Journal: BMC Genomics

Article Title: Circular and L50-like leaderless enterocins share a common ABC-transporter immunity gene

doi: 10.1186/s12864-023-09750-2

Figure Lengend Snippet: Comparison between different bacteriocin gene clusters. The figure shows the homologies found when comparing contigs from different species that contain the Mr10EFGH transporter associated with the structural genes of enterocins MR10A/B (yellow and gold arrows), AS-48 (green arrow), carnocyclin A (gray arrow), uberolysin enterocin family (blue arrow) and orphans in bacteriocins. Similarities are conserved only at the ABC transporter level. From top to bottom, GenBank accession nos. NZ_CP035137.1 ( Enterococcus faecium SRCM103341), NZ_KB944733.1 ( Enterococcus faecalis EnGen0369 39 − 5), NZ_AYLU01000046.1 ( Enterococcus faecalis AZ19), NZ_PJXM01000014.1 ( Enterococcus faecalis EN19), NZ_SRYT01000012.1 ( Enterococcus faecalis NM58_B2-5) and NZ_KE352861.1 ( Enterococcus faecalis LA3B-2)

Article Snippet: In some cases, these orphan genomes were the result of incomplete contigs, e.g., NZ_LWHF01000045.1 ( Enterococcus faecium strain 17OM39), NZ_PKMN01000041.1 ( Enterococcus faecalis strain EN788), or NZ_LDND01000117.1 ( Enterococcus faecium strain KACC15711); however, they were evident orphan transporters in some cases, i.e., GenBank accession no. NZ_CABHDR010000049.1 ( Enterococcus faecium strain 4928STDY7387731), GenBank accession no. NZ_KE352861.1 ( Enterococcus faecalis LA3B-2 Scaffold63), or GenBank accession no. NZ_KB946329.1 ( Enterococcus phoeniculicola ATCC BAA-412 acvKl-supercont1.7).

Techniques: Comparison

Journal: BMC Genomics

Article Title: Circular and L50-like leaderless enterocins share a common ABC-transporter immunity gene

doi: 10.1186/s12864-023-09750-2

Figure Lengend Snippet: Comparison between contigs containing the transporter Mr10EFGH associated with the same bacteriocin. Panel A shows the comparison between contigs that contain the ABC transporter associated with structural genes of the uberolysin enterocin family (blue arrow). Panel B shows the similarity between contigs that contain the ABC transporter associated with structural genes of MR10A/B enterocins (yellow and gold arrows). In both cases, the homologies are maintained at the gene cluster level. The similarity varies between 63% (light red) and 100% (dark red). From top to bottom, GenBank accession nos. are NZ_AYLU01000046.1 ( Enterococcus faecalis AZ19), NZ_KB944733.1 ( Enterococcus faecalis EnGen0369 39 − 5), NZ_PJXM01000014.1 ( Enterococcus faecalis EN19), NZ_CP035137.1 ( Enterococcus faecium SRCM103341), NZ_SEHG01000089.1 ( Enterococcus durans OSY-EGY), and NZ_SRYT01000012.1 ( Enterococcus faecalis NM58_B2-5)

Article Snippet: In some cases, these orphan genomes were the result of incomplete contigs, e.g., NZ_LWHF01000045.1 ( Enterococcus faecium strain 17OM39), NZ_PKMN01000041.1 ( Enterococcus faecalis strain EN788), or NZ_LDND01000117.1 ( Enterococcus faecium strain KACC15711); however, they were evident orphan transporters in some cases, i.e., GenBank accession no. NZ_CABHDR010000049.1 ( Enterococcus faecium strain 4928STDY7387731), GenBank accession no. NZ_KE352861.1 ( Enterococcus faecalis LA3B-2 Scaffold63), or GenBank accession no. NZ_KB946329.1 ( Enterococcus phoeniculicola ATCC BAA-412 acvKl-supercont1.7).

Techniques: Comparison

Journal: Yeast (Chichester, England)

Article Title: Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification

doi: 10.1002/yea.1672

Figure Lengend Snippet: Isolation of active mt nucleoids and identification of proteins associated with TAP–mtRNAP. (A) Purification of transcriptionally active mt nucleoids. Nucleoids were purified as described (see Materials and methods) and diluted as indicated. Transcription reactions were carried out for 20 min at 30 °C in the presence of 20 or 200 m m KCl or α-amanitin (AM). The labelled RNA products were resolved by electrophoresis in a 12% acrylamide denaturing gel, visualized by autoradiography and quantified by PhosphorImager analysis. The abundance of heterogeneous high molecular weight products under various conditions is shown; error bars represent standard error for mt nucleoid transcripts ( n = 3). (B) Rpo41p–TAP-associated proteins. Peptides eluted from the calmodulin–sepharose column were resolved on 4–12% bis-Tris NuPAGE Novex protein gels (Invitrogen) and visualized by silver staining (lane CE). Numbers indicate the molecular weights of protein markers in kDa (lane M). The bands from lane CE were submitted for LC–MS–MS analysis (Tufts University Core Facility). Arrows indicate silver-stained bands that match the molecular weight (MW) of major proteins identified in the gel slabs containing the bands. *Note that the actual MW of mature Pet127p may be smaller than predicted from its ORF as ∼90 N-terminal aa are likely to be absent in the mature form of Pet127p (Wiesenberger and Fox, ). (C) Example of LC–MS–MS spectra (Mss116p). Eleven peptides matching the Mss116p protein sequence were found as a result of an uninformed search against the S. cerevisiae protein database. S f , quality of match; TIC, total ion current; % TIC, signal or ion current for a given peptide; Sum TIC, sum of all TIC values for that protein identification—this exceeded the minimum required score in all cases. Additional information may be found at http://www.tucf.org . Complete LC–MS–MS scans are available upon request

