Journal: Cell communication and signaling : CCS

Article Title: BMX, a specific HDAC8 inhibitor, with TMZ for advanced CRC therapy: a novel synergic effect to elicit p53-, β-catenin- and MGMT-dependent apoptotic cell death.

doi: 10.1186/s12964-022-01007-x

Figure Lengend Snippet: Fig. 1 BMX, TMZ, oxaliplatin (Oxp) and doxorubicin (Dox) combination inhibited cell proliferation in CRC cells. (A) The proliferation of BMX, TMZ, Oxp, Dox, BMX plus TMZ, BMX plus Oxp or BMX plus Dox in HT29, HCT116 and RKO cells with various drug concentration. (B) Colony formation capability assay with different treatments of BMX, TMZ, Oxp, BMX plus TMZ, and BMX plus Oxp in HT29, HCT116 and RKO cells; the colonies were counted for quantification. (C) Cell cycle analysis after 48 h treatment with different concentrations of BMX alone or BMX combined with TMZ in HT29, HCT116, and RKO cells and the proportion of cells in each cell cycle phase. SubG1, cell with polyploid chromosome; > 4 N, polyploid cell. (D) Apoptosis analysis after 48 h treatment with different concentrations of BMX alone or BMX combined with TMZ and the apoptotic rate of cells in HT29, HCT116, and RKO cells. All results are shown as mean ± s.d. from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus control (HT29 cells); #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control (HCT116 cells); †p < 0.05, ††p < 0.01, †††p < 0.001 versus control (RKO cells)

Article Snippet: The cell proliferation of CRC cells was measured by CCK8 assay (Targetmol, Shanghai, China).

Techniques: Concentration Assay, Cell Cycle Assay, Control

Journal: Cell communication and signaling : CCS

Article Title: BMX, a specific HDAC8 inhibitor, with TMZ for advanced CRC therapy: a novel synergic effect to elicit p53-, β-catenin- and MGMT-dependent apoptotic cell death.

doi: 10.1186/s12964-022-01007-x

Figure Lengend Snippet: Fig. 5 BMX, VPA, SAHA, TMZ, Oxp, and Dox combination inhibited cell proliferation in CRC cells. (A) HDAC8 mRNA expression levels stimulated with BMX (5 µM), VPA (4 mM), SAHA (2 µM) with or without TMZ were determined using qRT-PCR assays. (B) HT29, HCT116, and RKO cells were treated with BMX with or without TMZ for 48 h. Then, cells were harvested for detection of acetyl-histone H3 (Lys9/Lys14), acetyl-histone H4 (Lys8), and HDAC8 by Western blot analysis. (C) The proliferation of BMX, VPA, SAHA with or without TMZ, Oxp (5 µM) and Dox (1 µM) in HT29, HCT116, and RKO cells with treatment durations were assayed using the CCK-8 method. (D) Colony formation capability assay with different treatments of BMX, VPA, SAHA with or without TMZ, Oxp and Dox in HT29, HCT116, and RKO cells; the colonies were counted for quantification. All results are shown as mean ± s.d. from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus control (HT29 cells); #p < 0.05, ##p < 0.01, ###p < 0.001 versus control (HCT116 cells); †p < 0.05, ††p < 0.01, †††p < 0.001 versus control (RKO cells)

Article Snippet: The cell proliferation of CRC cells was measured by CCK8 assay (Targetmol, Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Control

Journal: Cell communication and signaling : CCS

Article Title: BMX, a specific HDAC8 inhibitor, with TMZ for advanced CRC therapy: a novel synergic effect to elicit p53-, β-catenin- and MGMT-dependent apoptotic cell death.

doi: 10.1186/s12964-022-01007-x

Figure Lengend Snippet: Fig. 7 Autophagy expression levels stimulated with BMX and PCI-34051 in the presence or absence of TMZ. (A) HT29, HCT116, and RKO cells were treated with BMX and PCI-34051 with or without TMZ for 48 h. Then, cells were harvested for detection of acetyl-histone H3 (Lys9/Lys14), acetyl-histone H4 (Lys8), and HDAC8 by Western blot analysis. (B) The proliferation of BMX and PCI-34051 with or without TMZ in HT29, HCT116, and RKO cells. (C) Colony formation capability assay with different treatments of BMX and PCI-34051 with or without TMZ in HT29, HCT116, and RKO cells; the colonies were counted for quantification. (D, E) Expressions of cleaved caspase 3, cleaved PARP, LC3 and p62 proteins in HT29, HCT116, and RKO cells treated with BMX and PCI-34051 with or without TMZ for 48 h. All results are shown as mean ± s.d. from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus control (HT29 cells); #p < 0.05, ##p < 0.01, ###p < 0.001 versus control (HCT116 cells); †p < 0.05, ††p < 0.01, †††p < 0.001 versus control (RKO cells)

Article Snippet: The cell proliferation of CRC cells was measured by CCK8 assay (Targetmol, Shanghai, China).

