Journal: Scientific Reports

Article Title: DYNC2LI1 mutations broaden the clinical spectrum of dynein-2 defects

doi: 10.1038/srep11649

Figure Lengend Snippet: Immunofluorescence staining of 5 days starved control fibroblasts with antibodies against polyglutamylated tubulin (ciliary axoneme, red), DYNC2LI1 (green) and DAPI (DNA, blue in merge) identified localization of DYNC2LI1 to the basal body region ( a ) and to the transition zone ( b ) of the primary cilium, as well as to the centrosomes ( c ) during mitosis. Magnifications are shown in white boxes, white scale bars 10 μm.

Article Snippet: Alexa Fluor® 488 goat anti- rabbit IgG (green, 1:500) and Alexa Fluor® 594 goat anti-mouse IgG (red, 1:500) (Life Technologies) were used as secondary antibodies and cell nuclei were stained with DAPI (4′,6-Diamidin-2-phenylindol, 1:50000, Serva, Heidelberg, Germany).

Techniques: Immunofluorescence, Staining, Control

Journal: Scientific Reports

Article Title: DYNC2LI1 mutations broaden the clinical spectrum of dynein-2 defects

doi: 10.1038/srep11649

Figure Lengend Snippet: Immunofluorescence staining of 5 days starved fibroblasts (scrambled control vs. DYNC2LI1 siRNA) with antibodies against polyglutamylated tubulin (primary cilium, red), PCNT (green) and DAPI (DNA, blue in merge). ( a,b ) No significant differences in the number of cilium presenting cells were observed, 85% of control cells and 84% of DYNC2LI1 depleted cells showed primary cilium formation (p = 0.927 [χ2-test], white scale bars 20 μm). ( c,d ) The median cilia length was 1.83 μm in scrambled control cells, whereas cilia in knockdown cells were significantly shorter with median of 1.42 μm (p = 1.205 × 10 −6 [Mann-Whitney-U test]). Magnifications are shown in white boxes, white scale bars 10 μm).

Article Snippet: Alexa Fluor® 488 goat anti- rabbit IgG (green, 1:500) and Alexa Fluor® 594 goat anti-mouse IgG (red, 1:500) (Life Technologies) were used as secondary antibodies and cell nuclei were stained with DAPI (4′,6-Diamidin-2-phenylindol, 1:50000, Serva, Heidelberg, Germany).

Techniques: Immunofluorescence, Staining, Control, Knockdown, MANN-WHITNEY

Journal: Scientific Reports

Article Title: DYNC2LI1 mutations broaden the clinical spectrum of dynein-2 defects

doi: 10.1038/srep11649

Figure Lengend Snippet: Immunofluorescence staining of 5 days starved fibroblasts (scrambled control vs. DYNC2LI1 siRNA) with antibodies against polyglutamylated tubulin (primary cilium, red), PCNT/IFT57/IFT88 (green) and DAPI (DNA, blue in merge). ( a ) DYNC2LI1 depleted cells showed in addition to the reduced ciliary length further ciliary morphological abnormalities with bulbous tips in 14% of analyzed cilia. Scrambled control cells present normal cilia structure, but we observed as well malformations in 6% of measured control cilia (p = 0.028 [χ2-test]). ( b ) Scrambled controls and knockdown cells showed a localization of IFT57 at the basal body region of the primary cilium. In addition, knockdown of DYNC2LI1 resulted in an accumulation of IFT57 - component of IFT-B complex - in the bulbous ciliary tip (arrows). ( c ) IFT88 - another component of the IFT-B complex - is localized at the basal body region of the primary cilium in scrambled controls and knockdown cells. IFT88 is present at the ciliary tip in scrambled controls compared to a strong accumulation in the bulbous tip of DYNC2LI1 depleted cells (arrows). White scale bars 2.5 μm, three-dimensional reconstruction was performed using Imaris software (Bitplane, Zurich).

Article Snippet: Alexa Fluor® 488 goat anti- rabbit IgG (green, 1:500) and Alexa Fluor® 594 goat anti-mouse IgG (red, 1:500) (Life Technologies) were used as secondary antibodies and cell nuclei were stained with DAPI (4′,6-Diamidin-2-phenylindol, 1:50000, Serva, Heidelberg, Germany).