Article Snippet: The washes were combined, polypeptides were precipitated with acetone/TCA at −20 °C for 3 h and separated by electrophoresis in 4–12% Bis–Tris NuPAGE Novex protein gels in an MES buffer system (Invitrogen).

Techniques: Isolation, Purification, Electrophoresis, Autoradiography, High Molecular Weight, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Staining, Molecular Weight, Sequencing

Journal: Leukemia

Article Title: Base-edited CAR T cells for combinational therapy against T cell malignancies

doi: 10.1038/s41375-021-01282-6

Figure Lengend Snippet: A Schema of base editing for T cells employing 3rd generation codon optimised cytidine base deaminase (coBE3) fused to deactivated D10A Cas9 nickase and uracil glycosylase inhibitor (UGI) delivered as mRNA along with TRBC and CD7 single guide RNA (sgRNA). C->U->T conversion (G->A antisense strand) resulting in STOP codon. B Lentiviral transduction of edited cells from step 1 using 3rd generation lentiviral vectors. Lentiviral plasmid configuration of CD3ε targeting 2nd generation chimeric antigen receptor comprising OKT3 vL and vH scFv sequence fused to CD8 transmembrane domain (TM), 41BB co-stimulatory and CD3z activation domains under the control of a hPGK promoter. Lentiviral plasmid configuration of CD7 targeting 2nd generation CAR comprising 3A1e vL and vH scFv sequence fused to CD8TM-41BB-CD3z under the control of a hPGK. C coBE3 edited T cells devoid of shared antigens TCR/CD3 and CD7 surface receptors expressing either 3CAR or 7CAR evade fratricide and target T-ALL. BE: base editor; APOBEC: (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like); sgRNA: single guide RNA; PAM: protospacer adjacent motif; LTR: long terminal repeat; CMV: cytomegalovirus promoter; CAR: chimeric antigen receptor; cPPT: central polypurine track; U5: untranslated 5′ region; DU3: delta untranslated 3′ region; hPGK: human phosphoglycerate kinase promoter; vL: variable light chain; vH: variable heavy chain.

Article Snippet: Cells were stained with the following primary anti-human antibodies: mouse anti-human CD2 (clone LT2), mouse anti-human CD3 (clone BW264/56), mouse anti-human TCRαβ (clone BW242/412), mouse anti-human CD4 (clone VIT4), mouse anti-human CD8 (clone BW135/80), mouse anti-human CD45RA (T6D11), mouse anti-human CD62L (145/15), mouse anti-human CD19 (clone LT19), mouse anti-human CD56 (clone AF12-7H3), (all from Miltenyi Biotec), mouse anti-human CD7 (clone M-T701) (BD), mouse anti-human CD7 (clone 6B7) (BioLegend), mouse anti-human PD-1 (EH12.1) (BD), mouse anti-human TIM-3 (7D3) (BD), mouse anti-human LAG-3 (T47–530) (BD).

Techniques: Transduction, Plasmid Preparation, Sequencing, Activation Assay, Control, Expressing

Journal: Leukemia

Article Title: Base-edited CAR T cells for combinational therapy against T cell malignancies

doi: 10.1038/s41375-021-01282-6

Figure Lengend Snippet: A Phenotypic analysis of surface antigen CD3 and CD7 expression (top panel) and CAR expression (bottom panel) of CD3/CD28 activated peripheral blood mononuclear cells elcetroporated with sgRNA targeting TRBC and CD7 alongside coBE3 mRNA and subsequently transduced with 3CAR or 7CAR lentiviral vectors at MOI 5. Reduced TCR/CD3 and CD7 expression in edited groups and high level CAR expression with fratricide evasion (dotted red outline) was exhibited ( n = 4). Self-enrichment effects followed co-culture of 3CAR and 7CAR products ( n = 2) resulted in enriched TCR − CD7 − 3CAR/7CAR cells (red box). B Proportion of CD3 or CD7 surface antigen and CAR expression at end of 3CAR ( n = 4), 7CAR ( n = 4) production or untransduced (UTD) ( n = 4) cells. Error bars represent SEM across ( n = 4) donors. C Schematic of exonic regions within TRBC and CD7 genes. Red marking in exons 4 of TRBC and CD7 represent genomic translation stop sites followed by 5′ untranslated regions (white boxes). Red triangles with asterisk indicate position of base conversion resulting in premature stop codon formation. D Representative base EDITR output of Sanger sequencing results from mixed 3CAR/7CAR co-culture DNA PCR amplicons of TRBC and CD7 genomic loci. Sites of intended base conversion highlighted in red boxes. High frequency G > A (antisense) and C > T changes within editing window highlighted by red vs blue colour. E Percentage of G > A conversions throughout TRBC-targeting protospacer sequence (left), and C > T conversions throughout CD7-targeting protospacer sequence (right).

