Journal: Frontiers in Cell and Developmental Biology

Article Title: Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons

doi: 10.3389/fcell.2021.820105

Figure Lengend Snippet: Characterization of NEFL KO iPSC and motor neurons. (A) NEFL was knocked out from control iPSC with two guides targeting 5′UTR and Exon 1. iPSC clones were selected for differentiation. KO candidates were differentiated into motor neurons and absence of full-length NFL was confirmed by immunoblotting. Cell lines with no signal in immunoblotting were used for further assays. (B) NEFL KO and patient (PT) iPSC lines are pluripotent. Control (CTR), NEFL KO (KO1 and KO2) and PT iPSC express pluripotency markers NANOG (green) and TRA-1-60 (red), analyzed by immunocytochemistry (nuclei counterstain with DAPI, blue). Scale bar 50 µm. (C) KO and PT iPSC differentiate with similar efficiency as the isogenic control. Differentiation efficiency of Day 35 motor neurons was assayed by counting ISL1/2 (red) positive and TUJ1 (green) or NFM (not shown) positive cells in immunocytochemistry. DAPI counterstain (blue). Scale bar 50 µm. (D) Quantification of C. Average % of positive nuclei/cytoplasm with standard deviation (SD). Six replicates from three independent differentiations with 18 frames. One-way ANOVA followed with Dunnett’s multiple comparison post hoc test vs. CTR. p > .05. (E) NEFL , NEFM, and NEFH mRNA expression during differentiation. N = 2-3 per time-point. Normalized to ACTB expression and to day 10 expression of respective gene. (F) NFL, NFM, and TUBB3 protein levels during differentiation. Normalized to GAPDH signal intensity. N = 1 per time-point. (G) NFL measurement by Simoa from serum of control individuals and PT. Each dot indicates an individual sample [control values are the same as in ]. Nd = not detectable. Average with SD (pg/ml). (H) NFL measurement by Simoa from iPSC-MN culture media. Treatment with the neurotoxic drug vincristine (VIN), which induces axonal degeneration was used as a positive control. N.T = not treated. CTR n = 4 and KO1 n = 2. Average with SD (ng/ml). (I) Axonal growth is not affected by NFL loss. Masks of phase contrast images of day 21 motor neuron axons in a microfluidic device (Xona, 450 μm). Scale bar 100 µm. (J) Quantification of image I. axon area on axonal side of microfluidic device. Axonal growth and regrowth after mechanical axotomy is not affected by NFL loss. Average relative area of axonal coverage and SEM. n = 3 independent experiments/cell line with 5 frames analyzed per time point. Growth normalized to average day 15 axon area per experiment. X indicates axotomy. Two-way ANOVA followed with Tukey’s multiple comparison post hoc test p > .05. ( K ) Schematic of a microfluidic device used for axon analysis. # indicates abnormal karyotype.

Article Snippet: Membranes were blocked with 5%-nonfat dry milk (Valio) in Tris-base-solution with Tween20 0.1% (TBS-T) and incubated in primary antibodies with 3%-BSA (BioWest) TBS-T. Primary antibodies used in immunoblotting: mouse mAb NFL N-terminal 1:1000 (sc-390732, Santa Cruz), mouse mAb NFL 1:1000 (N5139, Sigma), rabbit mAb NFL C-terminal 1:1000 (MA5-14981, Thermo Fisher), rabbit pAb NFM 1:1000 (20664-1-AP, Proteintech), rabbit pAb NFH 1:1000 (pan-NFH, ab8135, Abcam), mouse mAb TUJ1 1:1000 (801,201, Biosite), rabbit pAb PRPH 1:1000 (AB1530, Merck), and mouse mAb INA 1:1000 (MAB5224, Merck) and secondary antibodies: Goat-anti-mouse (Jackson) 1:5000 or Goat-anti-rabbit (Jackson) 1:10,000.

