Journal:

Article Title: Exercise Decreases Myelin-Associated Glycoprotein Expression in the Spinal Cord and Positively Modulates Neuronal Growth

doi: 10.1002/glia.20521

Figure Lengend Snippet: Cdk5 kinase activity is increased in primary cortical neurons grown on myelin extracted from the spinal cord of exercised animals. (A) Primary cortical neurons were grown for 24 h on PDL or on increasing concentration (0.5–2 μg) of myelin extracted from either sedentary (sed-myelin) or exercised animals (ex-myelin). Cdk5-associated kinase activity was determined after immunoprecipitation with anti-cdk5 antibodies and by using histone H1 as a substrate. Panel A shows a representative experiment. (B) Quantification of the effect of 1 μg myelin on Histone H1-associated cdk5 kinase activity in cortical neurons. Data were obtained by PhosphorImager analysis of the auto-radiographs and are expressed as percentage of cdk5 activity found in neuronal cells grown on PDL. Average of four independent experiment ± SEM is shown, *P < 0.05 vs. PDL cultured neurons (Friedman Test followed by Dunn's Multiple Comparison Test). (C, D) Cortical neurons plated for 24 h on exercise-myelin display longer processes compared with cells plated on sedentary-myelin. Pictures of live neuronal cells were taken on a Zeiss inverted microscope (Scale bar = 50 μm)

Article Snippet: Lysates (50 μg) were immunoprecipitated with a saturating concentration of a rabbit polyclonal antibody against cdk5 (C8; Santa Cruz Biotechnology, Santa Cruz, CA) plus 20 μL of protein A-agarose (Santa Cruz Biotechnology) for 2 h at 4°C.

Techniques: Activity Assay, Concentration Assay, Immunoprecipitation, Cell Culture, Comparison, Inverted Microscopy

Journal:

Article Title: Exercise Decreases Myelin-Associated Glycoprotein Expression in the Spinal Cord and Positively Modulates Neuronal Growth

doi: 10.1002/glia.20521

Figure Lengend Snippet: Cdk5-associated kinase activity is greatly elevated by exercise in the lumbar enlargement of rat spinal cord. (A) Exercise is a positive modulator of cdk5 kinase activity in the rat lumbar spinal cord region. The animals were exposed to exercise for 7 days. Cdk5 activity was determined after immunoprecipitation with anti-cdk5 antibodies and using histone H1 as a substrate. Data were obtained by PhosphorImager analysis of the autoradiographs. Data are mean ± SEM and are expressed as percentage of cdk5 activity measured in sedentary animals (*P < 0.05 (Student's t-test); n = 3 animals/group). Insert shows representative gel bands. (B) The increase in cdk5-associated kinase activity is proportional to the amount of volunteer running. The graph shows the correlation between individual variability in running and the increase in cdk5 activity (p = 0.0011; r = 0.973). Changes in kinase activity were plotted as function of the amount of running distance for each animal that ran for 7 days (open triangles) or remained sedentary (filled triangles).

Article Snippet: Lysates (50 μg) were immunoprecipitated with a saturating concentration of a rabbit polyclonal antibody against cdk5 (C8; Santa Cruz Biotechnology, Santa Cruz, CA) plus 20 μL of protein A-agarose (Santa Cruz Biotechnology) for 2 h at 4°C.

Techniques: Activity Assay, Immunoprecipitation

Journal: Open Chemistry

Article Title: Phosphorylation of Pit-1 by cyclin-dependent kinase 5 at serine 126 is associated with cell proliferation and poor prognosis in prolactinomas

doi: 10.1515/chem-2021-0070

Figure Lengend Snippet: Figure 1: Both CDK5 inhibition and depletion affect the phosphorylation level of Ser126-Pit-1. (a) Bioinformatics analysis reveals that Ser126 Pit-1 was the only typical CDK5 potential phosphorylation site. (b) GH3 pituitary cells were transfected with WT-Pit-1 or Pit-1-126A. Western blotting shows that the anti-pS126-Pit-1 antibody can specifically recognize the phosphorylation of Ser126-Pit-1. (c). Western blot (left) and densitometry analysis (right) showed that S126-Pit-1 phosphorylation was reduced by roscovitine treatment at the indicated concentra- tions. The protein levels of Pit-1 was not clearly different. β-Actin served as a loading control. (d) Western blot showed that Ser126-Pit-1 phosphorylation was reduced by CDK5 siRNA treatment at the indicated concentrations. β-Actin served as a loading control. **, P < 0.01; ***, P < 0.001. The bar represents the mean ± SD.

Article Snippet: Primary antibodies anti-CDK5 (ab40773, 1/200) were obtained from Abcam (Cambridge, MA, USA).

Techniques: Inhibition, Phospho-proteomics, Transfection, Western Blot, Control

Journal: Open Chemistry

Article Title: Phosphorylation of Pit-1 by cyclin-dependent kinase 5 at serine 126 is associated with cell proliferation and poor prognosis in prolactinomas

doi: 10.1515/chem-2021-0070

Figure Lengend Snippet: Figure 2: pSer126-Pit-1 promotes GH3 cell proliferation and suppresses apoptosis. (a) Western blot showed pSer126-Pit-1 in GH3 cell with WT-Pit-1 or Pit-1-126A. (b) The colony formation test shows that the percentage of colonies formed was reduced in the Pit-1-126A groups compared with that of the Pit-1 groups. (c) The apoptosis rates of the Pit-1-126A groups were significantly higher than those of the Pit-1 groups. (d) Higher cell viability was observed in the Pit-1 groups. (e) The effect of CDK5 could be reversed by Pit-1 siRNA. **, P < 0.01; ***, P < 0.001. The bar represents the mean ± SD.

Article Snippet: Primary antibodies anti-CDK5 (ab40773, 1/200) were obtained from Abcam (Cambridge, MA, USA).