Article Snippet: Cells were stained with the following primary anti-human antibodies: mouse anti-human CD2 (clone LT2), mouse anti-human CD3 (clone BW264/56), mouse anti-human TCRαβ (clone BW242/412), mouse anti-human CD4 (clone VIT4), mouse anti-human CD8 (clone BW135/80), mouse anti-human CD45RA (T6D11), mouse anti-human CD62L (145/15), mouse anti-human CD19 (clone LT19), mouse anti-human CD56 (clone AF12-7H3), (all from Miltenyi Biotec), mouse anti-human CD7 (clone M-T701) (BD), mouse anti-human CD7 (clone 6B7) (BioLegend), mouse anti-human PD-1 (EH12.1) (BD), mouse anti-human TIM-3 (7D3) (BD), mouse anti-human LAG-3 (T47–530) (BD).

Techniques: Expressing, Transduction, Co-Culture Assay, Sequencing

Journal: Leukemia

Article Title: Base-edited CAR T cells for combinational therapy against T cell malignancies

doi: 10.1038/s41375-021-01282-6

Figure Lengend Snippet: A Experimental timeline of GFP + LUC + Jurkat T cell injection (Day 0) and effector T cell injection (Day 4) in n = 28 NOD/SCID/γc –/– (NSG) mice. Bioluminescent imaging (BLI) performed biweekly (Days 3–31). Organ harvest post mortem for flow-based characterisation (Day 31). B n = 28 NOD/SCID/γc –/– (NSG) mice were intravenously injected with 10 × 10 6 GFP + LUC + Jurkat T cells flow sorted for CD3 + CD7 + expression (Day 0) prior to receiving an IV infusion of effector T cells (Day 4). Mice received either 10 × 10 6 SpCas9 edited TCR − CD7 − 3CAR ( n = 3), 10 × 10 6 SpCas9 edited TCR − CD7 − 7CAR ( n = 3), 10 × 10 6 SpCas9 edited TCR − CD7 − 3CAR/7CAR ( n = 3), 10 × 10 6 coBE3 edited TCR−CD7 − 3CAR ( n = 5), 10 × 10 6 coBE3 edited TCR − CD7 − 7CAR ( n = 5), or 10 × 10 6 coBE3 edited TCR − CD7 − 3CAR/7CAR ( n = 5) effectors. Control mice received untransduced (UTD) cells ( n = 3) or PBS ( n = 1). Leukaemic progression was monitored by serial bioluminescent imaging (BLI) until day 31 when all groups were sacrificed. C Bioluminescence signal of each animal plotted as Average radiance [photons/s/cm 2 /sr]. Each line represents a different experimental group and each point on the line the mean of each group. Error bars represent median with interquartile range. Area under the curve was calculated for each experimental group and values were compared using a one-way ANOVA with Tukey multiple comparison post-hoc. D Number of CD2 + GFP − effector events or CD2 + GFP + tumour events per 1 × 10 4 acquired bone marrow events. Error bars represent SEM. *** P < 0.001 **** P < 0.0001.

Article Snippet: Cells were stained with the following primary anti-human antibodies: mouse anti-human CD2 (clone LT2), mouse anti-human CD3 (clone BW264/56), mouse anti-human TCRαβ (clone BW242/412), mouse anti-human CD4 (clone VIT4), mouse anti-human CD8 (clone BW135/80), mouse anti-human CD45RA (T6D11), mouse anti-human CD62L (145/15), mouse anti-human CD19 (clone LT19), mouse anti-human CD56 (clone AF12-7H3), (all from Miltenyi Biotec), mouse anti-human CD7 (clone M-T701) (BD), mouse anti-human CD7 (clone 6B7) (BioLegend), mouse anti-human PD-1 (EH12.1) (BD), mouse anti-human TIM-3 (7D3) (BD), mouse anti-human LAG-3 (T47–530) (BD).

