Journal: Nature Communications

Article Title: Integrative transcriptome-wide association analyses reveal PRKCG-linked GABAergic dysfunction in Fragile X-associated tremor/ataxia syndrome

doi: 10.1038/s41467-025-68163-9

Figure Lengend Snippet: a Venn diagrams showing the number of TWAS candidate genes overlapping with DEGs from bulk RNA-Seq on the cortex and cerebellum of the FXTAS mouse model. Bar plot showing the normalized read counts of shared genes in FXTAS and control mice from bulk RNA-Seq. DEGs from 3 to 4 months were selected with P < 0.01; DEGs from 6 to 7 months were selected with FDR < 0.05. Data are presented as mean ± SD; n = 3–4 biological replicates per group. b Venn diagrams showing the number of TWAS candidate genes overlapping with DEGs from TRAP-Seq on GABAergic neurons from cortex and cerebellum of conditional FXTAS model. Bar plot showing the normalized read counts of shared genes in FXTAS and control mice from TRAP-Seq. DEGs from 3 to 4 months were selected with P < 0.01. Data are presented as mean ± SD; n = 2-3 biological replicates per group. Two-sided Fisher’s exact test demonstrated significant overlap between TWAS candidates and TRAP-seq DEGs in cerebellar GABAergic neurons at 6–7 months ( P = 0.018; Supplementary Data ). c Quantitative RT-PCR analysis of Prkcg expression relative to Gapdh in the cortex at two stages. Three biological replicates were used in each group. Data are presented as mean ± SD; n = 3 biological replicates per condition. Statistical significance was assessed using a two-sided unpaired t -test with Welch’s correction. Significance is defined as P < 0.05. d Quantitative RT-PCR analysis of PRKCG expression relative to GAPDH in NPCs from FXTAS patients and controls. Data are presented as mean ± SD; n = 3 biological replicates per condition. Statistical significance was assessed using a one-sided unpaired t -test. Significance is defined as P < 0.05. e Scheme of the overexpression of human PRKCG in the FXTAS fly model. f Overexpression of hPRKCG and aPKC resulted in enhancement of neurotoxicity in the FXTAS fly model. g Cell viability assays following shRNA-mediated knockdown of Prkcg (left) and hPRKCG overexpression (right) in N2a cells expressing expanded CGG repeats. Data are presented as mean ± SD and statistical significance was assessed using a two-sided unpaired t -test with Welch’s correction. Significance is defined as P < 0.05.

Article Snippet: Full-length human PRKCG cDNA was obtained from a tagged ORF clone (OriGene Technologies, PRKCG Human Tagged ORF Clone, RG208502).

Techniques: RNA Sequencing, Control, Quantitative RT-PCR, Expressing, Over Expression, shRNA, Knockdown

Journal: Frontiers in Oncology

Article Title: Suppression of EGFR/PKC-δ/NF-κB Signaling Associated With Imipramine-Inhibited Progression of Non-Small Cell Lung Cancer

doi: 10.3389/fonc.2021.735183

Figure Lengend Snippet: The inhibition of EGFR/PKC-δ/NF-κB proteins phosphorylation and induction of apoptosis-related proteins by imipramine in CL1-5-F4/ NF-κB-luc2 bearing tumor. (A–C) The protein expression from IHC of EGFR (Try 1068), PKC-δ (Thr507), NF-κB (Ser536), cleaved caspase-3, -8, -9, MMP-9, XIAP, MCL-1 and their quantification bar chart are presented. (D, E) The tumor ex vivo Western blotting from each mice of cleaved caspase-3, -8, -9 and PARP-1 is presented. ( ** p < 0.01 vs . vehicle; scale bar =100 μm).

