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Figure Lengend Snippet: Δ eif4e1c mutants have impaired growth and poor survival. (A) Schematic of the CRISPR-mediated deletion of eif4e1c . Red rectangles represent coding exons, and the white box represents exons with untranslated regions. (B) Fish were grown together, genotyped at the indicated stage and immediately measured from jaw to the caudal fin bifurcation (3-month average wild type=1.82 cm, mutant=1.62 cm; Welch's t -test, P =0.0156; n =24 and 23, respectively; 6-month average wild type=2.48 cm, mutant=2.37 cm; Mann–Whitney, P =0.121; n =20 and 20, respectively). (C) After measuring length, fish were dried off as much as possible and weighed. The 8 wpf data (gray box) is shown on the right with a different scale for the y -axis. [8-week (blue) average wild type=35.75 mg, mutant=22.82 mg; Welch's t -test, * P =0.0392; n =24; 3-month (green) average wild type=146.5 mg, mutant=103.1 mg; Welch's t -test, ** P =0.0014; n =24 and 23, respectively; 6-month average (red) wild type=344.6 mg, mutant=261.7 mg; Welch's t -test, *** P =0.0002; n =20). (D) Uninjured heart ventricles from wild-type and mutant fish were sectioned and stained for muscle using antibodies to myosin heavy chain (MHC) and the cardiac transcription factor Mef2c to identify CM nuclei. Scale bar: 100 µm. Note that adjacent hearts have been removed from images for presentation purposes. (E) Numbers of Mef2c-positive cells were counted with MIPAR. The 8 wpf data (red box) is shown on the right with a different scale for the y -axis. [8-week (blue) average wild type=1078, mutant=848; Welch's t -test, * P =0.0383, n =17; 3-month (green) average wild type=6061, mutant=4557; Welch's t -test, ** P =0.0052, n =10 and 8, respectively). (F) Left: Western blot of whole-cell extracts from zebrafish hearts using an antibody directed towards canonical Eif4ea (top) and Tnnt2a (bottom). Right: Quantification of western blots with total Eif4ea levels normalized to the sarcomeric protein Tnnt2a as a measure of total cardiac mass (average increase=1.82; Welch's t -test, * P =0.0334, n =4). (G) Immunofluorescence of canonical Eif4ea/b (top, grayscale; bottom, green) and MHC (blue, bottom). Scale bar: 100 µm. For each panel, the lighter shade color is used for mutants, individual data points are represented by dots, and error bars represent mean±s.e.m. Mut, mutant; Wt, wild type.

Article Snippet: Primary antibodies used in this study were: anti-Mef2 (1:100, Abcam, ab197070), anti-hsEIF4E (1:100, Abcam, ab 33768), and anti-MF20 (1:100, Developmental Studies Hybridoma Bank, MF20).

Techniques: CRISPR, Mutagenesis, MANN-WHITNEY, Staining, Western Blot, Immunofluorescence

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Figure Lengend Snippet: Deletion of eif4e1c impairs CM proliferation in regenerating hearts. (A) Images of sectioned amputated ventricles (7 dpa) from wild-type and Δ eif4e1c mutant fish. Sections are stained for Mef2c (green) and EdU (red). Double-positive cells are highlighted in black below using a MIPAR software rendition. Scale bar: 100 µm. (B) Quantification of CM proliferation indices (Mef2/EdU double-positive over total Mef2 positive) in 7 dpa ventricles (average wild type=10.68%, mutant 6.02%; Welch's t -test, ** P =0.0054; n =11 and 13, respectively). Error bars represent mean±s.e.m. (C) Cryosections of fresh mutant and wild-type hearts were stained for Succinate dehydrogenase activity. Shown are uninjured hearts (top) and hearts that were ablated genetically using Z-CAT (7 days-post-incubation) (bottom). Scale bar: 20 µm. (D) The mean signal intensity was calculated per given area for Sdh-stained hearts (uninjured average wild type=7.47, mutant=7.57; n =6 and 8, respectively; regeneration average wild type=4.59, mutant=5.06; n =4). Welch's t -test was used to calculate significance (wild type versus mutant uninjured=0.781, wild type uninjured versus wild type regeneration **** P <0.0001, mutant uninjured versus mutant regeneration **** P <0.0001). For every replicate ( n =4) the mutant Sdh activity during regeneration (pink) was higher than the wild-type activity during regeneration (red). Error bars represent mean±s.e.m. (E) Diagram of caudal fin regeneration experiments. The blastema is the highly proliferative zone of growth that forms the new fin. (F) Caudal fins were amputated ∼50% in Δ eif4e1c mutants and their wild-type siblings and shown are images of fin regrowth 4 days later. (G) There was no significant difference between fin growth in wild type (blue) and mutant (light blue) fish (average wild type=0.849 mm, mutant 0.857 mm; Welch's t -test, P =0.861; n =30 and 26, respectively). Error bars represent mean±s.e.m. Mut, mutant; Wt, wild type.