Techniques: Injection, Imaging, Expressing, Control, Comparison

Journal: Leukemia

Article Title: Base-edited CAR T cells for combinational therapy against T cell malignancies

doi: 10.1038/s41375-021-01282-6

Figure Lengend Snippet: A NSG mice were infused with 1 × 10 7 GFP + LUC + Jurkat T cells modified to express mixed CD3 and/or CD7 surface antigens in groups of ( n = 5) CD3 − CD7 − , ( n = 4) CD3 + CD7 − , (n = 5) CD3 − CD7 + or ( n = 5) CD3 − CD7 − and imaged on day 3 prior to infusion of 1 × 10 7 TCR − CD7 − 3CAR/7CAR mixed effectors or untransduced (UTD) cells. Leukaemic progression monitored by serial BLI for 24 days and revealed disease progression in animals receiving untransduced T cells (3CAR − 7CAR − ) and in animals engrafted with antigen-negative (CD3 − CD7 − ) leukaemia. B Bioluminescence signal of each animal plotted as Average radiance [photons/s/cm 2 /sr]. Each line represents a different experimental group and each point on the line the mean of each group. Error bars represent SEM. Area under the curve was calculated for each experimental group and values were compared using a one-way ANOVA with Tukey multiple comparison post-hoc ****P < 0.0001. C Example of day 24 flow cytometry-based detection in bone marrow of mCD11b − /hCD45 + effector T cells (CD2 + GFP − ) in 3CAR/7CAR treated animals and residual leukaemia (CD2 + GFP + ) in antigen-negative and untransduced groups. D Frequency of hCD45 + CD2 + GFP − effector events or hCD45 + CD2 + GFP + Jurkat events per 10 4 acquired bone marrow events.

Article Snippet: Cells were stained with the following primary anti-human antibodies: mouse anti-human CD2 (clone LT2), mouse anti-human CD3 (clone BW264/56), mouse anti-human TCRαβ (clone BW242/412), mouse anti-human CD4 (clone VIT4), mouse anti-human CD8 (clone BW135/80), mouse anti-human CD45RA (T6D11), mouse anti-human CD62L (145/15), mouse anti-human CD19 (clone LT19), mouse anti-human CD56 (clone AF12-7H3), (all from Miltenyi Biotec), mouse anti-human CD7 (clone M-T701) (BD), mouse anti-human CD7 (clone 6B7) (BioLegend), mouse anti-human PD-1 (EH12.1) (BD), mouse anti-human TIM-3 (7D3) (BD), mouse anti-human LAG-3 (T47–530) (BD).

Techniques: Modification, Biomarker Discovery, Comparison, Flow Cytometry

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β-activated Kinase 1 Is an Essential Regulator of Myogenic Differentiation

doi: 10.1074/jbc.m109.064063

Figure Lengend Snippet: FIGURE 1. Expression of TAK1 in cultured myoblasts and the skeletal muscle of mice. A, C2C12 myoblasts were incubated in DM (2% horse serum in Dulbecco’s modified Eagle’s medium) for the indicated time periods, and the levels of TAK1, phospho-TAK1, TAB1, TAB2, and MyHCf were measured by Western blot. Representa- tive immunoblots and quantification from five independent experiments presented here show that TAK1, phosphorylated TAK1, TAB1, and TAB2 are highly expressed in C2C12 myoblasts, and their levels are reduced upon differentiation of myoblasts into myotubes. B, shown are protein levels of TAK1, phospho-TAK1, TAB1, and TAB2 in gastrocnemius muscle of mice of different ages. n 4 for each age group.

Article Snippet: Antibodies against phospho-I B (Ser32), phospho-Akt (Ser473), phospho-TAK1 (Ser412), phospho-AMPK (Thr172), and total TAK1, Akt, p38, AMP-activated protein kinase (AMPK), TAB1, and TAB2 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Cell Culture, Incubation, Modification, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β-activated Kinase 1 Is an Essential Regulator of Myogenic Differentiation

doi: 10.1074/jbc.m109.064063

Figure Lengend Snippet: FIGURE 2. Expression of TAK1 in regenerating TA muscle in vivo. A, TA muscle of 3-month-old C57BL6 mice was injected with saline alone or cardio- toxin as described under “Experimental Procedures.” After 5 days the TA mus- cle was isolated and processed for RNA isolation and measurement of mRNA levels for TAK1, myogenin, and MyoD by real-time-PCR. Data presented here show that the mRNA levels of TAK1 as well as myogenin and MyoD are signif- icantly increased in cardiotoxin-injected regenerating TA muscle compared with contralateral saline-injected TA muscle (n 3). *, p 0.01, value signif- icantly different from controls. B, representative immunoblots from two inde- pendentexperimentspresentedhereshowthattheproteinlevelsofTAK1are significantly increased in TA muscle 5 days after cardiotoxin injection. C, shown are protein levels of TAK1 in gastrocnemius muscle of 8-week-old wild-type and mdx mice measured by Western blot. The levels of TAK1 are noticeably higher in mdx mice compared with wild-type mice. There was no difference in the levels of unrelated protein actin.