Techniques: Control, Clone Assay, Western Blot, Immunocytochemistry, Standard Deviation, Comparison, Expressing, Positive Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons

doi: 10.3389/fcell.2021.820105

Figure Lengend Snippet: NFL protein is not induced to detectable levels by TRID or NMDi treatment in PT iPSC-MN. (A) Immunoblotting series (same blot sequentially analyzed with different antibodies) shows no detectable full-length NFL in treated patient motor neurons. Short and long exposure times shown with two separate NFL antibodies. Treatment between days 16–25 with 25 and 50 µM of amlexanox (AMX), 50 and 100 μg/ml of gentamicin (GEN) and 100 and 200 µM of PTC-124 (PTC). Asterisk indicates an unspecific band detected by the NFL antibody. Concentration as µM for AMX and PTC, and µg/ml for GEN. (B) Representative images of AMX toxicity in day 21 motor neurons (treated for 5 days). Motor neuron health is categorized to—no effect, + some neurite swellings, ++ swellings in most neurites, +++ neurite disruption and ++++ severe neurite disruption and cell death. Higher concentration causes more severe disruption. Scale bar 100 µm.

Article Snippet: Membranes were blocked with 5%-nonfat dry milk (Valio) in Tris-base-solution with Tween20 0.1% (TBS-T) and incubated in primary antibodies with 3%-BSA (BioWest) TBS-T. Primary antibodies used in immunoblotting: mouse mAb NFL N-terminal 1:1000 (sc-390732, Santa Cruz), mouse mAb NFL 1:1000 (N5139, Sigma), rabbit mAb NFL C-terminal 1:1000 (MA5-14981, Thermo Fisher), rabbit pAb NFM 1:1000 (20664-1-AP, Proteintech), rabbit pAb NFH 1:1000 (pan-NFH, ab8135, Abcam), mouse mAb TUJ1 1:1000 (801,201, Biosite), rabbit pAb PRPH 1:1000 (AB1530, Merck), and mouse mAb INA 1:1000 (MAB5224, Merck) and secondary antibodies: Goat-anti-mouse (Jackson) 1:5000 or Goat-anti-rabbit (Jackson) 1:10,000.

Techniques: Western Blot, Concentration Assay, Disruption

Journal: Cancers

Article Title: CDK7 Predicts Worse Outcome in Head and Neck Squamous-Cell Cancer

doi: 10.3390/cancers14030492

Figure Lengend Snippet: Expression patterns for ( A ) CDK7 and ( B ) pMED1 in our HNSCC cohort (magnification 20×, insert 30×). TMAs were immunohistochemically stained for CDK7 and pMED1. The cancer specimen showed a variation in protein expression. The expression of the proteins was homogenous within the patients’ samples, meaning there was little disparity within the staining of cells in a single core. In some HNSCCs the cancer cells showed no staining and accordingly protein expression was considered negative. Throughout the cohort a range of staining intensities was obtained, ( A , B ) illustrate samples of a negative, low, medium, and high protein-expressing tumor specimens.

Article Snippet: For the staining of CDK7 and pMED1, the following antibodies were used at the indicated dilution after successful control tissue staining according to data sheets: CDK7 (mouse monoclonal, CDK7 (MO1) Mouse mAb, 1:500, Cell Signaling, Danvers, MA, USA) and pMED1 (rabbit polyclonal, Anti-TRAP220/MED1 (phospho T1457) antibody, 1:100, Abcam, Cambridge, UK).

Techniques: Expressing, Staining

Journal: Cancers

Article Title: CDK7 Predicts Worse Outcome in Head and Neck Squamous-Cell Cancer

doi: 10.3390/cancers14030492

Figure Lengend Snippet: Overview of clinical characteristics of CDK7 high and low groups.

Article Snippet: For the staining of CDK7 and pMED1, the following antibodies were used at the indicated dilution after successful control tissue staining according to data sheets: CDK7 (mouse monoclonal, CDK7 (MO1) Mouse mAb, 1:500, Cell Signaling, Danvers, MA, USA) and pMED1 (rabbit polyclonal, Anti-TRAP220/MED1 (phospho T1457) antibody, 1:100, Abcam, Cambridge, UK).

Techniques: Expressing

Journal: Cancers

Article Title: CDK7 Predicts Worse Outcome in Head and Neck Squamous-Cell Cancer

doi: 10.3390/cancers14030492

Figure Lengend Snippet: Kaplan–Meier graphs with a p -value of log-rank test of ( A ) 5-year overall survival and ( B ) 5-year disease-free survival. The cohort was stratified into two groups based on the median expression of CDK7. Expression above the median was considered as high expression conversely expression below the median was considered as low expression. Upregulation of CDK7 correlated with a significantly shorter OS as well as DFS ( p = 0.037, p = 0.016).