Techniques: Expressing, Western Blot, Control

Journal: Journal of Extracellular Vesicles

Article Title: Calcified apoptotic vesicles from PROCR + fibroblasts initiate heterotopic ossification

doi: 10.1002/jev2.12425

Figure Lengend Snippet: Single‐cell RNA‐sequencing (scRNA‐seq) analysis identifies novel apoptotic‐preferential PROCR + fibroblasts in the early stage of HO. (a) The workflow depicting the collection and processing of specimens of sham and HO tendons for scRNA‐seq. (b) Dimension reduction presentation (via tSNE) of combined single‐cell transcriptome data from Achilles tendons of rats from the sham and HO groups after 1 and 3 weeks ( n = 14402). Each dot represents a single cell and is labelled with corresponding cell categories and is coloured according to its cell type identity. Clusters were generated using a resolution of 0.2 before subclustering into major cell types according to the Methods. The Seurat 3 R‐Package segregation grouped the cells into 6 distinct cell clusters. (c and d) Quantitative analysis of clusters based on the combined single‐cell transcriptome data in (b). (e) tSNE of fibroblast clusters (F1–F5). (f) Apoptosis score analyses of fibroblasts based on (e). (g) Bioinformatic analysis of PROCR + cell populations based on (e). (h) Representation analysis of GO categories showing different functions for PROCR + cells. (i) Representation analysis of KEGG categories showing different functions for PROCR + cells.

Article Snippet: To inhibit fibroblast apoptosis in the Achilles tendon, Z‐VAD‐FMK (T7020, TargetMol) dissolved in normal saline was injected into rats at 1 μg/rat, three times a week.

Techniques: Single Cell, RNA Sequencing, Generated

Journal: Stem Cells International

Article Title: Mesenchymal Stem Cells Inhibit Epithelial-to-Mesenchymal Transition by Modulating the IRE1 α Branch of the Endoplasmic Reticulum Stress Response

doi: 10.1155/2023/4483776

Figure Lengend Snippet: Suppression of ER stress contributed to MSC-mediated amelioration of EMT in A549 cells. (a) A549 cells were treated with 100 μ M TUDCA for different times. The protein expression levels of BiP, ATF6, ATF4, XBP-1s and XBP-1u were measured using western blotting and quantified using densitometry in ImageJ software ( n = 3, one-way ANOVA with Duncan's post hoc test). (b) A549 cells were treated with 10 ng/ml TGF- β 1 in the presence or absence of TUDCA for 72 hr. The protein expression levels of BiP, ATF6, ATF4, XBP-1s and XBP-1u were measured using western blotting and quantified using densitometry in ImageJ software ( n = 4, one-way ANOVA with Duncan's post hoc test). (c) A549 cells were treated with 100 μ M TUDCA for different times. The protein expression levels of E-cadherin and vimentin were measured using western blotting and quantified using densitometry in ImageJ software ( n = 3, one-way ANOVA with Duncan's post hoc test). (d) A549 cells were treated with 10 ng/ml TGF- β 1 in the presence or absence of TUDCA for 72 hr. The protein expression levels of E-cadherin and vimentin were measured using western blotting and quantified using densitometry in ImageJ software ( n = 4, one-way ANOVA with Duncan's post hoc test). The data are shown as the means ± SEMs ( ∗∗ P < 0.01, ∗ P < 0.05 vs. the control group; ## P < 0.05, # P < 0.05 vs. the TGF- β 1 group).

Article Snippet: A549 cells were treated with TGF- β 1 (10 ng/ml, 100-21, PeproTech, Rocky Hill, NJ, USA) for 72 hr with or without pretreatment with the ER stress inhibitor TUDCA (100 μ M, abs816166; Absin, Shanghai, China) or IRE1 α -specific inhibitor 4 μ 8c (10 μ M, T6363, Topscience, Shanghai, China) for 1 hr.

Techniques: Expressing, Western Blot, Software, Control

Journal: Clinical and Translational Medicine

Article Title: Elevation of ISG15 promotes diabetic kidney disease by modulating renal tubular epithelial cell pyroptosis

doi: 10.1002/ctm2.70337

Figure Lengend Snippet: ISG15 was involved in HG‐induced mitochondrial impairment and mtDNA release. (A) Representative TEM images of kidney tissues from WT and KO mice treated with STZ ( n = 6). (B) Western blot analysis ISG15/ISGylation expression in TECs treated with vehicle or HG ( n = 3). (C–E) Flow cytometry analysis and quantitative data depicting the mitochondrial membrane potential (C), mitochondrial mass (D) and mtROS (E) ( n = 3). (F and G) qPCR analysis the mtDNA ( Loop1‐3 and mt‐Nd4 ) copy number in the cytosolic compartments (F) and mitochondria (G) ( n = 3). TECs were treated with vehicle or C‐176 (10 µM), and then cultured in HG medium for 48 h. Results are expressed as the mean ± SD. * p < .05; ** p < .01; *** p < .001; ns, not significant.