Article Snippet: Antibodies against phospho-I B (Ser32), phospho-Akt (Ser473), phospho-TAK1 (Ser412), phospho-AMPK (Thr172), and total TAK1, Akt, p38, AMP-activated protein kinase (AMPK), TAB1, and TAB2 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, In Vivo, Injection, Saline, Isolation, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β-activated Kinase 1 Is an Essential Regulator of Myogenic Differentiation

doi: 10.1074/jbc.m109.064063

Figure Lengend Snippet: FIGURE3.RoleofTAK1inproliferationofC2C12myoblasts.A,C2C12myo- blasts (10,000 cells/well) were plated in 24-well plate and transfected with vector alone or TAK1 shRNA plasmid. Cells were selected in the presence of puromycin (1.5 g/ml), and the proliferation of myoblasts was measured using the BrdUrd uptake method as described under “Experimental Proce- dures.” Data presented here show that transfection of C2C12 myoblasts with TAK1 shRNA significantly reduced its protein levels and inhibited prolifera- tion. *, p 0.01, values significantly different from control shRNA transfected cells. B, stable transfection of C2C12 myoblasts with a dominant negative TAK1(dnTAK1)plasmidalsoinhibitedtheproliferationofC2C12myoblasts.#, p 0.01, values significantly different from control shRNA transfected myo- blasts. C, knockdown of TAK1 using RNAi technique did not affect the per- centage of apoptotic cells in C2C12 myoblasts assayed using the annexin V-EGFP apoptosis detection kit and fluorescence-activated cell sorting method.

Article Snippet: Antibodies against phospho-I B (Ser32), phospho-Akt (Ser473), phospho-TAK1 (Ser412), phospho-AMPK (Thr172), and total TAK1, Akt, p38, AMP-activated protein kinase (AMPK), TAB1, and TAB2 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Transfection, Plasmid Preparation, shRNA, Control, Stable Transfection, Dominant Negative Mutation, Knockdown, Fluorescence, FACS

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β-activated Kinase 1 Is an Essential Regulator of Myogenic Differentiation

doi: 10.1074/jbc.m109.064063

Figure Lengend Snippet: FIGURE4.InvolvementofTAK1indifferentiationofC2C12myoblasts.A,C2C12myoblastsweretransiently transfected with increasing amounts of dominant negative TAK1 (dnTAK1) plasmid along with either pSK-Luc or pMCK-Luc plasmid in a 1:10 ratio. After 24 h the cells were incubated in DM, and the luciferase activity in cell extracts was measured. Representative data from two independent experiments (each done in triplicate) presented here show that dnTAK1 inhibits the activation of both skeletal actin and muscle creatine kinase promoters in a dose-dependent manner. *, p 0.05, values significantly different from corresponding C2C12 cultures transfected with vector only. B, C2C12 myoblasts were transduced (multiplicity of infection 1:50) with control (Ad.Control) or dominant negative TAK1 (Ad.TAK1) adenoviral vectors for 24 h. The cells were then incubated in DM for indicated time intervals, and the expression of MyHCf was measured by Western blot. Representative immunoblots presented here show that dnTAK1 inhibits the expression of MyHCf without affecting the levels of an unrelated protein actin in C2C12 cultures. C, -fold difference is shown in the mRNA levels of Myf-5, MyoD, myogenin, and myocyte enhancer factor 2D (Mef2D) in Ad.control and Ad.dnTAK1- transduced C2C12 myoblasts 72 h after incubation in DM measured by real-time PCR technique. *, p 0.01, values significantly different from C2C12 myoblasts transduced with Ad.control vector. D, C2C12 myoblasts were transfected with control or either of the two TAK1 shRNA plasmids, each containing a different target sequence for TAK1 knockdown. The cells were selected in the presence of puromycin (1.6 g/ml) for 72–96 h followedbyincubationindifferentiationmediumfor72h.CKactivityincellextractswasmeasuredusingtheCK activityassaykit.ThelevelsofMyHCfandTAK1weremeasuredbyWesternblot.Datapresentedhereshowthat knockdown of TAK1 inhibits the expression of CK and MyHCf in C2C12 cultures. *, p 0.01, values significantly different from control shRNA transfected C2C12 myoblasts.

Article Snippet: Antibodies against phospho-I B (Ser32), phospho-Akt (Ser473), phospho-TAK1 (Ser412), phospho-AMPK (Thr172), and total TAK1, Akt, p38, AMP-activated protein kinase (AMPK), TAB1, and TAB2 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Transfection, Dominant Negative Mutation, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Activation Assay, Infection, Control, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Transduction, shRNA, Sequencing, Knockdown

