Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 4: FAIM deficiency suppressed the effects of PACAP on alleviating hepatic lipid accumulation in HFD-fed mice. (A) FAIM protein level of mice liver was subjected to western blot. Statistical analysis of FAIM/b-actin was shown in a bar chart (right) (*P < 0.05, **P < 0.01, ***P < 0.001 vs. CD group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. HFD group). (B) Schematic diagram of HFD mouse model with PACAP treatment (0.4 mg/kg) or control saline solution and lentivirus mediated Faim knockdown. (C) Hepatic Faim mRNA level was determined by qRT-PCR, n ¼ 3. (DeH) Modulation of Faim level in the liver influenced HFD-induced hepatic steatosis, (D) bodyweight, liver weight, and WAT weight; (E) liver TG and TC contents; (F) plasma TG, TC, HDL-c, and LDL-c levels; (G) fasting blood glucose level; blood glucose concentrations were measured on day 12 after lentivirus injection; (H) representative photomicrographs of Oil Red O and H&E staining of liver tissues in different groups (scale bars: 50 or 200 mM). (I and J) Ablation of Faim impaired glucose tolerance and insulin sensitivity in mice. IPGTT (I) and IPITT (J), blood glucose was measured at different time points after glucose or insulin injection (left), and quantification of the AUC (right). Data are presented as mean SEM, n ¼ 5 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Western Blot, Control, Saline, Knockdown, Quantitative RT-PCR, Clinical Proteomics, Injection, Staining

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 5: PACAP activates the FAIM-AMPK-IRb axis to govern the SREBP and lipid synthetic gene program. (A and B) PACAP activated AMPK-IRb signaling pathway and down- regulated the expression of lipogenesis genes in HFD-fed mice, such effects were suppressed following knockdown of Faim. Western blot analysis were used to detect the protein level of indicated antibodies. (C and D) The overexpression of FAIM activated AMPK-IRb signaling pathway, the protein levels of FAIM, p-AMPK, AMPK, p-IRb, IRb, and lipid synthesis genes SREBP1, SREBP2, FAS, SCD1, HMGCR were determined by western blot with quantifications. Data are presented as mean SEM, n ¼ 3 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Expressing, Knockdown, Western Blot, Over Expression

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 6: Knockdown of Faim inhibited the effects of PACAP on decreasing lipid accumulation and activating AMPK-IRb signaling pathway. AML12 cells were transiently transfected with Faim siRNA or scramble siRNA in cell culture medium containing 200 mM PA or 10% BSA (as control of PA solution) for 24 h, then the cells were treated with PACAP (1 mM) and Max.D.4 (10 mM) for another 24 h as indicated. (A) Representative for Oil Red O staining of AML12 (Scale bars, 50 mM). (B and C) Impacts of Faim knockdown on cellular TG and TC level (B), glucose uptake and glycogen content (C) of AML12. (D and E) Faim knockdown impaired the activation of AMPK-IRb pathway in AML12 treated with PACAP, the protein levels were analyzed by western blot and quantitative measurements (right). (F and G) AML12 were exposed to PA for 24 h, then treated with PACAP (1 mM), Max.D.4 (10 mM) and Compound C (10 mM) (F) or AICAR (0.5 mM) (G) for another 24 h. The protein level of FAIM was detected by western blot analysis and quantitative measurements (down). Data are presented as mean SEM. n ¼ 3 (&P < 0.05, &&P < 0.01, &&&P < 0.001 vs. control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. PA group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. PA þ PACAP group, OP < 0.05, OOP < 0.01, OOOP < 0.001 vs. PA þ PACAP þ Max.D.4 group).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Knockdown, Transfection, Cell Culture, Control, Staining, Activation Assay, Western Blot

