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

Journal: Biotechniques

Article Title: Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers

doi: 10.2144/btn-2021-0063

Figure Lengend Snippet: (A–C) Dose–response curves using double-bubble primer mix #8 and VIC-TqM probe #12 and decimal dilutions of SARS-CoV-2 synthetic RNA N. (A) Real-time amplification plot. C t values: 8 × 10 5 copies, 22.82; 8 × 10 4 copies, 26.27; 8 × 10 3 copies, 30.55; 8 × 10 2 copies, 32.51. (B) 5% agarose gel of the PCR amplification products. Lane 1: ultra-low-range ladder; lanes 2–5: 139-bp amplicons of 8 × 10 5 to 8 × 10 2 RNA copies per tube. Also included in the PCR reaction mix were 2× TqM buffer without UDP and iScript reverse-transcriptase. (C) Efficiency plot of the PCR reaction depicted in panel A. Slope = -3.3359 represents PCR reaction efficiency of 99.42%; R 2 = 0.9816. (D) One-tube RT-qPCR using double-bubble primer mix #10 from region 2 using SARS-CoV-2 synthetic RNA (2 × 10 4 copies per tube) and FAM-TqM probe #13. RT-qPCR was performed with standard conditions.

Article Snippet: For cDNA synthesis, iScript reverse-transcriptase kit was employed: 25°C, 5 min, 42°C, 15 min 95°C, 1 min, 4°C hold (BioRad, 1708891).

Techniques: Amplification, Agarose Gel Electrophoresis, Reverse Transcription, Quantitative RT-PCR

Journal: Biotechniques

Article Title: Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers

doi: 10.2144/btn-2021-0063

Figure Lengend Snippet: (A) qPCR using reverse-transcribed cDNA template and double-bubble (D-B) primer mix #10, FAM-TqM probe #13, TqM fast kit+ uracil-DNA glycosylase (UDG) assayed in duplicates. Insert depicts 5% agarose gel; lane 1: ultra-low-range (ULR) ladder; lanes 2 & 3: template cDNA; lanes 4 & 5: non-template control (NTC). C t average values: cDNA, 28.60; NTC, undetermined. (B) One-tube RT-qPCR using SARS-CoV-2 synthetic RNA N template and D-B primer mix #8, VIC-TqM probe #12, TqM fast kit without UDG iScript reverse transcriptase, assayed in duplicate. Insert depicts 5% agarose gel; lane 1: ULR ladder; lanes 2 and 3: template RNA; lanes 4 and 5: NTC. C t average values: RNA, 24.63, NTC, undetermined. (C) Duplex qPCR using SARS-CoV-2 synthetic RNA N template and TqM fast kit with UDG. C t values: primer 8, 26.13; primer 10, 32.19; duplex primers 8 + 10, 26.36 and 30.27, respectively; NTC, undetermined. (D) 5% agarose gel of samples shown in panel C. Lane 1: ULR ladder; lane 2: primer D-B mix #8 (amplicon 139 bp); lane 3: D-B primer mix #10 (amplicon 158 bp); lane 4: duplex of both primers #8 and #10 (amplicons 139 and 158 bp). Arrows (C & D) show duplex amplifications in the same tube with D-B primers #8 and #10. PCR was performed using fast conditions with 40 (A, C & D) and 30 (B) cycles.

Article Snippet: For cDNA synthesis, iScript reverse-transcriptase kit was employed: 25°C, 5 min, 42°C, 15 min 95°C, 1 min, 4°C hold (BioRad, 1708891).

Techniques: Reverse Transcription, Agarose Gel Electrophoresis, Control, Quantitative RT-PCR, Amplification

Journal: Biotechniques

Article Title: Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers

doi: 10.2144/btn-2021-0063

Figure Lengend Snippet: The reaction mixture contained hot-start double-bubble (D-B) primer mix #4, VIC-TqM probe #12, cost-effective non-hot-start Taq polymerase (FroggaBio), reverse transcriptase (iScript), synthetic SARS-CoV-2 RNA gene N as template, added dNTPs and ROX dye for internal calibration. The reaction was assembled at room temperature and subjected to real time RT-qPCR using the following fast conditions: 42°C, 15 min; 95°C,1 min; 30 cycles of 95°C, 1 s → 70°C, 20 s. The amplification plot and (insert) 5% agarose gel depict the amplification of SARS-CoV-2 RNA extracted from the nasopharyngeal swabs of patient S1 (lane 2) and negative patient N1 (lane 3), no-template controls (lanes 4 and 5) and SARS-CoV-2 gene N synthetic RNA as positive control (lane 6). Note the amplification in the real-time plot and the 115-bp band in the 5% agarose gel with SARS-CoV-2 virus or synthetic RNA (lanes 2 and 6), with no amplifications in the negative control N1 (lane 3) or NTC (lanes 4 and 5). Lane 1: ultra-low-range ladder. C t values: positive patient S1: 23.95; synthetic RNA: 23.84; negative patient N1 and NTC: undetermined.

Article Snippet: For cDNA synthesis, iScript reverse-transcriptase kit was employed: 25°C, 5 min, 42°C, 15 min 95°C, 1 min, 4°C hold (BioRad, 1708891).

Techniques: Reverse Transcription, Quantitative RT-PCR, Amplification, Agarose Gel Electrophoresis, Positive Control, Virus, Negative Control

Journal: Journal of biomedical science

Article Title: SLCO4A1-AS1 promotes colorectal tumourigenesis by regulating Cdk2/c-Myc signalling.

doi: 10.1186/s12929-022-00789-z

Figure Lengend Snippet: Fig. 2 Promoter hypomethylation promotes SLCO4A1-AS1 expression in CRC. a Schematic map illustrating a predicted CpG island and its DNA methylation probes in the promoter of SLCO4A1-AS1. TSS, transcription start site. b The β-value of methylation of SLCO4A1-AS1 was lower in tumour samples than in normal tissues according to the CRC dataset of TCGA. The β-value of methylation of SLCO4A1-AS1 was linearly related to SLCO4A1-AS1 expression in CRC tissues from TCGA (c) and cell lines from CCLE (d). e Relative expression of SLCO4A1-AS1 in CRC cell lines was measured using qRT-PCR (the left panel). The methylation levels of SLCO4A1-AS1 in CRC cell lines and leukocyte cells were determined by bisulfite sequencing PCR. A total of 5 individual clones were randomly picked for sequencing (the right panel). f The mean methylation levels of these CpG sites were negatively associated with the expression levels of SLCO4A1-AS1 in CRC cells. g SLCO4A1-AS1 expression in CRC cells treated with DNA methyltransferase inhibitor (5-aza-dC). h DNA methylation analyses of SLCO4A1-AS1 in paired CRC tissues and noncancerous tissues using methylation-specific PCR assay. N, adjacent noncancerous tissue; T, tumour tissue; M, DNA marker

Article Snippet: Genomic DNA was extracted from cancer cells or human leukocytes using a Genomic DNA Mini Preparation Kit (Beyotime, China) and then bisulfite-modified using an EpiJET Bisulfite Conversion Kit (Thermo Fisher, USA).

Techniques: Expressing, DNA Methylation Assay, Methylation, Quantitative RT-PCR, Methylation Sequencing, Clone Assay, Sequencing, Marker