Techniques: Western Blot

Journal: bioRxiv

Article Title: CA1i pyramidal neurons mediate the role of NMDA receptor subunit GluN3A in depressive behavior and D-serine anti-depression

doi: 10.1101/2024.04.24.590964

Figure Lengend Snippet: a Experimental scheme of CSDS modeling and behavioral tests. b Schematic of CSDS. c Example trajectory heatmap of SIT in control mice (left) and stress mice (right). d Social interaction ratio of SIT ( n = 9 mice per group). e Immobility time in the TST ( n = 8 mice per group). f Immobility time in the FST ( n = 6-7 mice per group). g Example trajectory heatmap of OFT in control mice (left) and stress mice (right). h The total distance traveled in OFT ( n = 7-8 mice per group). i The number of entries to centre area of OFT ( n = 6-7 mice per group). j The time in the centre area of OFT ( n = 6-7 mice per group). k Example trajectory heatmap of EPM in control mice (left) and stress mice (right). l The number of entries to open arms of EPM ( n = 9 mice per group). m Percentage of time spent in the open arms of EPM ( n = 9 mice per group). n Quantitative data of Grin3A mRNA in target brain area from control and stress mice ( n = 6-16 mice per group). o Quantitative data of GluN3A protein in target brain area from control and stress mice ( n = 4-7 mice per group). p Representative western blots for dissected target brain area. q Experimental scheme of behavioral studies. r Social interaction ratio of SIT ( n = 8 mice per group). s Immobility time in the TST ( n = 9 mice per group). t Immobility time in the FST ( n = 8 mice per group). u The total distance traveled in OFT ( n = 8 mice per group). v The entries of centre area in OFT ( n = 8 mice per group). w The time in the centre area of OFT ( n = 8 mice per group). x The number of entries to open arms of EPM ( n = 8 mice per group). y Percentage of time spent in the open arms of EPM ( n = 8 mice per group). * P < 0.05, *** P < 0.001.

Article Snippet: The PVDF membrane was blocked in TBST solution containing 5% milk powder and then incubated overnight at 4°C with diluted primary antibodies (5% milk powder TBST): Grin3A (1:500; Cat# orb694249, Biorbyt, UK); Gapdh (1:3000; Cat# 2118, Cell Signaling, USA).

Techniques: Control, Western Blot

Journal: bioRxiv

Article Title: Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer

doi: 10.1101/2022.01.09.475410

Figure Lengend Snippet: SDHB loss activates a succinate-Ca 2+ -calpain-Cdk5 cascade. (A) [Ca 2+ ] i activity reported by time lapse live-cell imaging of PC cells (WT vs . SDHB KO) loaded with Fluo-4 AM, images acquired pre- or post-stimulation with ionomycin (10 µM). Representative pseudo-colored images are shown with time-course of fluorescence intensity quantification as % [Ca 2+ ] i . (B) LC- MS quantitation of succinate in WT and KO cell extracts. (C) Quantitation of fluorescence intensity and photomicrographs showing effects of ionomycin on %[Ca 2+ ] i in WT hPheo1 cells treated with succinate (2 mM) or controls. (D) Time lapse measurement of [Ca 2+ ] i in SDHB KO cells pretreated with SUCNR1 antagonist, NF-56-EJ40, with representative images. (E) Schematic workflow of calpain sensor, and representative pseudocolor FRET map and normalized FRET index of wild type and SDHB KO hPheo1 cells. (F) Immunoblots of Cdk5, p25/p35 in WT and SDHB KO cells with quantitation. (G) 3D surface plot of immunostained cells comparing relative levels of p35/25 between WT and KO cells. (H-I) Representative confocal 3D photomicrographs and quantitation showing p35/25 expression in WT hPheo1 treated with increasing concentration of succinate as indicated. (J) Quantitative immunoblotting of Cdk5/p35/p25 levels in tumor lysates derived from WT (n=4) and KO (n=6) xenografts. Data are means ± SEM, *p < 0.05, **p < 0.01, ***p<0.001; n.s., non-significant compared by t -test and or one-way ANOVA. (K) Schematic illustrating mechanism of action mediated via succinate accumulation and concomitant disruption of Ca 2+ / Cdk5 homeostasis in chromaffin cells.

Article Snippet: Primary antibodies used included those for Cdk5 (Rockland# 200-301-163), -p35/25 (Cell Signaling#2680), ChrA (abcam#ab15160), anti-tyrosine hydroxylase (abcam#ab6211), and GFP (Cell signaling#2956).

Techniques: Activity Assay, Live Cell Imaging, Fluorescence, Liquid Chromatography with Mass Spectroscopy, Quantitation Assay, Western Blot, Expressing, Concentration Assay, Derivative Assay, Disruption

Journal: bioRxiv

Article Title: Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer

doi: 10.1101/2022.01.09.475410

Figure Lengend Snippet: Characterization of in vitro and in vivo model of hPheo1. (A) Immunoblot showing spectrin cleavage as a function of calpain activity in PC cells. (B) Quantitative immunoblots showing dose-dependent effect of dimethyl succinate (0, 0.5, 2.5, 5, 10 mM) on spectrin cleavage and p35/25 levels in WT hPheo1. (C) Plot comparing % tumor burden of WT vs. KO hPheo1 derived xenografts. (D) Quantitative immunoblotting of spectrin cleavage in extracts derived from WT (n=4) and KO (n=6) xenografts. (E) Immunostains for chromaffin cell neuroendocrine markers, TH, ChrA, Cdk5 and p35/25 in xenograft tissues. (F) Immunoblot quantification of TH, and ChrA in WT and KO tumor tissues as indicated. (G) Signaling schematic showing aberrant Cdk5 as a downstream effector in response to SDHB loss. n = 11 (C) and 4-6 (F), data are means ± S.E.M., *p < 0.05, **p < 0.01, ***p<0.001, n.s. non-significant, Student’s t -test or one way ANOVA.