Article Snippet: Primary antibodies used in this study were: anti-Mef2 (1:100, Abcam, ab197070), anti-hsEIF4E (1:100, Abcam, ab 33768), and anti-MF20 (1:100, Developmental Studies Hybridoma Bank, MF20).

Techniques: Mutagenesis, Staining, Software, Activity Assay, Incubation

Journal: Molecules

Article Title: L-Arginine Enhances Oral Keratinocyte Proliferation under High-Glucose Conditions via Upregulation of CYP1A1 , SKP2 , and SRSF5

doi: 10.3390/molecules28207020

Figure Lengend Snippet: L-Arginine enhances oral keratinocyte proliferation under high-glucose conditions. ( A ) TIGK cells were cultured in 4-chamber slides treated with 500 μM Arg under high-glucose conditions (48 mM). Cell images were taken 24 and 48 h after treatment and the number of cells was counted in each field. ( B ) Representative images of the cultured cells. ( C ) TIGK cells were cultured in 96-well plates. The proliferation was assessed using an MTS proliferation assay. OD490 values were normalized based on the untreated cell condition (100%). Scale bar: 100 µm. Con: Control, Arg: L-Arginine, * p < 0.05, # p < 0.01.

Article Snippet: Cell proliferation was examined using an MTS Cell Proliferation Assay Kit (Abcam, Waltham, MA USA, Catalog # ab197010), according to the manufacturer’s instructions, at 24, 72, and 96 h post-treatment for low-glucose (6 mM) conditions or 72 and 120 h post-treatment for high-glucose (48 mM) conditions.

Techniques: Cell Culture, Proliferation Assay

Journal: Molecules

Article Title: L-Arginine Enhances Oral Keratinocyte Proliferation under High-Glucose Conditions via Upregulation of CYP1A1 , SKP2 , and SRSF5

doi: 10.3390/molecules28207020

Figure Lengend Snippet: Knocking down CYP1A1 , SKP2 , or SRSF5 abolishes enhanced oral keratinocyte proliferation induced by L-Arginine treatment under high-glucose conditions. TIGK cells were transfected with 20 nM siRNAs of CYP1A1 , SKP2 , or SRSF5 and then treated with 500 μM Arg in a culture medium with high glucose (48 mM). ( A ) mRNA expression of CYP1A1 , SKP2 , or SRSF5 48 h after transfection. ( B ) The proliferation of TIGK cells after knocking down CYP1A1 , SKP2 , or SRSF5 assessed by cell count at 48 h and MTS assay at 72 and 120 h after transfection. ( C ) Representative images of cell density at 48 h after treatment. Scale bar: 100 µm. * p < 0.05, # p < 0.01.

Article Snippet: Cell proliferation was examined using an MTS Cell Proliferation Assay Kit (Abcam, Waltham, MA USA, Catalog # ab197010), according to the manufacturer’s instructions, at 24, 72, and 96 h post-treatment for low-glucose (6 mM) conditions or 72 and 120 h post-treatment for high-glucose (48 mM) conditions.

Techniques: Transfection, Expressing, Cell Counting, MTS Assay

Journal: bioRxiv

Article Title: Temperature elevation synergises with and enhances the type-I IFN-mediated restriction of MPXV

doi: 10.1101/2023.09.29.560106

Figure Lengend Snippet: (A) Quantitation of DAPI stained nuclei in mock treated HFt cells incubated at 33, 37, 38.5 or 40 °C for 48 h. (B) MTS cell viability assay of mock treated or puromycin (Puro, 1 μg/ml; positive control) treated HFt cells incubated at 37 or 40 °C for 24 h (as indicated). (C to E) Confluent HFt (human foreskin fibroblast), NOK (human normal oral keratinocyte), RPE (human retinal pigmented epithelial), Vero E6 (green monkey kidney epithelial), or CV-1 (green monkey kidney fibroblast) cell monolayers were infected with (C) HSV-1 (MOI 0.002 PFU/cell), (D) VACV (MOI 0.0005 PFU/cell), or (E) MPXV (MOI 0.001 PFU/cell) for 1 h at 37 °C prior to overlay and incubation at 33, 37, 38.5 or 40 °C. Infected cell monolayers were fixed at 48 h and stained with Coomassie Brilliant blue. (C) Quantitation of HSV-1 plaque counts in HFt cells over the temperature incubation range of analysis. Values were normalised to plaque counts at 37 °C. Means and SD shown; p -values shown, one sample two-tailed t test against a theoretical mean of 1. (D/E) Representative images of VACV or MPXV infected HFT, RPE, NOK, Vero E6 (Meso), and CV-1 cell monolayers stained with Coomassie Brilliant blue at 48 h post-infection (hpi). (A to E) N ≥3 independent biological experiments. Raw values presented in S9 data.

Article Snippet: Cell viability was measured using MTS reagent (Abcam, ab197010), as per the manufacturer’s guidelines, and absorbance measurements taken at OD=490 nm using a PHERAstar plate reader (BMG LABTECH).

Techniques: Quantitation Assay, Staining, Incubation, Viability Assay, Positive Control, Infection, Two Tailed Test