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 7: PACAP activates the PAC1-PKA-CREB signaling pathway to stimulate FAIM expression. (A) mRNA level of Faim in PA-induced AML12 treated with PACAP, determined by qRT-PCR (n ¼ 3). (B) The effect of PACAP on the activation of PKA and CREB in PA induced AML12 cells, the protein levels of PKA, CREB and FAIM, and phosphorylation of PKA and CREB were determined by western blot. (*P < 0.05, **P < 0.01, ***P < 0.001 vs. PA group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. PACAP (1 mM) group). (C) Schematic of the sequence of two CRE binding sites that CREB targets the WT or mutated promoter region of Faim mRNA. (DeF) Luciferase activities in AML12 transiently transfected with either wild type (WT), truncated, or mutant Faim promoter-luciferase reporter constructs. Then the cells were treated with PACAP (1 mM), or co- transfected with CREB cDNA or empty vectors. (G) Luciferase activities of FAIM WT reporter plasmids in AML12 cells with the treatment of KG-501 (a specific CREB inhibitor) and PACAP (1 mM) as indicated. Data are shown as mean SEM, n ¼ 3 or 5 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Expressing, Quantitative RT-PCR, Activation Assay, Phospho-proteomics, Western Blot, Sequencing, Binding Assay, Luciferase, Transfection, Mutagenesis, Construct

Journal: International Journal of Molecular Sciences

Article Title: miR-132-3p Modulates DUSP9-Dependent p38/JNK Signaling Pathways to Enhance Inflammation in the Amnion Leading to Labor

doi: 10.3390/ijms23031864

Figure Lengend Snippet: The role of p38 and JNK in DUSP9 siRNA-induced expression of proinflammatory cytokines and COX2 as well as PGE2 in WISH cells. WISH cells were transfected with si- DUSP9 or NC for 24 h, followed by 24 h of treatment with DMSO, p38 inhibitor SB203580 (10 μM), or JNK inhibitor SP600125 (20 μM). RT-qPCR analysis of the expression of IL-1β , TNF-α , COX2 ( A , G ), IL-6 , and IL-8 ( B , H ). Magnetic Luminex Assays of the secretion of IL-1β, TNF-α ( C , I ), IL-6, and IL-8 ( D , J ). Western blot analysis of COX2 expression ( E , K ). ELISA analysis of PGE2 level ( F , L ). Data were shown as mean ± SEM, n = 3. * p < 0.05, ** p < 0.01 vs. control (0), # p < 0.05, ## p < 0.01 vs. si- DUSP9 .

Article Snippet: To verify the involvement of p38 and JNK signaling pathways in the induction of IL-1β, IL-6, IL-8, TNF-α, and COX2 as well as PGE2 secretion by DUSP9 siRNA, the cells were transfected with NC or si- DUSP9 for 24 h, followed by 24 h of treatment with dimethyl sulfoxide (DMSO), p38 inhibitor SB203580 (10 μM, Selleck, Houston, TX, USA), or JNK inhibitor SP600125 (20 μM, Selleck, Houston, TX, USA).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Luminex, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: NRVMs were transduced with AdV5-CMV-CRYAB R120G -GFP and treated with 3 µM ruxolitinib or DMSO or 100 nM siRNAs targeting either JAK1 (siJak1), JAK2 (siJak2), STAT3 (siStat3) or scramble siRNA (scr). Thereafter, NRVMs were harvested or fixed after 4-6 days. A , Treatment with ruxolitinib or DMSO; Scale bar = 100 µm. B , Transfection with siJak1; Scale bar = 200 µm. C , Transfection with siJak2; Scale bar = 100 µm D , Transfection with siStat3, Scale bar = 20 µm. A , B , C , D , Western blots of protein extracts from treated NRVMs were stained with antibodies directed against indicated proteins. In the representative immunofluorescence images, aggregates are depicted in magenta (CRYAB R120G -GFP), cardiomyocytes in yellow (anti-cardiac troponin I), and nuclei in blue (DAPI). Quantification of aggregates in cardiomyocytes with NIS Elements or ImageJ software. Data were obtained from 1 (Western blot) or 2 (immunofluorescence) NRVM preparations with at least 3 wells per condition and at least 7 images per well for immunofluorescence. Data are depicted as mean ± SEM, and p-values were obtained with the unpaired Student’s t-test. Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Transduction, Transfection, Western Blot, Staining, Immunofluorescence, Software