Article Snippet: Primary antibodies used included those for Cdk5 (Rockland# 200-301-163), -p35/25 (Cell Signaling#2680), ChrA (abcam#ab15160), anti-tyrosine hydroxylase (abcam#ab6211), and GFP (Cell signaling#2956).

Techniques: In Vitro, In Vivo, Western Blot, Activity Assay, Derivative Assay

Journal: bioRxiv

Article Title: Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer

doi: 10.1101/2022.01.09.475410

Figure Lengend Snippet: SDHB and Cdk5 coactivator expression inversely correlate in human PC. (A) Box plots of Cdk5R1 ( i.e. , p35) expression in PC/PG tumors (n=179) vs. normal adrenal (n=3). (B) Correlation between SDHB and Cdk5R1 (p35), data quarried from cBioportal PCPG PanCancer atlas; (R is Spearman coefficient -0.41; p=6.78e). (C) Quantitation and representative blots showing protein levels of SDHB, Cdk5, and p35/25 in human sporadic PC tumors (n=11) compared with normal medulla (n=5). (D) Expression analysis of SDHB, Cdk5, p35/25 in human SDHB mutant tumors (n=7) vs. normal medulla (n=7). (E) HE and immunostains of Cdk5 and p35/p25 in human PC, scale bar = 50 µm. (F) Histological assessment of Cdk5, and p35/25 in tissue microarray sections of adrenocortical adenoma (ACA, n=40), and PC ( n=30); quantification presented as optical density; *p<0.05, **p < 0.01, ***p < 0.001 compared using t -test with Welch’s correction; n.s., non-significant.

Article Snippet: Primary antibodies used included those for Cdk5 (Rockland# 200-301-163), -p35/25 (Cell Signaling#2680), ChrA (abcam#ab15160), anti-tyrosine hydroxylase (abcam#ab6211), and GFP (Cell signaling#2956).

Techniques: Expressing, Quantitation Assay, Mutagenesis, Microarray

Journal: bioRxiv

Article Title: Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer

doi: 10.1101/2022.01.09.475410

Figure Lengend Snippet: Gene expression analysis of Cdk5/SDHB signaling components in human PCPG dataset. (A) Box plot showing distribution of gene expression values for PC (n=84) and normal adrenal tissues (n=5) following normalization (NCBI/GEO/GSE-19422). X and Y-axis represents GEO samples and expression values. (B) Plots comparing gene expression values of Cdk5 signaling components with that of SDHB, SDHA, SDHC, and SDHD. (C) Table presents the statistical significance following analysis of gene expression values comparing Cdk5 signaling components with that of SDHB, SDHA, SDHC, or SDHD as indicated. Data are means ± SD ***p<0.001, n.s. non-significant, Tukey’s multiple comparisons test.

Article Snippet: Primary antibodies used included those for Cdk5 (Rockland# 200-301-163), -p35/25 (Cell Signaling#2680), ChrA (abcam#ab15160), anti-tyrosine hydroxylase (abcam#ab6211), and GFP (Cell signaling#2956).

Techniques: Gene Expression, Expressing

Journal: bioRxiv

Article Title: Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer

doi: 10.1101/2022.01.09.475410

Figure Lengend Snippet: Aberrant Cdk5 develops PC in bi-transgenic mice. (A) Schematic of PiggyBac expression system designed to engineer single-copy PNMT–tTA (gene-1) crossed with Tet-Op- p25GFP (gene-2) to generate Dox ON/OFF bi-transgenic model. (B) T2-weighted representative MRI coronal images indicating temporal changes in adrenal gland size in WT (PNMT-tTA) vs. p25-ON (PNMT-tTA×p25GFP) mice. (C) MRI quantitation of adrenal size in p25-ON mice (n=11) compared to WT (n=6). (D) Immunoblots showing expression of p25-GFP, Cdk5, TH, and ChrA in tissue lysates derived from p25-ON or WT adrenal glands as indicated. (E) Histological assessment of NE markers in WT adrenals or PC tissue sections derived from p25-ON mice, scale bar 100 µM. (F-H) Measurements of plasma metanephrines (F), nor-metanephrines (G) and tail-cuff blood pressure (H); (n=6-11), *p < 0.05, **p < 0.01 compared by using t -test with Welch’s correction.

Article Snippet: Primary antibodies used included those for Cdk5 (Rockland# 200-301-163), -p35/25 (Cell Signaling#2680), ChrA (abcam#ab15160), anti-tyrosine hydroxylase (abcam#ab6211), and GFP (Cell signaling#2956).

Techniques: Transgenic Assay, Expressing, Quantitation Assay, Western Blot, Derivative Assay, Clinical Proteomics

Journal: bioRxiv

Article Title: Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer

doi: 10.1101/2022.01.09.475410

Figure Lengend Snippet: Characterization of functional Cdk5 targets in human PC. (A) Schematic showing NE tumors from p25-ON/OFF model analyzed for potential Cdk5 phosphorylation sites in PC. Function of phosphosite ratio (log2 fold change) between growing (p25-ON) and arrested (p25- OFF) tumors is plotted against p values. (B) Table showing Cdk5 phosphotargets downregulated in growing p25-ON tumors. (C) Graphical presentation of network tree highlighting enriched pathways associated with downregulated Cdk5 targets. (D) Table summarizing novel phosphosites selected to evaluate their effects on PC cellular proliferation. (E) Effects of phospho- and dephospho-mimetics (D/E/A) on cell growth was determined by dual fluorescence acridine orange/propidium iodide (AO/PI) viability staining, WT-wild-type; EV-empty vector. (F) Schematic of AMP-activated protein kinase heterotrimeric complex composed of α-, β- and γ-subunits. Novel phosphorylation site, Ser65 (S65) is located in PRKAG2 subunit of trimeric complex. (G) Immunoblot and quantification of phospho-Thr172 (P-T172) AMPK catalytic phosphorylation in response to PRKAG2 S65D/E/A phosphosite mutant expression, n=4. Values are means ± SEM, *p<0.05, **p<0.01, ***p<0.001, n.s. non-significant, one-way ANOVA multiple comparisons with Tukey’s method. (H) Quantitative immunoblotting of P-S65 PRKAG2 and -T172 AMPKα in human PC (n=11) vs. Normal adrenal medulla (n=4). Values are means ± SEM, *p<0.05, Student’s t -test. (I) Correlation plot between P-S65 PRKAG2 and -T172 AMPK in PC patients; r is Spearman’s correlation coefficient, **p<0.01.