Techniques: Immunofluorescence, Staining, Control, Knockdown, Software

Journal: Cancer Medicine

Article Title: Ketamine induces apoptosis in lung adenocarcinoma cells by regulating the expression of CD 69

doi: 10.1002/cam4.1288

Figure Lengend Snippet: Effects of ketamine on cell growth inhibition of A549 cells. (A) Detection of CD 69 mRNA level in A549 cells treated with 0, 1, 10, and 100 μ m ketamine for 24 h, respectively. Data represent mean ± SD of three independent experiments ( n = 4, * P < 0.05, ** P < 0.01, *** P < 0.001). (B) The CD 69 level detected by western blot analysis. (C) Expression of CD69 in the normal ( n = 51) and cancer ( n = 501) tissues. P = 1.31e–29. (D) CD69 expression was lower in the cancer tissues compared with the normal tissues in same patients. P = 7.90e−17. (E) RSEM analysis showed downregulation of the expression level of CD 69 in cancer tissues compared with normal tissues and associated with the T stage.

Article Snippet: The protein was transferred onto the PVDF membrane (Bio‐Rad, USA) and incubated with primary antibody: CD69 antibody (sc‐373799, Santa Cruz, USA).

Techniques: Inhibition, Western Blot, Expressing

Journal: Advanced Science

Article Title: Epigenetic Regulation of DAPK1 and Netrin‐1 Drives Diabetic Encephalopathy

doi: 10.1002/advs.202502535

Figure Lengend Snippet: DAPK1 levels were increased in the brain of diabetic mice with diabetic encephalopathy. A) Schematic overview of diabetic mouse modeling and cognitive‐related behavior assessments. B,C) Representative swimming paths on day 7, B) latency to reach the platform from days 1–7, C) swimming paths on day 9 and number of crossing the platform region on day 9 in the Morris water maze task for diabetic groups (6 month old STZ‐treated mice and db/db mice) and control groups (6 month old SCB‐treated mice and db/m mice). n = 10–12 per group. D) Western blot analysis and E) quantification of DAPK1 protein expression relative to β‐actin in the hippocampus of diabetic and control groups. n = 6 per group. F) Representative images and G) quantitative analysis of immunohistochemical staining for DAPK1 in hippocampal subregions (CA1, DG, and CA3) of diabetic and control mice. n = 6 slices from 3 mice per group. H) Distribution of DAPK1 (red) in excitatory pyramidal neurons (CaMKII, green) and its absence in inhibitory neurons (GAD1, green). Nuclei were stained with DAPI (blue). Higher‐magnification images of the regions marked with white squares in the CA1 area are shown on the right. I) Colocalization analysis of DAPK1 with CaMKII (up) and GAD1 (down), showing overlay levels and Pearson's correlation coefficient. J) Representative images and K) qualification of microglia (IBA1, red) and astrocytic (GFAP, green) activation in the hippocampus of diabetic and control groups. n = 6 slices from three mice per group. L) Representative images and M) quantification of apoptotic cells in the hippocampus of diabetic and control groups, as indicated by TUNEL‐positive staining (green). Nuclei were stained with DAPI (blue). The regions marked with white squares in the CA1 area are shown at higher magnification on the right. The percentage of apoptotic cells was calculated with the number of TUNEL‐positive cells divided by the total number of DAPI‐labeled cells in the same microscopic field. n = 4 slices from three mice per group. N,O) Electrophysiological recordings of N) field excitatory postsynaptic potential (fEPSP) slope to evaluate the LTP at the CA3‐CA1 synapse. n = 9 cells from three mice per group. Data are presented as mean ± S.E.M., and statistical analysis was performed using an unpaired Student's t ‐test or one‐way or two‐way analysis of variance (ANOVA), unless otherwise specified. ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking with 5% nonfat milk for 30 min, the membranes were incubated with primary antibodies overnight at 4 °C to DAPK1 (rabbit, 1:1000, No.25136‐1‐AP, Proteintech), DAPK1 (mouse, 1:1000, No.67815‐1‐Ig, Proteintech), β‐actin (mouse, 1:800, No.66009‐1‐Ig, Proteintech), HNF1A polyclonal antibody (rabbit, 1:1000, No. 22426‐1‐AP, Proteintech), Phospho‐HNF1A (Ser247) Ab (rabbit, 1:1000, AF7045, Affinity), or Netrin‐1 (rabbit, 1:500, DF8579, Affinity), followed by washes with Tris‐buffered saline (PBS)‐Tween 20.