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β-activated Kinase 1 Is an Essential Regulator of Myogenic Differentiation

doi: 10.1074/jbc.m109.064063

Figure Lengend Snippet: FIGURE 5. Deletion of TAK1 inhibits MyoD-induced differentiation in fibroblasts. TAK1/ and TAK1/ MEF were transduced with Ad.MyoD for 24 h at multiplicity of infection 50. The cells were then incubated in DM for different time intervals. A, myotube formation was measured after 48 h of incubation in DM by performing immunofluorescence using MF20 antibody. Nuclei were stained with 4,6-diamidino-2-phenylindole. The top photomicrographs (green fluorescence protein) show equal transduction of TAK1/ and TAK1/ MEF by Ad.MyoD vector. The bottom photomicrographs show that myotube formation (red color) is significantly reduced in TAK1/ MEF cultures compared with TAK1/. B, Western blot showed that the levels of MyHCf were reduced in TAK1/ MEF compared with TAK1/ after incubation in DM. Immunoblots also show equal levels of MyoD protein in both TAK1/ and TAK1/ MEF and the presence of truncated TAK1 protein in TAK1/ MEF. Wt, wild type. C, levels of CK were also found to be significantly reduced in TAK1/ MEF compared with TAK1/ MEF after 24 and 48 h of incubation in DM. *, p 0.01, values significantly different from TAK1/ MEF at corresponding time point. D, TAK1/ and TAK1/ MEFs were transfected with pcDNA3-MyoD plasmid along with pSK-Luc plasmid in a 1:10 ratio for 24 h. Cells were then incubated in DM for 48 h, and the activation of skeletal -actin promoter was monitored by measuring luciferase activity. Data presented here show a significant reduction in the activation of skeletal actin promoter in TAK1/ MEF compared with TAK1/ MEF. #, p 0.01, value significantly different from TAK1/ MEF.

Article Snippet: Antibodies against phospho-I B (Ser32), phospho-Akt (Ser473), phospho-TAK1 (Ser412), phospho-AMPK (Thr172), and total TAK1, Akt, p38, AMP-activated protein kinase (AMPK), TAB1, and TAB2 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Transduction, Infection, Incubation, Immunofluorescence, Staining, Fluorescence, Plasmid Preparation, Western Blot, Transfection, Activation Assay, Luciferase, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β-activated Kinase 1 Is an Essential Regulator of Myogenic Differentiation

doi: 10.1074/jbc.m109.064063

Figure Lengend Snippet: FIGURE 6. TAK1 regulates myogenic differentiation through the activation of p38 MAPK. A, TAK1/ and TAK1/ MEF were transduced with Ad.MyoD for 24h followed by incubation in DM for the indicated time intervals. Analysis of cell extracts by Western blot showed that the phosphorylation of p38 MAPK protein was completely blocked in TAK1/ MEF compared with TAK1/. There was no difference in total p38, phosphorylated AMPK, and total or phosphor- ylated IB levels between TAK1/ and TAK1/ MEFs. B, C2C12 myoblasts were stably transfected with vector alone or plasmid expressing either dominant negativeTAK1(dnTAK1)orTAK1shRNAsandincubatedindifferentiationmediumfor72h.Thelevelsoftotalandphospho-p38MAPKandTAK1weremeasured by Western blot. Representative immunoblots presented here show that overexpression of either dnTAK1 protein or TAK1 shRNAs inhibited the levels of phosphorylated p38 in C2C12 cultures. C, TAK1/ and TAK1/ MEF were transiently transfected with either pcDNA3-MyoD alone or with pcDNA3-caMKK6 plasmid for 24 h followed by incubation in differentiation medium for additional 72 h. Myotube formation was monitored by immunocytochemistry using MF-20 antibody and 4,6-diamidino-2-phenylindole. Representative photomicrographs presented here show that transfection with caMKK6 restored the myotube formation in TAK1/ MEF cultures. D, shown is quantification of the differentiation index in TAK1/ and TAK1/MEF cultures transfected with MyoD along with caMKK6 or without caMKK6. *, p 0.01, values significantly different from TAK1/ MEF cultures without caMKK6. E, levels of CK measured using a CK activity assay kit and MyHCf and phospho-p38 protein (Western blot) were also found to be significantly increased in TAK1/ MEF transfected with caMKK6.*,p0.05,valuessignificantlydifferentfromTAK1/MEFswithoutcaMKK6.#,p0.05,valuessignificantlydifferentfromTAK1/MEFstransfected with no caMKK6.

Article Snippet: Antibodies against phospho-I B (Ser32), phospho-Akt (Ser473), phospho-TAK1 (Ser412), phospho-AMPK (Thr172), and total TAK1, Akt, p38, AMP-activated protein kinase (AMPK), TAB1, and TAB2 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Transduction, Incubation, Western Blot, Phospho-proteomics, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Over Expression, Immunocytochemistry, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β-activated Kinase 1 Is an Essential Regulator of Myogenic Differentiation