Article Snippet: Primary antibodies used included those for Cdk5 (Rockland# 200-301-163), -p35/25 (Cell Signaling#2680), ChrA (abcam#ab15160), anti-tyrosine hydroxylase (abcam#ab6211), and GFP (Cell signaling#2956).

Techniques: Functional Assay, Phospho-proteomics, Fluorescence, Staining, Plasmid Preparation, Western Blot, Mutagenesis, Expressing

Journal: bioRxiv

Article Title: Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer

doi: 10.1101/2022.01.09.475410

Figure Lengend Snippet: Generation of phospho-Ser65 PRKAG2 antibody. (A) Clustal omega alignment shows conservation of Ser65 phosphorylation site in human and mouse PRKAG2 amino acid sequence. (A) Dot blots shows selective reactivity of anti-serum for phospho-PRKAG2 peptide. (C) Immunoblots of AMPK holo-complex (α1β1γ2) comprised of WT or S65A PRKAG2 phosphorylated by GSK3β in vitro, demonstrating specificity of affinity purified antibody for P-S65 PRKAG2 (WT) and not the S65A form. (D) The mRNA expression of PRKAG2 and AMPKα in PC vs. normal adrenals. Data derived from Gene Expression Profiling Interactive Analysis (GEPIA) PCPG dataset, p-value cutoff 0.05. (E) NetPhos3.1 analysis suggests CDK5 and GSK3β as possible kinases that phosphorylates Ser65 PRKAG2. (F-G) IP kinase assay performed to phosphorylate purified PRKAG2 (WT, S65D, S65A) incubated with immunoprecipitated GSK3 (left) in the presence of cold ATP at 30°C for 60 min. The reaction products were probed with antibody to phospho-Ser65 PRKAG2 (right), CB=Coomassie blue stained protein. (H). Immunoblots densitometry shows differential expression levels of P-GSK3 in WT vs . SDHB KO cells.

Article Snippet: Primary antibodies used included those for Cdk5 (Rockland# 200-301-163), -p35/25 (Cell Signaling#2680), ChrA (abcam#ab15160), anti-tyrosine hydroxylase (abcam#ab6211), and GFP (Cell signaling#2956).

Techniques: Phospho-proteomics, Sequencing, Western Blot, In Vitro, Affinity Purification, Expressing, Derivative Assay, Gene Expression, IP-Kinase Assay, Purification, Incubation, Immunoprecipitation, Staining, Quantitative Proteomics

Journal: bioRxiv

Article Title: Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer

doi: 10.1101/2022.01.09.475410

Figure Lengend Snippet: Cdk5/GSK3 signaling regulates P-PRKAG2/P-AMPK. (A) In vitro phosphorylation of recombinant PRKAG2 by GSK3, with time-dependent 32 P incorporation and Coomassie stained protein (CBB) shown with stoichiometry. (B) Immunoblots showing GSK3 but not Cdk5 phosphorylates Ser65 PRKAG2 in vitro . (C) Immunoblots of recombinant AMPK holoenzyme trimeric complex (α1β1γ2) phosphorylated by GSK3β. (D) Effects of ectopic expression of wildtype (WT) vs. kinase dead (KD) Cdk5 vector on the levels of indicated phosphorylation sites in PC cells. (E) Dose-dependent effects of GSK3 (SB216763, 24 h) vs. CDK5 inhibition (CP681301, 24 h) on SDHB KO hPheo1 cell viability. (F) Plot showing dose dependent effect of AMPK activator AICAR on cell viability treated for 24 h. (G) Time-dependent effects of AMPK activators, Metformin (20 mM), 2-Deoxy-D-glucose (2-DG, 20 mM), and AMPK inhibitor, Compound C (10 μM) on KO hPheo1 cell viability. n=3, values are mean ± SEM, *p<0.05, ** p < 0.01 compared by one-way ANOVA. (H) Schematic of the potential signaling mechanism where aberrant CDK5/p25/GSK3β crosstalk regulates phospho-dynamics between S-65 PRKAG2 and T-172 AMPKα.

Article Snippet: Primary antibodies used included those for Cdk5 (Rockland# 200-301-163), -p35/25 (Cell Signaling#2680), ChrA (abcam#ab15160), anti-tyrosine hydroxylase (abcam#ab6211), and GFP (Cell signaling#2956).

Techniques: In Vitro, Phospho-proteomics, Recombinant, Staining, Western Blot, Expressing, Plasmid Preparation, Inhibition

Journal: bioRxiv

Article Title: Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer

doi: 10.1101/2022.01.09.475410

Figure Lengend Snippet: Targeting Cdk5 to regulate the P-PRKAG2/P-AMPK cascade. (A) Chemical structures of Cdk5 inhibitors, Roscovitine, CR8 and MRT3-007. (B) IC50 heatmap showing in vitro kinase profile of CR8, Roscovitine, MRT3-124, and MRT3-007. (C) Dose-response effects of Cdk5 inhibitors on cell viability (left) and colony formation (right) of hPheo1. n=4, data are means ± S.E.M. (D) Glycolytic profile of SDHB KO cells treated with or without MRT3-007 ([Cdk5 in ], 25 nM for 12 h, (left). Plots comparing basal glycolysis rate, glycolytic capacity, and glycolytic reserve between control versus MRT3-007 (Cdk5 in ) (right). (E) Bioenergetic phenotype of KO cells in response to MRT3-007. Data are means ± SD, *p < 0.05, Student t -test, n=2 (10 replicates per group). (F) Immunoblot quantification showing time dependent effect of MRT3-007 (Cdk5 in ) on phosphorylation states of P-S65 PRKAG2, P-T172 AMPK and P-S79 ACC respectively. n=3, values presented as fold change normalized with time=0. Data are mean ± S.E,M. (G-I) Immunoblot and densitometric analysis presents impact of GSK3 inhibitor, SB216763 (5 µM, pretreatment 10 h) on Cdk5 in -induced phosphorylations as indicated. (J) hPheo1 pre-incubated with or without compound C (CC, 10 µM), representative blot and quantitation comparing effects of Cdk5 in alone or in combination with CC as indicated.