Article Snippet: Primary antibodies against Matrix metalloproteinase-9 (MMP-9) (AB19016, Millipore), vascular endothelial growth factor (VEGF) (ab1316, Abcam, Cambridge, UK), EGFR (Try 1068) (#2234, Cell signaling, Danvers, MA, USA), EGFR (E-AB-63555, Elabscience, Houston, TX, USA), PKC-δ (Thr507) (E-AB-20968, Elabscience), PKC-δ (E-AB-14675, Elabscience), NF-κB p65 (Ser536) (E-AB-70335, Elabscience), NF-κB p65 (E-AB-22066, Elabscience), cell leukemia-1 (MCL-1) (BV-438, BioVision), cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (cFLIP) (D16A8, Cell signaling), X-linked inhibitor of apoptosis protein (XIAP) (PA5-29253, Thermo Fisher Scientific), Fas (E-AB-40063, Elabscience), Fas ligand (FasL) (E-AB-31410, Elabscience), cleaved caspase-3 (E-AB-30004, Elabscience), cleaved caspase-8 (E-AB-22107, Elabscience), cleaved caspase-9 (#9505, Cell Signaling Technology), PARP-1 (#9532, Cell Signaling Technology) and β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, Texas, Waltam, MA, USA) for Western blotting were purchased from different companies as listed.

Techniques: Inhibition, Phospho-proteomics, Expressing, Ex Vivo, Western Blot

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ( A ) IP3R blocking inhibits PLCE1-induced signal transduction in the axis of PKC/GSK-3β/β-Catenin. ECA-109 cells were transfected with the plasmid expressing Plce1 for 6 hr and then treated with 2-APB (10 µM) for additional 18 hr. ( B ) [Ca 2+ ] blocking represses signal transduction in the axis of PKC/GSK-3β/β-Catenin induced by PLCE1. ECA-109 cells were transfected with the plasmid expressing Plce1 for 6 hr and then treated with BAPTA-AM (10 µM) for additional 18 hr. ( C ) PKC inhibition blocks PLCE1 stimulated signal transduction in the axis of PKC/GSK-3β/β-Catenin. ECA-109 cells were transfected with the plasmid expressing Plce1 . 6 hr post-transfection, cells were treated with 100 nM of Midostaurin for additional 18 hr. ( D ) PKC inhibition represses PLCE1-induced nuclear translocation of β-Catenin in ECA-109 cells. Cells were transfected with the plasmid expressing PLCE1. 6 hr post-transfection, 2-APB (10 µM), BAPTA-AM (10 µM), or Midostaurin (100 nM) was added in culture medium, and cells were cultured for additional 18 hr. Scale bar = 10 µm. Immunofluorescence was used to examine subcellular distribution of β-Catenin. ( E ) TAK1 counteracts PLCE1-induced signal transduction in the axis of PKC/GSK-3β/β-Catenin. ECA-109 cells were transfected with the plasmids expressing Plce1 or Map3k7 as indicated for 24 hr. ( F ) TAK1 reduces PLCE1-induced nuclear distribution of β-Catenin in ECA-109 cells. Cells were transfected with the plasmids expressing Plce1 or Map3k7 as indicated. Scale bar = 10 µm. ( G ) Dominant negative TAK1 (K63W) fails to block signal transduction in the axis of PKC/GSK-3β/β-Catenin/MMP2 induced by PLCE1. ECA-109 cells were transfected with the plasmids expressing Plce1 or mutated Map3k7 (TAK1 K63W) for 24 hr. ( H ) TAK1 has no effect on PLCE1 S1060A-induced signal transduction in the axis of PKC/GSK-3β/β-Catenin. ECA-109 cells were transfected with the plasmids expressing PLCE1 S1060A or TAK1 for 24 hr. Protein levels were analyzed by western blot, and Actin was used as a loading control. Representative blots were shown. Figure 5—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 5—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Blocking Assay, Transduction, Transfection, Plasmid Preparation, Expressing, Inhibition, Translocation Assay, Cell Culture, Immunofluorescence, Dominant Negative Mutation, Western Blot, Control

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ( A–F ) Quantified data for the western blots as shown in . n=3 biologically independent replicates. Data are presented as mean ± SD. Statistical significance was tested by two-tailed one-way ANOVA test. *p<0.05, **p<0.01, and ***p<0.001. Figure 5—figure supplement 7—source data 1. TAK1 mitigates PLCE1-induced signal transduction in the axis of PKC/GSK-3β/β-Catenin.