doi: 10.1074/jbc.m109.064063

Figure Lengend Snippet: FIGURE 7. Role of TAK1 in activation of Akt during myogenic differen- tiation. A, TAK1/ and TAK1/ MEF were transduced with Ad.MyoD for 24 h followed by incubation in DM for different time intervals. Levels of phosphorylated and total Akt were measured by Western blotting. Data presented here show that the phosphorylation of Akt is blocked in TAK1/ MEF compared with TAK1/ MEF upon incubation in DM. B, C2C12 myoblasts were transfected with vector alone or dnTAK1 and incubated in differentiation medium for 72 h. Representative immunob- lots show that the overexpression of dnTAK1 inhibits the levels of phos- phorylated Akt in C2C12 cultures. C, TAK1/ and TAK1/ MEF were transiently transfected with either pcDNA3-MyoD alone or with pUSEamp-caAkt plasmid for 24 h followed by incubation in DM for 72 h. Data presented here show that the expression of caAkt did not affect with the levels of CK or MyHCf in TAK1/ MEF. D, TAK1/ and TAK1/ MEF were transiently transfected with either pcDNA3-MyoD alone or with pcDNA3-caMKK6 plasmid followed by incubation in differentiation medium for 72 h. A representative immunoblot presented here shows that transfec- tionwithcaMKK6increasedthelevelsofphosphorylatedAktinTAK1/MEF.

Article Snippet: Antibodies against phospho-I B (Ser32), phospho-Akt (Ser473), phospho-TAK1 (Ser412), phospho-AMPK (Thr172), and total TAK1, Akt, p38, AMP-activated protein kinase (AMPK), TAB1, and TAB2 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Transduction, Incubation, Western Blot, Phospho-proteomics, Transfection, Plasmid Preparation, Over Expression, Expressing

Journal: Journal of Biological Chemistry

Article Title: Transforming Growth Factor-β-activated Kinase 1 Is an Essential Regulator of Myogenic Differentiation

doi: 10.1074/jbc.m109.064063

Figure Lengend Snippet: FIGURE 8. IGF-I augments myogenesis through the activation of TAK1. TAK1/ and TAK1/ MEF were transduced with Ad.MyoD for 24 h followed by incubation in DM with or without IGF-I (100 ng/ml). A, data presented here show that IGF-I significantly increased the levels of CK in TAK1/ but not TAK1/ MEF. *, p 0.01, values significantly different compared with TAK1/ MEF incubated without IGF-I. B, Western blot anal- ysis showed that there was no increase in the levels of either MyHCf or myogenin between IGF-1-treated or untreated TAK1/ MEF. C, TAK1/ and TAK1/ MEF were treated with IGF-I (100 ng/ml) for the indicated time periods, and the levels of phosphorylated or total Akt and p38 MAPK were measured by Western blot. Representative immunoblots presented here show that IGF-I-induced phosphorylation of p38 MAPK (but not Akt) was blocked in TAK1/ MEF compared with TAK1/ MEF.

Article Snippet: Antibodies against phospho-I B (Ser32), phospho-Akt (Ser473), phospho-TAK1 (Ser412), phospho-AMPK (Thr172), and total TAK1, Akt, p38, AMP-activated protein kinase (AMPK), TAB1, and TAB2 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Transduction, Incubation, Western Blot, Phospho-proteomics

Journal: Cancers

Article Title: The Immunomodulatory Effect of Different FLT3 Inhibitors on Dendritic Cells

doi: 10.3390/cancers16213719

Figure Lengend Snippet: Effect of FLT3 inhibition on DC differentiation and surface expression of costimulatory molecules. DCs generated from murine bone marrow cells were differentiated in the presence of 100 nM gilteritinib, midostaurin or quizartinib and were gated using forward and sideward scatter. Dead cells were excluded by life/dead staining. Proportion of double-positive CD11c+/CD11b+ cells is markedly reduced after differentiation in the presence of midostaurin ( a ). Human PBMCs were cultured to obtain DCs as described. Midostaurin-induced monocyte differentiation indicated by reduced CD1a and increased CD14 expression in the presence of midostaurin ( p < 0.0001) and gilteritinib ( p < 0.05) ( b ). Differentiation of murine bone marrow cells in the presence of midostaurin (M1 = 1 nM; M10 = 10 nM; M100 = 100 nM), gilteritinib (G1 = 1 nM; G10 = 10 nM; G100 = 100 nM) or quizartinib (Q1 = 1 nM; Q10 = 10 nM; Q100 = 100 nM) in different concentrations did not induce apoptosis ( c ). The mean fluorescence intensity of CD40 and CD86 on bmDCs after LPS stimulation was reduced in a dose-dependent manner by midostaurin ( p < 0.0001) and gilteritinib ( p < 0.05) exposure ( d ). MoDCs showed reduced CD80, CD83 and CD86 expression after midostaurin treatment with 100 nM. The effect was less pronounced after gilteritinib (100 nM) and only minimal after quizartinib (100 nM) treatment. MFI and rates of positive cells are depicted in the right upper corner ( e ). ns not significant, * p < 0.05, **** = p < 0.0001.

Article Snippet: Briefly, the medium was changed every second day, and the different FLT3i, midostaurin, gilteritinib and quizartinib (all purchased from Selleckchem, Houston, TX, USA), were added at specific concentrations ranging from 1 to 100 nM every other day for the whole differentiation period of 6 days to mimic clinical routine, where continuous TKI treatment affects DC differentiation and development as well.