Article Snippet: To inhibit mitochondrial reactive oxygen species (mtROS), NLRP3 or STING, cells were pretreated with the mitochondria‐specific antioxidant Mito‐TEMPO (25 μM; TargetMol, China), NLRP3‐specific inhibitor MCC950 (2 μM; TargetMol) or STING‐specific inhibitor C‐176 (10 μM; TargetMol) for 1.5 h before exposure to 40 mM HG.

Techniques: Western Blot, Expressing, Flow Cytometry, Membrane, Cell Culture

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: A Novel Ubiquitin Ligase Adaptor PTPRN Suppresses Seizure Susceptibility through Endocytosis of Na V 1.2 Sodium Channels.

doi: 10.1002/advs.202400560

Figure Lengend Snippet: Figure 5. PTPRN promotes NaV1.2 channel internalization through ubiquitin-dependent endocytosis. A) Schematic of the effect of inhibitors on the trafficking and ubiquitination of proteins. B) Left panel: Immunoblot analysis of cell surface biotinylation performed in primary cortical neurons infected with lentivirus containing shRNA-PTPRN#3 or nonsilencing lentivirus for 7 days. Total lysates (total) and biotinylated fractions (membrane) were an- alyzed by western blot analysis. Right panel: Quantification of NaV1.2 total expression and surface expression. n = 3, *p < 0.05, two-way ANOVA with Bonferroni’s multiple-comparisons test. C) Current density versus voltage relationship for HEK-293T cells expressing NaV1.2 and PTPRN in a 1:2 ratio (NaV1.2 + PTPRN) or NaV1.2 and empty vector (NaV1.2), with or without treatment using dynasore (80 μm, 2 h). n = 21 for all samples except NaV1.2 + PTPRN plus dynasore (n = 24). D) Graph depicting voltage-dependence of activation for NaV1.2 channels described in (C). The lines are the best-fitted Boltzmann curves. E) Current density versus voltage relationship for HEK-293T cells expressing NaV1.2 and PTPRN in a 1:2 ratio (NaV1.2 + PTPRN) or NaV1.2 and empty vector (NaV1.2), with or without treatment using TAK-243 (1 μm, 2 h). n = 18 and 26 for untreated NaV1.2 and NaV1.2 + PTPRN, respectively, and n = 20 and 26 for NaV1.2 and NaV1.2 + PTPRN plus TAK-243, respectively. F) Graph depicting voltage dependence of activation for NaV1.2 channels described in (E). The lines are the best-fitted Boltzmann curves. G) Current density versus voltage relationship for HEK-293T cells expressing NaV1.2 and PTPRN in a 1:2 ratio (NaV1.2 + PTPRN) or NaV1.2 and empty vector (NaV1.2), with or without treatment with Heclin (20 μm, 2 h). n = 18 and 26 for NaV1.2 and NaV1.2 + PTPRN plus Heclin, respectively, and n = 19 for both samples without treatment. H) Graph depicting voltage dependence of activation for NaV1.2 channels described in (G). The lines are the best-fitted Boltzmann curves. I) Workflow showing experimental procedures for purification of PTPRN intracellular fragment (aa 601–979, PTPRN-cyto) and TagRFP and whole cell patch-clamp recordings. J) Left panel:

Article Snippet: The resultant materials were then subjected to western blot analysis Trafficking Inhibition: To investigate the mechanism of the regulation on NaV1.2 by PTPRN, 80 μm Dynasore (Abcam), [54] 1 μm TAK243 (TargetMol),[35] 20 μm Pitstop2 (GLPBIO),[55] or 50 μm Heclin (GLPBIO)[48c] were added into the medium 2 h before the electrophysiology recording.