Article Snippet: Primary antibodies used included those for Cdk5 (Rockland# 200-301-163), -p35/25 (Cell Signaling#2680), ChrA (abcam#ab15160), anti-tyrosine hydroxylase (abcam#ab6211), and GFP (Cell signaling#2956).

Techniques: In Vitro, Control, Western Blot, Phospho-proteomics, Incubation, Quantitation Assay

Journal: bioRxiv

Article Title: Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer

doi: 10.1101/2022.01.09.475410

Figure Lengend Snippet: Selectivity assessment of MRT3-007 (Cdk5 in ). (A) Heatmap shows gene expression of Cdk5, Cdk2 and Cdk1 in human PCPG dataset analyzed in GEPIA (left). Bar graphs compares Cdk5/Cdk2 transcript levels in normal adrenals vs. PC tumors (right). (B) Growth inhibitory effects of Cdk5 in (MRT3-007) compared to that of the Cdk2 inhibitor (CVT-313) on hPheo1. (C) Dose- dependent effect of MRT3-007 on different cancer cell types including WT and SDHB KO hPheo1, MDAMB-231, HepG2, H1184, and HeLa (left) expressing high and low levels of Cdk5/p35/25 as indicated by immunoblot in right. Note MRT3-007 was least effective in cells expressing lowest levels of p25 (MDAMB-231, HepG2, and H184). Data are means ± S.E.M., n=3. (D) IP kinase assays with immunoblot for histone H1 phosphorylation (p-H1) with Cdk5 (left) and Cdk2 (right) immunoprecipitated from hPheo1 cell lysates treated with vehicle (-) vs. MRT3-007 (+) for 12 h. Data are means ± S.E.M., **p<0.01, n.s. non-significant, Student’s t -test, n=2. Coomassie blue (CB) staining shows total histone H1 protein levels. (E) Immunoblot quantitation confirming elevation of p25 in Dox OFF cells (left) as shown with effect of MRT3-007 (Cdk5 in ) on cell viability of p25-ON (Dox OFF) vs. p25-OFF (Dox ON) NE cells (right). Data are means ± S.E.M., ***p<0.001, Student’s t -test, n=3.

Article Snippet: Primary antibodies used included those for Cdk5 (Rockland# 200-301-163), -p35/25 (Cell Signaling#2680), ChrA (abcam#ab15160), anti-tyrosine hydroxylase (abcam#ab6211), and GFP (Cell signaling#2956).

Techniques: Gene Expression, Expressing, Western Blot, Phospho-proteomics, Immunoprecipitation, Staining, Quantitation Assay

Journal: bioRxiv

Article Title: Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer

doi: 10.1101/2022.01.09.475410

Figure Lengend Snippet: Cdk5 in induces senescence–like phenotypic characteristics. Immunoblots quantification showing time dependent effect of Cdk5 in ( i.e MRT3-007) on phosphorylation states or levels of (A) P-S15 p53, (B) p16 INK4a ,(C) p27 Kip , (D) P-S139 H2AX. Values presented as fold change normalized with time=0. n=3, data are means ± S.E,M. (E) Scanning electron microscopy (SEM) images of hPheo1 cell morphology in control vs. Cdk5 in (Indolinone A). (F) Imaging of proliferating cells and those treated with Cdk5 in (MRT3-007, 20 nM, 48 h) for common senescent markers. Representative confocal photomicrographs and quantitation shows phalloidin stain of F-actin, p16 INK4a (inset is senescence-associated-β-galactosidase; β-gal), p27 Kip and P-H2Ax (S139) respectively; scale=136 µM. (G) Representative DNA histogram (left) showing cell cycle profile of KO cells treated with vehicle or Cdk5 in (20 nM); histograms analysis by MODfit LT 3.0 (right) presents cell cycle distribution in G1 (red), early-S (yellow), and late-S/G2/M (green); n=2, Data are means ± SD. (H) Confocal images of Ki67 staining and bar graph show number of Ki67- positive cells.

Article Snippet: Primary antibodies used included those for Cdk5 (Rockland# 200-301-163), -p35/25 (Cell Signaling#2680), ChrA (abcam#ab15160), anti-tyrosine hydroxylase (abcam#ab6211), and GFP (Cell signaling#2956).

Techniques: Western Blot, Phospho-proteomics, Electron Microscopy, Control, Imaging, Quantitation Assay, Staining

Journal: bioRxiv

Article Title: Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer

doi: 10.1101/2022.01.09.475410

Figure Lengend Snippet: Inhibition of p53 or AMPK abrogate effects of Cdk5 in (MRT3-007). (A) Representative blots and densitometry detects P-p53 (S16) and p27 Kip upon Cdk5 in treatment in presence or absence of P53 inhibitor PFT-α (10 µM, 10 h pre-treatment). (B) Representative phase contrast microphotographs showing effects of AMPK inhibitor, Compound C (CC, 10 µM) on Cdk5 in induced morphological changes. (C) Representative histograms and quantitation indicating cell cycle profile of KO cells expressing either WT, S65D or S65A phosphomutants of PRKAG2.