Article Snippet: For the staining of CDK7 and pMED1, the following antibodies were used at the indicated dilution after successful control tissue staining according to data sheets: CDK7 (mouse monoclonal, CDK7 (MO1) Mouse mAb, 1:500, Cell Signaling, Danvers, MA, USA) and pMED1 (rabbit polyclonal, Anti-TRAP220/MED1 (phospho T1457) antibody, 1:100, Abcam, Cambridge, UK).

Techniques: Expressing

Journal: Cancers

Article Title: CDK7 Predicts Worse Outcome in Head and Neck Squamous-Cell Cancer

doi: 10.3390/cancers14030492

Figure Lengend Snippet: Cox regression analysis of CDK7 expression. * p < 0.05.

Article Snippet: For the staining of CDK7 and pMED1, the following antibodies were used at the indicated dilution after successful control tissue staining according to data sheets: CDK7 (mouse monoclonal, CDK7 (MO1) Mouse mAb, 1:500, Cell Signaling, Danvers, MA, USA) and pMED1 (rabbit polyclonal, Anti-TRAP220/MED1 (phospho T1457) antibody, 1:100, Abcam, Cambridge, UK).

Techniques: Expressing

Journal: Cancers

Article Title: CDK7 Predicts Worse Outcome in Head and Neck Squamous-Cell Cancer

doi: 10.3390/cancers14030492

Figure Lengend Snippet: Comparison of CDK7 expression regarding different clinicopathological parameters.

Article Snippet: For the staining of CDK7 and pMED1, the following antibodies were used at the indicated dilution after successful control tissue staining according to data sheets: CDK7 (mouse monoclonal, CDK7 (MO1) Mouse mAb, 1:500, Cell Signaling, Danvers, MA, USA) and pMED1 (rabbit polyclonal, Anti-TRAP220/MED1 (phospho T1457) antibody, 1:100, Abcam, Cambridge, UK).

Techniques: Comparison, Expressing

Journal: Cancers

Article Title: CDK7 Predicts Worse Outcome in Head and Neck Squamous-Cell Cancer

doi: 10.3390/cancers14030492

Figure Lengend Snippet: ( A ) Boxplot showing the expression of CDK7 in different HNSCC tumor sites. No significant difference was observed between the locations. ( B ) Boxplot comparing the expression of CDK7 in PTs, LNs, DMs, and RDs. CDK7 expression in LNs was significantly lower compared to PTs ( p < 0.0001, adjusted). Other comparisons did not reach significance ( p > 0.05). **** p < 0.0001, ● Outliers.

Article Snippet: For the staining of CDK7 and pMED1, the following antibodies were used at the indicated dilution after successful control tissue staining according to data sheets: CDK7 (mouse monoclonal, CDK7 (MO1) Mouse mAb, 1:500, Cell Signaling, Danvers, MA, USA) and pMED1 (rabbit polyclonal, Anti-TRAP220/MED1 (phospho T1457) antibody, 1:100, Abcam, Cambridge, UK).

Techniques: Expressing

Journal: Cancers

Article Title: CDK7 Predicts Worse Outcome in Head and Neck Squamous-Cell Cancer

doi: 10.3390/cancers14030492

Figure Lengend Snippet: CDK7 expression in different tumor immune profiles. Cold tumors had few to no immune cells; in excluded tumors, immune cells were only present in the tumor stroma; and in hot tumors, immune cells were diffusely infiltrating in between the tumor cells. Compared to cold and excluded tumors, hot tumors harbored a significantly higher CDK7 expression (hot vs. cold p = 0.045, hot vs. excluded p = 0.044), while cold and excluded tumors showed no significant difference ( p = 0.91). * p < 0.05, ● Outliers.

Article Snippet: For the staining of CDK7 and pMED1, the following antibodies were used at the indicated dilution after successful control tissue staining according to data sheets: CDK7 (mouse monoclonal, CDK7 (MO1) Mouse mAb, 1:500, Cell Signaling, Danvers, MA, USA) and pMED1 (rabbit polyclonal, Anti-TRAP220/MED1 (phospho T1457) antibody, 1:100, Abcam, Cambridge, UK).