Techniques: Control, Western Blot, Expressing, Immunohistochemical staining, Staining, Activation Assay, TUNEL Assay, Labeling

Journal: Advanced Science

Article Title: Epigenetic Regulation of DAPK1 and Netrin‐1 Drives Diabetic Encephalopathy

doi: 10.1002/advs.202502535

Figure Lengend Snippet: Knockdown hippocampus DAPK1 in excitatory neurons rescued diabetic encephalopathy in diabetes. A) Schematic of the strategy for conditional DAPK1 kinase domain knockout in hippocampal excitatory neurons using CaMKII‐CreERT and tamoxifen (TAM) induction in diabetic mice. Western blot analysis of DAPK1 and β‐actin protein expression in the hippocampus of STZ/KD f/f and STZ/KD −/− groups. n = 2 per group. B) Representative path traces to the hidden platform on day 7 and latency over 7 days in different groups. n = 10 per group. C) Swimming paths on day 9 and number of crossing the platform region on day 9 in the MWM. n = 10 per group. D) Immunofluorescence staining of IBA1 (red) and GFAP (green) in the hippocampus, showing changes in microglial and astrocytic activation across groups. n = 6 slices from three mice per group. E) TUNEL staining of hippocampal sections for apoptosis detection, with DAPI counterstaining; apoptotic cells appear green in images. F) Quantification of TUNEL‐positive cells in the hippocampus, showing reduced apoptosis in DAPK1 −/− mice. n = 6 slices from three mice per group. G) Electrophysiological recordings illustrating normalized field excitatory postsynaptic potential (fEPSP) slopes in CA3–CA1 synapses, with reduced long‐term potentiation (LTP) impairment in DAPK1 −/− mice. H) Summary of fEPSP slope changes among groups, highlighting improved synaptic function in DAPK1 −/− mice. n = 9 cells from three mice per group. I) Representative traces (up) and quantification (down) of paired‐pulse facilitation (PPF) recorded from hippocampal slices. n = 10 cells from three mice per group. J) Representative traces (up) and quantification (down) of AMPA/NMDA current ratio measured in CA1 neurons. n = 10 cells from three mice per group. K) Representative transmission electron microscopy (TEM) images showing hippocampal synapses in the stratum radiatum. L) Quantification of postsynaptic density (PSD) length from TEM images. n = 130–150 synapses from 3 to 5 mice per group. Data are shown as mean ± S.E.M., and statistical analysis was performed using one‐way or two‐way analysis of variance (ANOVA). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking with 5% nonfat milk for 30 min, the membranes were incubated with primary antibodies overnight at 4 °C to DAPK1 (rabbit, 1:1000, No.25136‐1‐AP, Proteintech), DAPK1 (mouse, 1:1000, No.67815‐1‐Ig, Proteintech), β‐actin (mouse, 1:800, No.66009‐1‐Ig, Proteintech), HNF1A polyclonal antibody (rabbit, 1:1000, No. 22426‐1‐AP, Proteintech), Phospho‐HNF1A (Ser247) Ab (rabbit, 1:1000, AF7045, Affinity), or Netrin‐1 (rabbit, 1:500, DF8579, Affinity), followed by washes with Tris‐buffered saline (PBS)‐Tween 20.