Article Snippet: Primary antibodies used included those for Cdk5 (Rockland# 200-301-163), -p35/25 (Cell Signaling#2680), ChrA (abcam#ab15160), anti-tyrosine hydroxylase (abcam#ab6211), and GFP (Cell signaling#2956).

Techniques: Inhibition, Quantitation Assay, Expressing

Journal: bioRxiv

Article Title: Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer

doi: 10.1101/2022.01.09.475410

Figure Lengend Snippet: (A) Maximum tolerated dose (MTD) of Cdk5 in MRT3-007 in C57BL6 mice expressed as body weight (% of original). Mice injected with escalating doses of 0.75, 1, 2, or 5 mg/kg MRT3- 007, respectively (n=5). (B) Time-dependent effect of vehicle or MRT3-007 (0.5 mg/kg) on body weight of SDHB KO xenografts (n=4).

Article Snippet: Primary antibodies used included those for Cdk5 (Rockland# 200-301-163), -p35/25 (Cell Signaling#2680), ChrA (abcam#ab15160), anti-tyrosine hydroxylase (abcam#ab6211), and GFP (Cell signaling#2956).

Techniques: Injection

Journal: bioRxiv

Article Title: Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer

doi: 10.1101/2022.01.09.475410

Figure Lengend Snippet: Cdk5 inhibition attenuates in vivo tumor growth. (A-B) Tumors in mice implanted with SDHB KO xenografts and treated with vehicle (C, n=8) or Cdk5 in (T, n=10) (0.5 mg/Kg) were analyzed for volume (A) and cell proliferation (Ki67, B). (C) Efficacy of Cdk5 inhibition tested in a metastatic allograft PC model, examined via in vivo bioluminescent imaging. Representative images of vivisected liver (top) and tumor spread indicated by histological analysis and photon flux quantitation (bottom). (D-H) Quantitative immunoblots of phosphorylation sites and proteins indicated in xenograft tumor lysates. n = 5-8, Data are means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 compared by Student’s t -test.

Article Snippet: Primary antibodies used included those for Cdk5 (Rockland# 200-301-163), -p35/25 (Cell Signaling#2680), ChrA (abcam#ab15160), anti-tyrosine hydroxylase (abcam#ab6211), and GFP (Cell signaling#2956).

Techniques: Inhibition, In Vivo, Imaging, Quantitation Assay, Western Blot, Phospho-proteomics

Journal: Cellular & Molecular Biology Letters

Article Title: CDK5 regulates PPARγ/NF-κB signaling to exacerbate obesity-related osteoarthritis via modulating macrophage polarization and chondrocyte apoptosis

doi: 10.1186/s11658-025-00822-7

Figure Lengend Snippet: Increased severity of OA lesions and elevated CDK5 levels in obese patients with OA. Synovial and cartilage samples were collected from six participants without OA (control), six nonobese patients with OA (Thin-OA), and six obese patients with OA (Obesity-OA). A H&E staining was performed on cartilage tissues to evaluate the severity of cartilage degradation and OA pathology. B The severity of articular cartilage damage was evaluated using the Osteoarthritis Research Society International (OARSI) scoring system. C H&E staining was performed on synovial tissue samples to evaluate the severity of synovial inflammation and hyperplasia. D The quantification of synovitis score was evaluated the synovitis severity of synovial tissues. N = 6. E , F Immunoblotting was performed to examine CDK5 levels in synovial and cartilage tissues. N = 3 (biological replicates). ** p < 0.01; # p < 0.05

Article Snippet: Membranes were subjected to 1-h blocking at room temperature (RT) using 5% skim milk in Tris-buffered saline with Tween 20 (TBS-T), followed by an overnight incubation at 4 °C using primary antibodies against CDK5 (10430-1-AP, Proteintech, Wuhan, China), reduced glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (60004-1-IG, Proteintech), PPARγ (16643-1-AP, Proteintech), p-PPARγ (BS-4888R, Bioss, Woburn, USA), p65 (BF8005, Affinity, Changzhou, China), p-p65 (AF2006, Affinity), Aggrecan (DF7561, Affinity), collagen II (AF0135, Affinity), cleaved caspase-3 (25128-1-AP, Proteintech), Bax (50599-2-Ig, Proteintech), and Bcl2 (26593-1-AP, Proteintech).

Techniques: Control, Staining, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: CDK5 regulates PPARγ/NF-κB signaling to exacerbate obesity-related osteoarthritis via modulating macrophage polarization and chondrocyte apoptosis

doi: 10.1186/s11658-025-00822-7

Figure Lengend Snippet: HFD-induced obesity exacerbates DMM-induced OA and M1 macrophage infiltration in mice. Mice were randomly allocated into four groups: Sham, HFD + Sham, DMM, and HFD + DMM groups, and the OA model was established in mice as described; normal diet or HFD was administered to model mice. A H&E staining was performed on cartilage tissue samples to evaluate the severity of cartilage degradation and OA pathology. B Safranin O/Fast Green staining was performed on cartilage tissues to assess proteoglycan loss and cartilage matrix degradation. C IHC staining for cleaved caspase-3 was conducted on cartilage tissue samples to evaluate apoptosis. D IF staining was performed to detect INOS + cells in synovial tissues to assess M1 macrophage infiltration. E The severity of articular cartilage damage was evaluated using the OARSI scoring system. F Quantification of cleaved caspase-3 positive cell intensity based on the IHC staining of the cartilage tissues. G Quantification of INOS fluorescence intensity based on the IF staining of the cartilage tissues. H , I Immunoblotting was performed to determine CDK5 levels in cartilage and synovial tissues. N = 6 (biological replicates) for A – G and N = 3 (biological replicates) for H and I . ** p < 0.01; # p < 0.05, ## p < 0.01

Article Snippet: Membranes were subjected to 1-h blocking at room temperature (RT) using 5% skim milk in Tris-buffered saline with Tween 20 (TBS-T), followed by an overnight incubation at 4 °C using primary antibodies against CDK5 (10430-1-AP, Proteintech, Wuhan, China), reduced glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (60004-1-IG, Proteintech), PPARγ (16643-1-AP, Proteintech), p-PPARγ (BS-4888R, Bioss, Woburn, USA), p65 (BF8005, Affinity, Changzhou, China), p-p65 (AF2006, Affinity), Aggrecan (DF7561, Affinity), collagen II (AF0135, Affinity), cleaved caspase-3 (25128-1-AP, Proteintech), Bax (50599-2-Ig, Proteintech), and Bcl2 (26593-1-AP, Proteintech).