Techniques: Inhibition, Expressing, Generated, Staining, Cell Culture, Fluorescence

Journal: Cancers

Article Title: The Immunomodulatory Effect of Different FLT3 Inhibitors on Dendritic Cells

doi: 10.3390/cancers16213719

Figure Lengend Snippet: FTL3 inhibition suppresses cytokine secretion by dendritic cells. After 6 days of differentiation in the presence of FLT3i at 100 nM concentration, cytokine release of bmDCs was measured 18 h after LPS stimulation. IL-12 levels were only reduced after midostaurin treatment ( p < 0.01), while IL-6 ( p < 0.01 for midostaurin and p < 0.001 for gilteritinib) and CCL-2 levels ( p < 0.0001 for both FLT3i) were also negatively affected by gilteritinib treatment. TNFα secretion was not affected. The generation of bmDCs is illustrated. Created with BioRender.com/j07f023 ( a ). For moDCs, midostaurin (100 nM) and gilteritinib (100 nM) treatment reduces TNFα ( p < 0.0001), IL-6 ( p < 0.01) and IL-12 levels ( p < 0.0001), quizartinib (100 nM) treatment did not affect TNFα levels but lowered IL-12 ( p < 0.01) and IL-6 ( p < 0.05) levels. Schematic illustration of moDC generation created with BioRender.com/u60g276 ( b ). ns not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Article Snippet: Briefly, the medium was changed every second day, and the different FLT3i, midostaurin, gilteritinib and quizartinib (all purchased from Selleckchem, Houston, TX, USA), were added at specific concentrations ranging from 1 to 100 nM every other day for the whole differentiation period of 6 days to mimic clinical routine, where continuous TKI treatment affects DC differentiation and development as well.

Techniques: Inhibition, Concentration Assay

Journal: Cancers

Article Title: The Immunomodulatory Effect of Different FLT3 Inhibitors on Dendritic Cells

doi: 10.3390/cancers16213719

Figure Lengend Snippet: RNA sequencing reveals inhibition of important immune response pathways by FLT3i, which is supported by protein expression analysis. BmDCs were cultured in the presence of DMSO or FLT3i. After 6 days, cells were stimulated with LPS, when indicated, and RNA was harvested 18 h later. Using hierarchical clustering, the top 20 differentially expressed genes found in RNA sequencing analysis reveal specific clustering of DMSO control samples and after exposure to midostaurin but not for other treatment groups. Expression values are depicted from low (blue) to high (red) ( a ). Concomitantly, principal component analysis (PCA) based on all analyzed gene sets also showed high similarity of midaustaurin-treated samples ( b ). Log-fold change in gene expression of midostaurin-, gilteritinib- and quizartinib-treated moDCs after LPS maturation is depicted in a heatmap. Up- (blue) and downregulation (red) are color-coded ( c ). To prove the inhibition of relevant pathways, protein was extracted from human moDCs after FLT3i treatment and LPS stimulation. Stat3 and Stat5 phosphorylation was impaired by midostaurin treatment ( d ). Inhibition of Akt phosphorylation was observed after midostaurin and quizartinib treatment ( e ). The NFκB pathway was inhibited by midostaurin and gilteritinib treatment as cRel and relB expression were reduced. Quizartinib did not affect relB or cRel expression ( f ). The uncropped bolts are shown in .

Article Snippet: Briefly, the medium was changed every second day, and the different FLT3i, midostaurin, gilteritinib and quizartinib (all purchased from Selleckchem, Houston, TX, USA), were added at specific concentrations ranging from 1 to 100 nM every other day for the whole differentiation period of 6 days to mimic clinical routine, where continuous TKI treatment affects DC differentiation and development as well.

Techniques: RNA Sequencing, Inhibition, Expressing, Cell Culture, Control, Gene Expression, Phospho-proteomics

Journal: Cancers

Article Title: The Immunomodulatory Effect of Different FLT3 Inhibitors on Dendritic Cells

doi: 10.3390/cancers16213719

Figure Lengend Snippet: KEGG analysis of midostaurin-treated bmDCs (selected pathways).

Article Snippet: Briefly, the medium was changed every second day, and the different FLT3i, midostaurin, gilteritinib and quizartinib (all purchased from Selleckchem, Houston, TX, USA), were added at specific concentrations ranging from 1 to 100 nM every other day for the whole differentiation period of 6 days to mimic clinical routine, where continuous TKI treatment affects DC differentiation and development as well.

Techniques: Cell Differentiation

Journal: The EMBO Journal

Article Title: LARP1 binds ribosomes and TOP mRNAs in repressed complexes

doi: 10.1038/s44318-024-00294-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Samples were boiled at 95 °C for 5 min and loaded into 4–12% Criterion XT-Bis-Tris polyacrylamide gels (Bio-Rad 3450125).

Techniques: Over Expression, Recombinant, Luciferase, Sequencing, Protease Inhibitor, SYBR Green Assay, BIA-KA, Membrane, Blocking Assay, Reporter Assay, Lysis, Marker, Software, Chromatography, Amplification, Imaging