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Western Blot, Two Tailed Test, Transduction

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: Each mouse was intravenously injected with 1×10 6 ECA-109 cells diluted in 100 µl PBS. Mice were treated with Takinib at the dosage of 50 mg/kg/day for 15 days, mice in control group were received vehicle (corn oil). Eight weeks later, mice were sacrificed, and the lungs and livers from each group were collected and photographed. ( A ) Typical images of specimens. ( B ) Hematoxylin and eosin staining of metastatic nodules in lungs. ( C ) The number of nodules in lungs. ( D ) Takinib treatment induces signal transduction in the axis of PKC/GSK-3β/β-Catenin. Protein levels were analyzed by western blot, and Actin was used as a loading control. n=3 biologically independent replicates. ( E ) Quantitative analysis of the western blot data shown in ( D ). Data are presented as mean ± SD. Statistical significance was tested by unpaired Student’s t-test. * p < 0.05 , ** p<0.01, and *** p<0.001 . Figure 6—source data 1. Inhibition of TAK1 by Takinib promotes esophageal squamous cell carcinoma (ESCC) metastasis in nude mice. Figure 6—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 6—source data 3. Original files for western blot analysis displayed in .

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Injection, Control, Staining, Transduction, Western Blot, Inhibition

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet: ECA-109 cells were transduced with lentivirus bearing PLCE1 shRNA (LV-shPLCE1) or NC shRNA (LV-shPLCE1 NC). Each mouse was intravenously injected with the LV transduced cells (1×10 6 cells/mouse). Eight weeks later, mice were sacrificed, and the lungs and livers from each group were collected and photographed. ( A ) Typical images of lung specimens. ( B ) Hematoxylin and eosin staining of metastatic nodules in lungs. ( C ) The number of nodules in lungs. ( D ) PLCE1 knockdown represses signal transduction in the axis of PKC/GSK-3β/β-Catenin. Protein levels were analyzed by western blot, and Actin was used as a loading control. n=3 biologically independent replicates. ( E ) Quantitative analysis of the western blot data shown in ( D ). Data are presented as mean ± SD. Statistical significance was tested by unpaired Student’s t-test. ** p<0.01 and *** p<0.001 . Figure 7—source data 1. PLCE1 knockdown inhibits esophageal squamous cell carcinoma (ESCC) metastasis in nude mice. Figure 7—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 7—source data 3. Original files for western blot analysis displayed in .

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Transduction, shRNA, Injection, Staining, Knockdown, Western Blot, Control

Journal: eLife

Article Title: TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

doi: 10.7554/eLife.97373

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal antibodies against TAK1 (#5206), phospho-TAK1 (Ser412, #9339), PKCα (#2056), phospho-PKC (pan) (gamma Thr514) (#38938), GSK-3β (#9315), phospho-GSK-3β (Ser9) (#5558), β-Catenin (#8480), phospho-β-Catenin (Ser33/37/Thr41) (#9561), MMP-2 (#87809), mouse monoclonal antibodies against Actin (#3700), Myc-Tag (Sepharose Bead Conjugate) (#55464), Epithelial-Mesenchymal Transition (EMT) antibody sample kit (#9782), phosphor-IKK (#2078), IKK (#61294), phosphor-JNK (#4668), JNK (#9252), phospho-ERK (#4370), ERK (#9102), phospho-P38 MAPK (#9211), P38 MPAK (#9212), and normal rabbit IgG (#2729) were from Cell Signaling Technology (Beverley, MA, USA).

Techniques: Transfection, Construct, shRNA, Recombinant, Plasmid Preparation, Sequencing, Activity Assay, Enzyme-linked Immunosorbent Assay, Software