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: A , Heart weight-to-body weight ratio (HW/BW) in 1-, 4- and 7-month-old CRYAB p.Arg120Gly transgenic (R120G) and non-transgenic (NTG) mice. B , Ejection fraction (EF) in 7-month-old R120G and NTG mice. C , Representative Western blots of phosphorylated (P-)STAT3, STAT3 and indicated control in 1-, 4- and 7-month-old R120G and NTG mice. D , P-STAT3/STAT3 quantification of Western blots from 1-, 4- and 7-month-old R120G and NTG mice. E , Stat3 mRNA level determined by RT-qPCR from 7-month-old R120G and NTG mice. Western blot quantification was performed with Image Lab software. Data are depicted as mean ± SEM, and p-values were obtained with the unpaired Student’s t-test. Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Transgenic Assay, Western Blot, Control, Quantitative RT-PCR, Software

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: CRYAB p.Arg120Gly transgenic (R120G) and non-transgenic (NTG) mice treated for 3 weeks with 75 mg/kg ruxolitinib (ruxo) or vehicle (veh) twice-daily oral gavage. Transthoracic echocardiography was performed at the start (before, 21-week-old) and the end of the treatment (veh or ruxo, 24-week-old). A , Scheme of experimental outline. B , WB and quantification of phosphorylated (P-) STAT3, STAT3 and indicated controls at the end of treatment. WB quantification was performed with Image Lab software. C, Ejection fraction (EF) and left ventricular mass-to-body weight ratio (LVM/BW) before and after (veh/ruxo) treatment. D , Heart weight-to-body weight ratio (HW/BW) and body weight (BW) at the end of the treatment. E , Representative images and quantification of R120G and NTG mouse heart sections after vehicle or ruxolitinib treatment. CRYAB is depicted in green, cardiomyocytes in purple (anti-cardiac troponin I) and nuclei in blue (DAPI). Scale bar = 10 µm. Quantification of aggregates in cardiomyocytes with NIS Elements software. At least 5 images of 3 mice per group were analyzed. Data are depicted as mean ± SEM, and p-values were obtained with the one- way ( B ) or two-way ANOVA and Tukey’s multiple comparisons post-hoc analysis ( C, D ) or unpaired Student’s t-test ( E ). Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Transgenic Assay, Software

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: Heterozygous (het) or homozygous (hom) Jak1 knockout (KO) was induced in MCM-transgenic (TG) mice crossed with CRYAB p.Arg120Gly TG (R120G) or non-transgenic (NTG) mice with tamoxifen chow. Transthoracic echocardiography was performed 26 weeks and hearts were extracted. A , Scheme of experimental outline. B , Jak1 mRNA levels determined by RT-qPCR. C , JAK1 protein levels determined by Western blot and normalized to ACTN2. Western blot quantification was performed with Image Lab software D, Ejection fraction (EF) and left ventricular mass-to-body weight ratio (LVM/BW). E , Heart weight-to-body weight ratio (HW/BW) and body weight (BW). F , Representative images and quantification of mouse heart sections. Aggregates are depicted in green, cardiomyocytes in purple (anti-cardiac troponin I) and nuclei in blue (DAPI). Scale bar = 50 µm. Quantification of aggregates in cardiomyocytes with NIS Elements software. At least 5 images of 3 mice per group were analyzed. Data are depicted as mean ± SEM, and p-values were obtained with the one-way ( B ) or two- way ANOVA ( D , E ) with Tukey’s multiple comparisons post-hoc analysis or unpaired Student’s t-test ( C , F ). Abbreviation: ns, non-significant.

