells. c Co-immunoprecipitation of exogenously expressed Trim32-GFP with the Gli1 truncated mutants. d Schematic drawing of full-length Trim32 (Trim32-wt) and deletion mutants. Trim32 contains a RING finger (R), two B-boxes (B), a coiled-coil region (Coil), and an NHL domain (NHL). e Co-immunoprecipitation of exogenously expressed Flag-tagged Gli1 with the Trim32 truncated mutants shown as in d

Article Snippet: The human Trim32 cDNA and GLi1 cDNA plasmid were ordered from OriGene (#RC201289L2, #RC201110).

Techniques: In Vivo, Binding Assay, Expressing, Transfection, Immunoprecipitation

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 promotes Gli1 ubiquitination and degradation. a HEK293T cells transfected with Gli1 and increasing doses of Trim32 were collected for immunoblotting with the indicated antibodies. b Quantification of Gli1 and Trim32 protein levels (normalized to actin) from a. The means ± SEM from three experiments are shown. c Cells stably expressing scramble RNA or shRNA targeting Trim32 (#1, #2) were harvested for immunoblotting with the indicated antibodies. d HEK293T cells were transfected with plasmids encoding Flag-tagged Gli1 and control GFP vector or Trim32 or Trim32-ΔR, treated with 20 µg/ml cycloheximide (CHX) and harvested at the indicated times points. The levels of Gli1 and Trim32 in the lysates were investigated by immunoblotting. e Quantification of Gli1 remaining protein levels (normalized to actin) from d. The means ± SEM from three experiments are shown. f Cells were transfected with scramble RNA or shRNA targeting Trim32 (#1), treated with 20 µg/ml cycloheximide (CHX), collected at the indicated time points, and then immunoblotted with the indicated antibodies. g Quantification of Gli1 protein levels (normalized to actin) from f. The means ± SEM from three experiments are shown. h Cells transfected with Gli1 or Trim32 were treated with or without 10 µM MG132 for 8 h, collected, and then immunoblotted with the indicated antibodies. Graph representing quantification of Gli1 protein levels (normalized to actin). i Anti-ubiquitin immunoblotting of immunoprecipitated exogenous Gli1 in HEK293T cells transfected with the indicated plasmids. j The effect of Trim32-wt or Trim32 deletion mutant (Trim32-ΔR) to the activation of Gli-RE-luciferase induced by Gli1 in HEK293T cells. The means ± SD from three experiments are shown. Double asterisks indicate P < 0.01

Article Snippet: The human Trim32 cDNA and GLi1 cDNA plasmid were ordered from OriGene (#RC201289L2, #RC201110).

Techniques: Transfection, Western Blot, Stable Transfection, Expressing, shRNA, Plasmid Preparation, Immunoprecipitation, Mutagenesis, Activation Assay, Luciferase

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 knockout increases the incidence of MB in Ptch1+/− mice. Quantitative PCR mRNA (a; mean ± SD; n = 9) and protein levels (b; n = 3) of Trim32 in mouse medulloblastomas (MB) from Ptch1+/− mice and normal cerebella. c RNA expression level of Trim32 negatively correlated with Gli1 (n = 51) in human SHH MB samples. Data are analyzed from Oncomine database. d Kaplan–Meier analysis of MB incidence in 63 Ptch1+/−/Trim32wt mice (gray line) versus 17 Ptch1+/−/Trim32KO mice (black line). Ptch1+/−/Trim32wt mice and Ptch1+/−/Trim32KO mice, obtained by interbreeding for at least three generations the progeny of Ptch1 heterozygous and Trim32 knock-out mice, were then monitored for the onset of medulloblastoma; (triple asterisks indicate P < 0.001, Logrank test). e Expression of the Math1-GFP protein (GFP, green) in the Math1-GFP mouse medulloblastomas and adjacent cerebellar cortices sections from Ptch1+/−/Trim32wt mice and Ptch1+/−/Trim32KO mice. Nuclei were counterstained with DAPI (blue). The scale bar represents 200 µm. MB medulloblastoma, IGL internal granule layer. RT-qPCR analysis of SHH target genes (f), Trim32 (g), the granule neuronal progenitor marker Maht1 (h), and the differentiated granule cell markers including Tuj1 and NeuN (i) in adult mouse cerebellums and medulloblastomas from Ptch1+/−/Trim32wt mice and Ptch1+/−/Trim32KO mice. Data are expressed as means ± SD (n = 3). An asterisk indicates P < 0.05, double asterisks indicate P < 0.01, and triple asterisks indicate P < 0.001. j Immunoblotting analysis of Trim32, Gli1, Ccnd2, MycN, and NeuN in MB from Ptch1+/−/Trim32wt mice and Ptch1+/−/Trim32KO mice

Article Snippet: The human Trim32 cDNA and GLi1 cDNA plasmid were ordered from OriGene (#RC201289L2, #RC201110).

Techniques: Knock-Out, Real-time Polymerase Chain Reaction, RNA Expression, Expressing, Quantitative RT-PCR, Marker, Western Blot

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: mRNA expression analysis

Article Snippet: The human Trim32 cDNA and GLi1 cDNA plasmid were ordered from OriGene (#RC201289L2, #RC201110).

Techniques: Expressing

Journal: Frontiers in Psychiatry

Article Title: Histone deacetylase inhibitors mitigate antipsychotic risperidone-induced motor side effects in aged mice and in a mouse model of Alzheimer’s disease

doi: 10.3389/fpsyt.2022.1020831

Figure Lengend Snippet: Effects of histone deacetylase (HDAC) inhibitors on histone acetylation at the 5htr2a and Drd2 gene promoters in the striatum in young and aged mice. (A) No significant differences of H3K27ac binding at the 5htr2a promoter between young and aged group was found, but a significant increase in H3K27ac binding at the 5ht2a promoter in the striatum was found in aged and young mice co-treated with RIS and VPA or MS-275 compared to those treated with RIS alone. (B) H3K27ac binding at the Drd2 promoter was significantly decreased in aged mice as compared to young mice in Veh treated group. However, H3K27ac binding at the Drd2 promoter in the striatum was observed to be significantly increased in aged mice co-treated with RIS and HDAC inhibitors VPA or MS-275 when compared to those treated with Veh alone. Data are presented as mean ± SEM, ( n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Blots were blocked for 1-hour with 5% non-fat dry milk (Biorad, 1706404) and incubated using 1:1,000 5HT2A (Boster Biological, PA1373), 1:500 D2R (Millipore, AB5084P), or 1:1000 B-actin (Santa Cruz, sc-47778) overnight at 4°C.

Techniques: Histone Deacetylase Assay, Binding Assay

Journal: Journal of Microbiology and Biotechnology

Article Title: The Effect of Lactobacillus gasseri BNR17 on Postmenopausal Symptoms in Ovariectomized Rats

doi: 10.4014/jmb.2105.05032

Figure Lengend Snippet: Serum estradiol (E2) ( A ), Calcitonin ( B ), Osteocalcin ( C ), and 5-HT-2A ( D ) were measured after 14 weeks of initial administration ( n = 6~8 rats per group). Sophora japonica fruit extract (SJFE) treatment was used to create the positive control.

Article Snippet: The concentrations of estradiol (E2, CSB-E05110r), osteocalcin (CSB-E05129r), calcitonin (CSB-E05132r), estradiol (CSB-E05110r), and Serotonin 2A (5-HT-2A, CSB-E14956r) were assessed using an ELISA kit (CUSABIO, China).

Techniques: Positive Control