Techniques: Expressing

Journal: Cancers

Article Title: CDK7 Predicts Worse Outcome in Head and Neck Squamous-Cell Cancer

doi: 10.3390/cancers14030492

Figure Lengend Snippet: Scatter plot of CDK7 and pMED1 expression in tissue of ( A ) PTs, ( B ) LNs, ( C ) DMs, and ( D ) RDs. A trendline is shown. Irrespective of the tissue type CDK7 and pMED1 levels showed a significant correlation (DMs p = 0.0019, the rest p < 0.001), while the PCC ranged from 0.39 for LNs to 0.71 for DMs.

Article Snippet: For the staining of CDK7 and pMED1, the following antibodies were used at the indicated dilution after successful control tissue staining according to data sheets: CDK7 (mouse monoclonal, CDK7 (MO1) Mouse mAb, 1:500, Cell Signaling, Danvers, MA, USA) and pMED1 (rabbit polyclonal, Anti-TRAP220/MED1 (phospho T1457) antibody, 1:100, Abcam, Cambridge, UK).

Techniques: Expressing

Journal: Cancers

Article Title: CDK7 Predicts Worse Outcome in Head and Neck Squamous-Cell Cancer

doi: 10.3390/cancers14030492

Figure Lengend Snippet: Kaplan–Meier graphs with p -values acquired from log-rank tests of ( A ) 5-year OS and ( B ) 5-year DFS. The cohort was stratified into four groups based on CDK7 and pMED1 expression. The expression above the median for the respective protein was considered as a high expression, the expression below the median as low expression. Based on this classification, four groups were formed to display all possible combinations of expression patterns. The log-rank tests revealed no significant difference in 5-year OS or 5-year DFS for the four groups. Nonetheless, we observed that curves grouped by CDK7 expression. There was a trend of shorter 5-year OS and 5-year DFS for both groups with high CDK7 expression compared to the two groups with low CDK7 expression. This effect seemed to be predominant and independent of the pMED1 expression level.

Article Snippet: For the staining of CDK7 and pMED1, the following antibodies were used at the indicated dilution after successful control tissue staining according to data sheets: CDK7 (mouse monoclonal, CDK7 (MO1) Mouse mAb, 1:500, Cell Signaling, Danvers, MA, USA) and pMED1 (rabbit polyclonal, Anti-TRAP220/MED1 (phospho T1457) antibody, 1:100, Abcam, Cambridge, UK).

Techniques: Expressing

Journal: Pediatric research

Article Title: Preterm birth alters the feeding-induced activation of Akt signaling in the muscle of neonatal piglets.

doi: 10.1038/s41390-022-02382-4

Figure Lengend Snippet: Fig. 2 Preterm birth attenuates the phosphorylation of Akt1 and Akt2, but not Akt3. The phosphorylation of individual Akt isoform was analyzed by immunoprecipitation followed by western blot. a–c Phosphorylated Akt1 at Ser472, Akt2 at Ser473, and Akt3 at Ser474. Phosphorylation values were corrected by their protein abundance in the immunoprecipitates. For Akt1 and Akt2, there was a significant interaction (P < 0.01) between GAB and STATE. Representative blots are shown. Data are expressed as mean ± SEM; n = 21–25/group (*P < 0.05 and **P < 0.01). AU arbitrary units.

Article Snippet: The primary antibodies that were used were: phospho-pan-/Akt (Ser472/473/474) (1:1000; #ab192623; Abcam, Cambridge, MA); pan-Akt, Akt1, Akt2, Akt3 (Rabbit mAb #4685, Rabbit mAb #75692, Rabbit mAb #3063, and Mouse mAb #8018), FAK (total, #3285 and Tyr397, #8556), mTOR (total, #2972), PTEN (total, #9552 and Ser308, #9551), PP2A (total, #2260) and PDK1 (total, #3062 and Ser241, #3438) (1:1000; Cell Signaling Technology); PP2A (Tyr307, # AF3989-SP; 1:1000; R&D System, Minneapolis, MN); Vinculin (#66305-1-lg; 1:4000); PHLPP (#676640-1-lg), Ubl4A (#14253-1-AP), IP6K1 (#12057-2-AP), Hsp90 (#13171-1-AP) and GAPDH (#60004-1-lg) (1:1000; Proteintech, Rosemont, IL); phospho-mTOR (Ser2481, #651701) (1:1000; BioLegend).

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Quantitative Proteomics