Article Snippet: RT-qPCR was then performed with TaqMan gene expression assays (Thermo Fisher Scientific; Psmd1 - Rn01400483_m1, Jak1 - Mm00600614_m1, Jak2 -Mm01208489_m1, Stat1 - Mm01257286_m1, Stat3 - Mm01219775_m1, Tyk2 -Mm00444469_m1, 18s - Hs03003631_g1) and SsoAdvancedTM Universal Probes Supermix (BioRad #1725281).

Techniques: Knock-Out, Transgenic Assay, Quantitative RT-PCR, Western Blot, Software

Journal: bioRxiv

Article Title: Quantitative study of the somitogenetic wavefront in zebrafish

doi: 10.1101/419705

Figure Lengend Snippet: (a) A Clock and Wavefront model: antagonistic gradient of Fgf8 (originating from the posterior PSM, green) and RA (originating from the somites, violet) define a wavefront which interacts with a particular phase of the segmentation clock (in the PSM, red) to generate somites at periodic times and positions. (b) Kymograph of somitogenesis from 7 to 20 somites. The tail elongates at a constant rate V tail while the PSM shrinks at a roughly constant rate V PSM resulting in a somite wavefront propagating at a rate V front = V tail -V PSM .

Article Snippet: RT-qPCR was performed using the aforementioned cDNAs with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assay: Dr03119263_m1(rpl13a) and Dr03105657_m1(fgf8) (Applied Biosystems).

Techniques:

Journal: bioRxiv

Article Title: Quantitative study of the somitogenetic wavefront in zebrafish

doi: 10.1101/419705

Figure Lengend Snippet: Molecular models of the determination wavefront. (a) The Goldbeter, Gonze and Pourquié (G 2 P) model assumes that RA directly affects the translation of Fgf8 mRNA into protein, while Fgf8 represses RA via the activation of its degradation enzyme Cyp26. (b) The modified version of the G 2 P model (mG 2 P) proposed here takes into account the observed positive feedbacks of RA on Fgf8 and of Fgf8 on RaldH and the mutual inhibition of RA on MapK (via the RA-mediated activation of Mkp3) and of MapK on RA (via the MapK-controlled activation of Cyp26). (c) Both models predict a bistability of MapK activity for a certain positional range (in grey).

Article Snippet: RT-qPCR was performed using the aforementioned cDNAs with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assay: Dr03119263_m1(rpl13a) and Dr03105657_m1(fgf8) (Applied Biosystems).

Techniques: Activation Assay, Modification, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: Quantitative study of the somitogenetic wavefront in zebrafish

doi: 10.1101/419705

Figure Lengend Snippet: Time of appearance of somites (t=0 at 7 somites stage). Notice the linearity of the plot, i.e. the regularity of the period of somitogenesis, in all the conditions studied here: WT (n=8), DEAB (an inhibitor of RaldH; with (n=14) or without (n=12) external RA), morpholinos against Fgf8 (MO-Fgf8; n=16), BCI (an inhibitor of Mkp3; n=16) or activation of exogenous Fgf8 (n=17).

Article Snippet: RT-qPCR was performed using the aforementioned cDNAs with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assay: Dr03119263_m1(rpl13a) and Dr03105657_m1(fgf8) (Applied Biosystems).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Quantitative study of the somitogenetic wavefront in zebrafish

doi: 10.1101/419705

Figure Lengend Snippet: (a) Staining of phosphorylated MapK by antibodies against this active form in WT embryos at 10 and 15 somites. Notice the smaller domain of activity at 15s as compared to 10s. (b) The data in was quantified by measuring the fluorescence intensity in a single embryo along the antero-posterior axis and averaged over n=17 (10s) and n=21 (15s) embryos. The averaged data is compared to simulations of the mG 2 P model with the parameters of assuming an exponential decrease (see ) between 7s and 10s (or 15s) of the mRNA Fgf8. The x and y-scales were chosen to fit the data at 10s. The simulation results are in qualitative agreement with the data, even though the latter might not be a perfect reflection of the MapK activity level (which depends on the efficiencies of staining and washing).