Techniques: Staining, Immunohistochemistry, Fluorescence, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: CDK5 regulates PPARγ/NF-κB signaling to exacerbate obesity-related osteoarthritis via modulating macrophage polarization and chondrocyte apoptosis

doi: 10.1186/s11658-025-00822-7

Figure Lengend Snippet: CDK5 knockdown inhibits obesity-related osteoarthritis cartilage damage and modulates the PPARγ/NF-κB pathway. Mice were randomly allocated into three groups: HFD + DMM, HFD + DMM + lv-shNC, and HFD + DMM + lv-shCDK5 groups. A H&E staining of cartilage tissue from different groups of mice. B The severity of articular cartilage damage was evaluated using the OARSI scoring system. C Safranin O/Fast Green staining of cartilage tissue from different groups of mice. D IHC staining of cleaved caspase-3 in mouse cartilage tissue. E Quantification of cleaved caspase-3-positive cell intensity based on the IHC staining of the cartilage tissues. F Immunoblotting of aggrecan and collagen II protein levels in mouse cartilage tissue. G Immunoblotting of PPARγ, p-PPARγ (Ser273), p65, and p-p65 protein levels in cartilage tissue from different groups of mice. N = 6 (biological replicates) for A – D and N = 3 (biological replicates) for F and G . ** p < 0.01

Article Snippet: Membranes were subjected to 1-h blocking at room temperature (RT) using 5% skim milk in Tris-buffered saline with Tween 20 (TBS-T), followed by an overnight incubation at 4 °C using primary antibodies against CDK5 (10430-1-AP, Proteintech, Wuhan, China), reduced glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (60004-1-IG, Proteintech), PPARγ (16643-1-AP, Proteintech), p-PPARγ (BS-4888R, Bioss, Woburn, USA), p65 (BF8005, Affinity, Changzhou, China), p-p65 (AF2006, Affinity), Aggrecan (DF7561, Affinity), collagen II (AF0135, Affinity), cleaved caspase-3 (25128-1-AP, Proteintech), Bax (50599-2-Ig, Proteintech), and Bcl2 (26593-1-AP, Proteintech).

Techniques: Knockdown, Staining, Immunohistochemistry, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: CDK5 regulates PPARγ/NF-κB signaling to exacerbate obesity-related osteoarthritis via modulating macrophage polarization and chondrocyte apoptosis

doi: 10.1186/s11658-025-00822-7

Figure Lengend Snippet: CDK5 knockdown inhibits M1 macrophage infiltration and modulates the PPARγ/NF-κB pathway in synovial tissues of obesity-related OA mice. A IF staining of INOS levels in mouse synovial tissues. B Quantification of INOS fluorescence intensity based on IF staining of the synovial tissues. C Immunoblotting of PPARγ, p-PPARγ (Ser273), p65, and p-p65 protein levels in synovial tissue from different groups of mice. N = 6 (biological replicates) for A and B and N = 3 (biological replicates) for C . ** p < 0.01

Article Snippet: Membranes were subjected to 1-h blocking at room temperature (RT) using 5% skim milk in Tris-buffered saline with Tween 20 (TBS-T), followed by an overnight incubation at 4 °C using primary antibodies against CDK5 (10430-1-AP, Proteintech, Wuhan, China), reduced glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (60004-1-IG, Proteintech), PPARγ (16643-1-AP, Proteintech), p-PPARγ (BS-4888R, Bioss, Woburn, USA), p65 (BF8005, Affinity, Changzhou, China), p-p65 (AF2006, Affinity), Aggrecan (DF7561, Affinity), collagen II (AF0135, Affinity), cleaved caspase-3 (25128-1-AP, Proteintech), Bax (50599-2-Ig, Proteintech), and Bcl2 (26593-1-AP, Proteintech).

Techniques: Knockdown, Staining, Fluorescence, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: CDK5 regulates PPARγ/NF-κB signaling to exacerbate obesity-related osteoarthritis via modulating macrophage polarization and chondrocyte apoptosis

doi: 10.1186/s11658-025-00822-7

Figure Lengend Snippet: CDK5 affects PA-induced macrophage polarization. Mouse macrophage RAW264.7 cells were transfected with siCDK5 or CDK5 OE. A qRT-PCR was performed to examine INOS and TNF-α (M1 polarization markers), Arg-1, and IL-10 (M2 polarization markers) mRNA levels in macrophages. B FC analysis of INOS + macrophages to evaluate macrophage M1 polarization. C FC analysis of CD206 + macrophages to evaluate macrophage M2 polarization. D Calculation of INOS/CD206 levels to determine macrophage polarization. E Immunoblotting was performed to determine PPARγ, p-PPARγ (Ser273), p-p65, and p65 protein levels in macrophages. All N = 3 (biological replicates). * p < 0.05, ** p < 0.01; ## p < 0.01; && p < 0.01

Article Snippet: Membranes were subjected to 1-h blocking at room temperature (RT) using 5% skim milk in Tris-buffered saline with Tween 20 (TBS-T), followed by an overnight incubation at 4 °C using primary antibodies against CDK5 (10430-1-AP, Proteintech, Wuhan, China), reduced glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (60004-1-IG, Proteintech), PPARγ (16643-1-AP, Proteintech), p-PPARγ (BS-4888R, Bioss, Woburn, USA), p65 (BF8005, Affinity, Changzhou, China), p-p65 (AF2006, Affinity), Aggrecan (DF7561, Affinity), collagen II (AF0135, Affinity), cleaved caspase-3 (25128-1-AP, Proteintech), Bax (50599-2-Ig, Proteintech), and Bcl2 (26593-1-AP, Proteintech).

