fgf1 nm 010197 mouse  (OriGene)

Journal: bioRxiv

Article Title: Impact of Bmal1 rescue and time-restricted feeding on liver and muscle proteomes during the active phase in mice

doi: 10.1101/2023.06.12.544652

Figure Lengend Snippet: (A) Abundance of fibroblast growth factor 1 (FGF1) protein in liver. AL – ad libitum feeding; TRF – time-restricted feeding. Two-way ANOVA with Tukey’s multiple comparisons test, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, n=4. (B) mRNA levels of FGF isoforms in WT (Alfp- and Hsa-Cre+) liver, n=3. ZT – zeitgeber time; ZT0 = lights on, ZT12 = lights off. (C) Western blot for FGF1 in liver at the indicated diurnal time points. Biological replicates are shown (bottom) and quantified (top). One-way ANOVA with Fisher’s LSD test, *p<0.05, ** p<0.01, n=3. Liver-RE = Bmal1 stopFL/stopFL ; Alfp -Cre tg/0 , hepatocyte-specific reconstitution of Bmal1 . (D and E) Knockdown or overexpression of Fgf1 in AML12 hepatocytes. si-Ctrl = scrambled sequence; si-Fgf1 #1 and #2 both target Fgf1 but by different sequences. pCMV6-Empty = control vector without Fgf1 open reading frame clone. Left, qPCR, Student’s t-test, ** p<0.01, *** p<0.001, n=3. Middle, oxygen consumption rate in AML12 hepatocytes. Arrow indicates application of 0.5 uM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and 1 uM oligomycin. Right, quantification of middle. Basal and stressed values are averages of data points before and after drug application, respectively. Two-way ANOVA with Dunnett’s post-hoc test, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns – not significant, n=5-6. (F) mRNA levels of Fgf receptor (FGFR) isoforms in WT (Alfp- and Hsa-Cre+) liver. (G) Measurement of oxygen consumption rate as in D and E. AML12 cells were treated for 24 h with vehicle control (Veh) or the indicated concentration of the pan FGFR inhibitor AZD4547. Two-way ANOVA with Tukey’s post-hoc test, * p<0.05, *** p<0.001, **** p<0.0001, ns – not significant, n=5-6.

Article Snippet: For overexpression experiments, cells were transfected with 100 ng Fgf1 (NM_010197) Mouse Tagged ORF Clone (ORIGENE, MR201152) or control empty vector using Lipofectamine 3000 Transfection Reagent (Invitrogen, #L3000001) according to the manufacturer’s instructions.

Techniques: Western Blot, Knockdown, Over Expression, Sequencing, Control, Plasmid Preparation, Concentration Assay

Journal: bioRxiv

Article Title: Impact of Bmal1 rescue and time-restricted feeding on liver and muscle proteomes during the active phase in mice

doi: 10.1101/2023.06.12.544652

Figure Lengend Snippet: (A) Western blot analysis of whole cell lysates from liver harvested at the indicated diurnal time points. ZT – zeitgeber time; ZT0 = lights on, ZT12 = lights off. Right, quantification of n=5 biological replicates. (B) Example western blot showing protein expression of FGF1 in AML12 hepatocytes and liver whole cell lysate. Total protein from gel. Ug - indicates the amount of protein loaded. (C) Change in the rate (left, extracellular acidification; right, oxygen consumption) from basal to stressed conditions in AML12 hepatocytes with control transfection or overexpression of Fgf1 . Student’s t-test, ** p<0.01, *** p<0.001, n=5-6. (D) Left, extracellular acidification rate in AML12 hepatocytes with control transfection or overexpression of Fgf1 . Arrow indicates application of 0.5 uM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and 1 uM oligomycin. Right, quantification of left. Basal and stressed values are averages of data points before and after drug application, respectively. Two-way ANOVA with Sidak’s post-hoc test, **** p<0.0001, ns – not significant, n=5-6. (E) Quantification of FGF1 protein in WT serum by ELISA. (F) Example western blot showing the relative expression of FGF1 across metabolic tissues. Total protein from gel. Muscle is gastrocnemius. Brain is cortex. (G) Example western blot showing the presence of FGF1 in chemically-defined media (bubbled with carbogen) incubated ex vivo for 45 min with a whole liver lobe. Albumin is a known liver-secreted protein and is included as a positive control. Total protein from gel.

Article Snippet: For overexpression experiments, cells were transfected with 100 ng Fgf1 (NM_010197) Mouse Tagged ORF Clone (ORIGENE, MR201152) or control empty vector using Lipofectamine 3000 Transfection Reagent (Invitrogen, #L3000001) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Control, Transfection, Over Expression, Enzyme-linked Immunosorbent Assay, Incubation, Ex Vivo, Positive Control