Article Snippet: RT-qPCR was performed using the aforementioned cDNAs with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assay: Dr03119263_m1(rpl13a) and Dr03105657_m1(fgf8) (Applied Biosystems).

Techniques: Staining, Activity Assay, Fluorescence

Journal: bioRxiv

Article Title: Quantitative study of the somitogenetic wavefront in zebrafish

doi: 10.1101/419705

Figure Lengend Snippet: (a) Variation with time of Fgf8 concentration versus somite stage and fit to an exponential decay past 5 somites stage. (b) PSM shortening from 7 somites stage (dots and error bars on mean; n=8) and results (continuous line) of a simulation of the mG 2 P model (with the displayed parameters and assuming an exponential decay of Fgf8 with the timescale measured in (a)). Details in Supp.Mat.

Article Snippet: RT-qPCR was performed using the aforementioned cDNAs with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assay: Dr03119263_m1(rpl13a) and Dr03105657_m1(fgf8) (Applied Biosystems).

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Quantitative study of the somitogenetic wavefront in zebrafish

doi: 10.1101/419705

Figure Lengend Snippet: Rates of PSM shrinkage (V PSM ), tail growth (V tail ) and wavefront velocity (V front ) in embryos growing (a) without or with morpholinos against Fgf8 (MO-Fgf8; dots and error bars on mean; n=16) injected at one-cell stage or (b) in which an exogenous source of Fgf8 was turned on (n=17). While V PSM is unaffected by MO-Fgf8 and slightly decreases upon over-expression of Fgf8 (in the strong phenotype 2 embryos), V tail decreases in both conditions, resulting in an overall decrease of V front . Continuous lines: simulations of the mG 2 P model with a 60% decrease in Fgf8 mRNA (due to interference with MO-Fgf8; γ=0.8 instead of γ=2 in model, see Supp.Mat.) qualitatively reproduce the data (continuous line in (a)). Similarly simulations with an increasing Fgf8 mRNA of about 2% of the measured increase (see Fig.S7) seem to reproduce the data observed in the strong phenotype 2 case (see Supp.Mat. Fig.S5).

Article Snippet: RT-qPCR was performed using the aforementioned cDNAs with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assay: Dr03119263_m1(rpl13a) and Dr03105657_m1(fgf8) (Applied Biosystems).

Techniques: Injection, Over Expression

Journal: bioRxiv

Article Title: Quantitative study of the somitogenetic wavefront in zebrafish

doi: 10.1101/419705

Figure Lengend Snippet: (a,c) MapK activity at steady-state from a simulation of mG 2 P model with the parameters shown in the figures and linear gradients of RaldH (parameter α) and Fgf8 mRNA (parameter γ). Notice the existence of a bistability window (between distances: −40 and −32 in (c)). (b,d) Variation with time of the distance from the tail end (at 0) of the rightmost bistability boundary (red vertical line in (a,c)): assuming linear decrease with time of Fgf8 mRNA (red curve; t ∼ 1 - mF0(t)/mF(0)) or exponential decrease with time of Fgf8 mRNA (blue curve, mF0(t) = mF0(0) e -t ). (e,f) Results of simulations with linear gradients of Cyp26 (parameter β) and Fgf8 mRNA (parameter γ). Notice that the MapK activity levels off at the tail end though a bistability window is still present. (g,h) Results of simulations with a linear gradient of Fgf8 mRNA (parameter γ) only. Notice the increasing MapK activity level and the increased PSM shrinkage rate at the tail end (even assuming an exponential decay with time of FgF8 mRNA).

Article Snippet: RT-qPCR was performed using the aforementioned cDNAs with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assay: Dr03119263_m1(rpl13a) and Dr03105657_m1(fgf8) (Applied Biosystems).

Techniques: Activity Assay