Techniques: Transfection, Quantitative RT-PCR, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: CDK5 regulates PPARγ/NF-κB signaling to exacerbate obesity-related osteoarthritis via modulating macrophage polarization and chondrocyte apoptosis

doi: 10.1186/s11658-025-00822-7

Figure Lengend Snippet: CDK5 affects PA-induced chondrocyte damage. Mouse chondrocytes were transfected with siCDK5 or CDK5 OE. A FC was performed to determine cell apoptosis. B Immunoblotting was performed to determine the protein levels of cleaved-caspase3, Bax, and Bcl2. C Immunoblotting was performed to determine PPARγ, p-PPARγ (Ser273), p-p65, and p65 protein levels in chondrocytes. All N = 3 (biological replicates). ** p < 0.01; ## p < 0.01; && p < 0.01

Article Snippet: Membranes were subjected to 1-h blocking at room temperature (RT) using 5% skim milk in Tris-buffered saline with Tween 20 (TBS-T), followed by an overnight incubation at 4 °C using primary antibodies against CDK5 (10430-1-AP, Proteintech, Wuhan, China), reduced glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (60004-1-IG, Proteintech), PPARγ (16643-1-AP, Proteintech), p-PPARγ (BS-4888R, Bioss, Woburn, USA), p65 (BF8005, Affinity, Changzhou, China), p-p65 (AF2006, Affinity), Aggrecan (DF7561, Affinity), collagen II (AF0135, Affinity), cleaved caspase-3 (25128-1-AP, Proteintech), Bax (50599-2-Ig, Proteintech), and Bcl2 (26593-1-AP, Proteintech).

Techniques: Transfection, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: CDK5 regulates PPARγ/NF-κB signaling to exacerbate obesity-related osteoarthritis via modulating macrophage polarization and chondrocyte apoptosis

doi: 10.1186/s11658-025-00822-7

Figure Lengend Snippet: PPARγ/NF-κB signaling mediates effects of CDK5 on PA-induced macrophage polarization. Under PA induction, mouse macrophage RAW264.7 cells were transfected with CDK5 OE with or without treatment with Rosi (a PPARγ agonist inhibiting PPARγ phosphorylation at Ser273). A qRT-PCR was performed to examine INOS and TNF-α (M1 polarization markers), Arg-1, and IL-10 (M2 polarization markers) mRNA levels in macrophages. B FC analysis of INOS + macrophages to evaluate macrophage M1 polarization. C FC analysis of CD206 + macrophages to evaluate macrophage M2 polarization. D Calculation of INOS/CD206 levels to determine macrophage polarization. E Immunoblotting was performed to determine PPARγ, p-PPARγ (Ser273), p-p65, and p65 protein levels in macrophages. All N = 3 (biological replicates). ** p < 0.01; ## p < 0.01

Article Snippet: Membranes were subjected to 1-h blocking at room temperature (RT) using 5% skim milk in Tris-buffered saline with Tween 20 (TBS-T), followed by an overnight incubation at 4 °C using primary antibodies against CDK5 (10430-1-AP, Proteintech, Wuhan, China), reduced glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (60004-1-IG, Proteintech), PPARγ (16643-1-AP, Proteintech), p-PPARγ (BS-4888R, Bioss, Woburn, USA), p65 (BF8005, Affinity, Changzhou, China), p-p65 (AF2006, Affinity), Aggrecan (DF7561, Affinity), collagen II (AF0135, Affinity), cleaved caspase-3 (25128-1-AP, Proteintech), Bax (50599-2-Ig, Proteintech), and Bcl2 (26593-1-AP, Proteintech).

Techniques: Transfection, Phospho-proteomics, Quantitative RT-PCR, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: CDK5 regulates PPARγ/NF-κB signaling to exacerbate obesity-related osteoarthritis via modulating macrophage polarization and chondrocyte apoptosis

doi: 10.1186/s11658-025-00822-7

Figure Lengend Snippet: PPARγ/NF-κB signaling mediates effects of CDK5 on PA-induced chondrocyte damage. Under PA treatment, mouse chondrocytes were transfected with CDK5 OE with or without treatment with Rosi (a PPARγ agonist inhibiting PPARγ phosphorylation at Ser273). A FC was performed to determine cell apoptosis. B Immunoblotting was performed to determine the protein levels of cleaved caspase-3, Bax, and Bcl2. C Immunoblotting was performed to determine PPARγ, p-PPARγ (Ser273), p-p65, and p65 protein levels in chondrocytes. D The levels of inflammatory factors INOS, TNF-α, and IL-6 within chondrocytes were detected by qRT-PCR. All N = 3 (biological replicates). ** p < 0.01; ## p < 0.01

Article Snippet: Membranes were subjected to 1-h blocking at room temperature (RT) using 5% skim milk in Tris-buffered saline with Tween 20 (TBS-T), followed by an overnight incubation at 4 °C using primary antibodies against CDK5 (10430-1-AP, Proteintech, Wuhan, China), reduced glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (60004-1-IG, Proteintech), PPARγ (16643-1-AP, Proteintech), p-PPARγ (BS-4888R, Bioss, Woburn, USA), p65 (BF8005, Affinity, Changzhou, China), p-p65 (AF2006, Affinity), Aggrecan (DF7561, Affinity), collagen II (AF0135, Affinity), cleaved caspase-3 (25128-1-AP, Proteintech), Bax (50599-2-Ig, Proteintech), and Bcl2 (26593-1-AP, Proteintech).

Techniques: Transfection, Phospho-proteomics, Western Blot, Quantitative RT-PCR