Techniques: Knockdown, Knock-Out, Western Blot, Expressing, Immunofluorescence, Staining, Activation Assay, TUNEL Assay, Transmission Assay, Electron Microscopy

Journal: Advanced Science

Article Title: Epigenetic Regulation of DAPK1 and Netrin‐1 Drives Diabetic Encephalopathy

doi: 10.1002/advs.202502535

Figure Lengend Snippet: The elevation of DAPK1 is driven by suppression of miR‐216a‐5p. A) Relative mRNA levels of DAPK1 in hippocampus of control (SCB and db/m) and diabetic (STZ and db/db) mice, showing no significant differences across groups. n = 5. B) Heatmap of miRNA expression profiles in different groups. C) Diagram to display the conserved binding site in DAPK1 3′UTR to the miR‐216a‐5p. The mutant sequence in 3′UTR of DAPK1 for luciferase analysis was provided at the bottom. D) Luciferase reporter assay of wild‐type (WT) and mutant (MUT) DAPK1 3′UTR constructs in cells transfected with miR‐216a‐5p mimic or scramble control. n = 6 per group. E,F) N2a cells were transfected with miR‐216a‐5p agomir or antagomir. E) The cell lysates were collected, and the protein levels of DAPK1 were then detected by western blot (WB) after 48 h, F) with quantification. n = 4 per group. G,H) Immunofluorescent staining showing DAPK1 (green) and miR‐216a‐5p (red) in the hippocampus of db/m and db/db mice, with DAPI counterstaining (blue). Correlation analysis between miR‐216a‐5p and DAPK1 levels, showing a significant negative relationship ( R 2 = 0.7465, p < 0.001). n = 15 slices per group from five mice. Data are presented as mean ± S.E.M., and statistical analysis was performed using an unpaired Student's t ‐test or linear regression. * p < 0.05, ** p < 0.01, and **** p <0.0001.

Article Snippet: After blocking with 5% nonfat milk for 30 min, the membranes were incubated with primary antibodies overnight at 4 °C to DAPK1 (rabbit, 1:1000, No.25136‐1‐AP, Proteintech), DAPK1 (mouse, 1:1000, No.67815‐1‐Ig, Proteintech), β‐actin (mouse, 1:800, No.66009‐1‐Ig, Proteintech), HNF1A polyclonal antibody (rabbit, 1:1000, No. 22426‐1‐AP, Proteintech), Phospho‐HNF1A (Ser247) Ab (rabbit, 1:1000, AF7045, Affinity), or Netrin‐1 (rabbit, 1:500, DF8579, Affinity), followed by washes with Tris‐buffered saline (PBS)‐Tween 20.

Techniques: Control, Expressing, Binding Assay, Mutagenesis, Sequencing, Luciferase, Reporter Assay, Construct, Transfection, Western Blot, Staining

Journal: Advanced Science

Article Title: Epigenetic Regulation of DAPK1 and Netrin‐1 Drives Diabetic Encephalopathy