Techniques: Ubiquitin Proteomics, Western Blot, Infection, shRNA, Membrane, Expressing, Plasmid Preparation, Activation Assay, Patch Clamp

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: A Novel Ubiquitin Ligase Adaptor PTPRN Suppresses Seizure Susceptibility through Endocytosis of Na V 1.2 Sodium Channels.

doi: 10.1002/advs.202400560

Figure Lengend Snippet: Figure 7. PTPRN-NaV1.2 axis regulates neuronal intrinsic excitability and seizure susceptibility. A) Schematic diagram showing stereotaxic injection of shRNA-Scn2a AAV in adult PTPRN-KO mice. B) Infection of AAV expressing shRNA-Scn2a and EGFP in the hippocampal DG region 14 days after injection. Scale bar, 125 μm. C) Validation of Scn2a knockdown efficiency by RT-PCR analysis of Scn2a in hippocampal tissues obtained from mice infected with AAV-containing shRNA-Scn2a or control AAV for 14 days. The levels of mRNA were normalized to those of 𝛽-Actin. n = 3, **p < 0.01, unpaired two-tailed Student’s t-test. D) Top panel: Validation of Scn2a knockdown efficiency by western blot analysis using hippocampi from mice infected with AAV-containing shRNA-Scn2a or control AAV for 14 days. Bottom panel: Quantification of the results by normalizing the protein levels of NaV1.2 to those of 𝛽-Actin. n = 3, *p < 0.05, unpaired two-tailed Student’s t-test. E) Left panel: Representative current-clamp recordings of DG granule cells from PTPRN-WT and PTPRN-KO mice infected with AAV containing shRNA-Scn2a or control AAV for 14 days. For comparison, the cells were held at -80 mV of membrane potential. Right panel: The mean number of APs generated in response to depolarizing current pulses. *p < 0.05, ***p < 0.001, ****p < 0.0001, ####p < 0.0001, two-way ANOVA with Bonferroni’s multiple-comparisons test. F) Typical APs of DG granule cells from mice described in (E). G) Phase plane plots associated with APs in (F). H) The average AP threshold (left) and peak rising phase dV/dt (right). *p < 0.05, ####p < 0.0001, $$$$p < 0.0001, one-way ANOVA with Bonferroni’s multiple-comparisons test. I) Graph depicting the seizure progression in PTPRN-WT and PTPRN-KO mice infected with AAV containing shRNA-Scn2a or control AAV for 14 days, illustrated as mean maximum seizure stage reached by 15, 30, 45, 60, and 75 min after KA administration. n = 6, $p < 0.05, *p < 0.05, ***p < 0.001, ****p < 0.0001, ###p < 0.001, $$$p < 0.001, two-way ANOVA with Bonferroni’s multiple-comparisons test. J) Graph showing time taken to reach each stage of seizure after administration of KA in mice described in (I). n = 6, $$p < 0.01, ****p < 0.0001, two-way ANOVA with Bonferroni’s multiple-comparisons test. K) Number of class V seizures within 1 h after KA administration. n = 6, ***p < 0.001, ###p < 0.001, one-way ANOVA with Bonferroni’s multiple-comparisons test. L) Incidence of maximum seizure stage reached during the course of the experiments in (I). n = 6, ****p < 0.0001, ####p < 0.0001, $$$$p < 0.0001, Chi-square test. Data are represented as mean ± s.e.m.

Article Snippet: The resultant materials were then subjected to western blot analysis Trafficking Inhibition: To investigate the mechanism of the regulation on NaV1.2 by PTPRN, 80 μm Dynasore (Abcam), [54] 1 μm TAK243 (TargetMol),[35] 20 μm Pitstop2 (GLPBIO),[55] or 50 μm Heclin (GLPBIO)[48c] were added into the medium 2 h before the electrophysiology recording.

Techniques: Injection, shRNA, Infection, Expressing, Biomarker Discovery, Knockdown, Reverse Transcription Polymerase Chain Reaction, Control, Two Tailed Test, Western Blot, Comparison, Membrane, Generated

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: A Novel Ubiquitin Ligase Adaptor PTPRN Suppresses Seizure Susceptibility through Endocytosis of Na V 1.2 Sodium Channels.

doi: 10.1002/advs.202400560

Figure Lengend Snippet: Figure 8. Proposed mechanism for PTPRN regulation of NaV1.2 channels and neuronal intrinsic plasticity. Elevated neuronal activity augments the recruitment of NEDD4L by PTPRN to NaV1.2 sodium channels. This interaction promotes NEDD4L-mediated ubiquitination, subsequently leading to the endocytosis of NaV1.2 channels. The presented regulatory mechanism provides a feedback response to heightened activity within the nervous system, thereby aiding the tuning of neuronal intrinsic plasticity.

Article Snippet: The resultant materials were then subjected to western blot analysis Trafficking Inhibition: To investigate the mechanism of the regulation on NaV1.2 by PTPRN, 80 μm Dynasore (Abcam), [54] 1 μm TAK243 (TargetMol),[35] 20 μm Pitstop2 (GLPBIO),[55] or 50 μm Heclin (GLPBIO)[48c] were added into the medium 2 h before the electrophysiology recording.

Techniques: Activity Assay, Ubiquitin Proteomics