doi: 10.1002/advs.202502535

Figure Lengend Snippet: Knockdown of DAPK1 in excitatory neurons restores Netrin‐1 (Ntn1) expression. A) Heatmap showing the relative mRNA levels of various neurotrophic factors, including Ntn1, in db/db mice compared to controls. n = 3 per group. B) Western blot analysis of Ntn1 protein levels in the hippocampus of db/m and db/db mice, with quantification showing a significant reduction of Ntn1 in db/db mice. n = 5 per group. C) Western blot analysis of Ntn1 in DAPK1‐KD +/− and DAPK1‐KD −/− diabetic mice treated with tamoxifen (TAM), with quantification indicating restoration of Ntn1 levels following DAPK1 knockdown. n = 5 per group. D) Immunofluorescent staining for DAPK1 (green) and Ntn1 (red) in the hippocampus of different groups, with merged images showing colocalization. E) Quantitative analysis of Ntn1 levels in different group mice, demonstrating increased Ntn1 expression following DAPK1 kinase domain knockdown. n = 6 slices per group from three mice. F) Correlation analysis between the relative protein levels of DAPK1 and Ntn1 ( R 2 = 0.4808, p < 0.001). n = 6 per group. G) Schematic of the dual‐luciferase reporter assay construct used to assess Ntn1 promoter activity in response to DAPK1 kinase domain knockdown. H) Relative luciferase activity in cells after cotransfection with the reporter vector and the control or effector vector for 48 h. I) Western blot analysis of HNF1A and Ntn1 levels in control and DAPK1‐overexpressing (oe‐DAPK1) cells, with J) quantification showing a decrease in HNF1A and Ntn1 levels upon DAPK1 overexpression. n = 3 per group. K) Chromatin immunoprecipitation (ChIP) assay was performed to assess the binding of HNF1A to the Ntn1 promoter in HEK293T cells transfected with either DAPK1 vector, wild‐type HNF1A, or phosphorylation‐deficient mutant HNF1A‐S249A. Immunoprecipitated DNA was quantified using qPCR targeting the Ntn1 promoter region. n = 3 per group. L) Luciferase reporter assay showing the transcriptional activity of wild‐type HNF1A and its phosphorylation mutants (S249A and S249D) on the Ntn1 promoter. HEK293T cells were co‐transfected with the Ntn1 promoter‐luciferase construct and the indicated HNF1A variants. Luciferase activity was measured 24 post‐transfection and normalized to Renilla luciferase. n = 5 per group. Data are presented as mean ± S.E.M., and statistical analysis was performed using an unpaired Student's t ‐test or one‐way or two‐way analysis of variance (ANOVA), unless otherwise specified. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking with 5% nonfat milk for 30 min, the membranes were incubated with primary antibodies overnight at 4 °C to DAPK1 (rabbit, 1:1000, No.25136‐1‐AP, Proteintech), DAPK1 (mouse, 1:1000, No.67815‐1‐Ig, Proteintech), β‐actin (mouse, 1:800, No.66009‐1‐Ig, Proteintech), HNF1A polyclonal antibody (rabbit, 1:1000, No. 22426‐1‐AP, Proteintech), Phospho‐HNF1A (Ser247) Ab (rabbit, 1:1000, AF7045, Affinity), or Netrin‐1 (rabbit, 1:500, DF8579, Affinity), followed by washes with Tris‐buffered saline (PBS)‐Tween 20.

Techniques: Knockdown, Expressing, Western Blot, Staining, Luciferase, Reporter Assay, Construct, Activity Assay, Cotransfection, Plasmid Preparation, Control, Over Expression, Chromatin Immunoprecipitation, Binding Assay, Transfection, Phospho-proteomics, Mutagenesis, Immunoprecipitation

Journal: iScience

Article Title: MBNL2 promotes aging-related cardiac fibrosis via inhibited SUMOylation of Krüppel-like factor4

doi: 10.1016/j.isci.2024.110163

Figure Lengend Snippet: Inhibition of MBNL2 activated SUMOylation of KLF4 (A–D) Representative graphs of SUMO1/2/3 protein in Young vs. Aged, Control vs. H 2 O 2 , oe-NC vs. oe-MBNL2, sh-NC vs. sh-MBNL2. (E–G) Representative and statistical graphs of SENP1 and SUMO1 protein. (H–J) Representative and statistical graphs of KLF4 and SENP1 protein. (K) Representative immunoblots of KLF4-SUMOylation after incubation with sh-MBNL2 or/and H 2 O 2 in CFs ( n = 3). (L–O) Western blot analysis and quantification of TGF-β1, T-SMAD3 and P-SMAD3 protein levels (n = 3–6, data are expressed as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01 vs. the control group, # p < 0.05, ## p < 0.01 vs. the H 2 O 2 group, & p < 0.05, && p < 0.01 vs. the H 2 O 2 +sh-MBNL2 group).

Article Snippet: Anti-SUMO1 antibody , Proteintech , Cat# 10329-1-AP; RRID:AB_2286872.

Techniques: Inhibition, Control, Western Blot, Incubation

Journal: iScience

Article Title: MBNL2 promotes aging-related cardiac fibrosis via inhibited SUMOylation of Krüppel-like factor4

doi: 10.1016/j.isci.2024.110163

Figure Lengend Snippet:

Article Snippet: Anti-SUMO1 antibody , Proteintech , Cat# 10329-1-AP; RRID:AB_2286872.

Techniques: Recombinant, Staining, cDNA Synthesis, SYBR Green Assay, Magnetic Beads, Plasmid Preparation, Extraction, Microarray, Software