Journal: Journal of Biological Chemistry

Article Title: Transgenic Expression of MicroRNA-185 Causes a Developmental Arrest of T Cells by Targeting Multiple Genes Including Mzb1

doi: 10.1074/jbc.m113.503532

Figure Lengend Snippet: FIGURE 5. miR-185 targets a number of genes in developing thymocytes. A, relative mRNA levels of Mzb1, Nfatc3, and Camk4 in DN3 thymocytes, normalized to the endogenous Gapdh levels, were determined by real-time quantitative PCR. WT values were set to 1. Data shown are of the mean S.E. of at least three independent experiments performed in triplicates. Bars are representative of WT (white), Tg-25 (light gray), Tg-35 (dark gray), and Tg-6 (black) mice. (n.s. nonsignificant, *, p 0.05, **, p 0.01, ***, p 0.001; versus the threshold set as 1; one sample Student’s t test) B, immunoblot analysis of Mzb1 and NFATc3 expression in miR-185 Tg-6 DN3 thymocytes when compared with the wild type control. -Actin was used as the endogenous control. C, Mzb1 protein expression levels in human thymocytes obtained from five normal individuals (C1–C5) and four patients with 22q11.2 deletion syndrome (P1–P4). -Actin was used as the endogenous control. Band intensities of Mzb1 and -actin were measured using the ImageJ software. The Mzb1/-actin ratio was calculated by dividing the band intensity of Mzb1 to that of the -actin for each sample. Relative Mzb1 levels were then determined for each experiment (Exp #1–Exp. #3.) indicated as a group. This was done by normalizing the Mzb1/-actin ratio for each sample relative to the first control sample. The first control sample was set as 1 in each of three independent experiments. D, the Mzb1 CDS and 3-UTR each contain one putative miR-185 binding site. The diagram shows conserved miR-185 base pairing with human and murine Mzb1 mRNA. Mutated Mzb1 sequences are underlined. E, miR-185 directly targets Mzb1 CDS. A representative blot was shown from HEK293T cells transfected with the plasmid (pEF1) containing either wild type Mzb1 CDS-Myc or mutant Mzb1 CDS-Myc fusion, along with theemptyvector(white)orpCDNA3.1/miR-185(black).AGFP-expressingplasmid(pEGFP)wasusedasthetransfectioncontrol.BandintensitiesofMycandGFP were calculated for each lane using the ImageJ software. Relative Myc levels in the wild type Mzb1 or mutant Mzb1 transfectants were determined by normalizing the Myc/GFP ratio of pCDNA3.1/miR-185 to that of the pCDNA3.1 control, which was set as 1. F, graph shows the mean S.E. of relative Myc/GFP levels from four independent experiments performed in at least duplicates (n.s. nonsignificant, *, p 0.05, **, p 0.01, ***, p 0.001; two-tailed unpaired Student’s t test). G, the Mzb1 3-UTR is a direct target of miR-185. H, validation of additional miR-185 targets. G–H, luciferase activity was normalized to the -galactosidase of COS-1 cells transfected with the luciferase plasmids containing the indicated 3-UTR, along with either the empty vector or pCDNA3.1-miR-185. Normalized luciferase activity of the pCDNA3.1/miR-185 (black) transfectant was determined relative to that of the empty pCDNA3.1 vector (white), which was set as 1. Btk 3-UTR, a previously validated target of miR-185, was used as a positive control. Data shown are of the mean S.E. from four independent experiments performed in at least triplicates (*, p 0.05, **, p 0.01, ***, p 0.001; two-tailed unpaired Student’s t test).

Article Snippet: Immunoblotting was performed as described previously with the following antibodies: Mzb1 (11454-1-AP, Proteintech), NFATc3 (SC-8321, Santa Cruz Biotechnology), -actin (4967, Cell Signaling), GFP (632380, Clontech), Myc epitope (2272, Cell Signaling), anti-rabbit HRP-conjugated secondary antibody, and anti-mouse IgGHRP-conjugated secondary antibody (19).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Control, Software, Binding Assay, Transfection, Plasmid Preparation, Mutagenesis, Two Tailed Test, Biomarker Discovery, Luciferase, Activity Assay, Positive Control

Journal: Journal of Biological Chemistry

Article Title: Transgenic Expression of MicroRNA-185 Causes a Developmental Arrest of T Cells by Targeting Multiple Genes Including Mzb1

doi: 10.1074/jbc.m113.503532

Figure Lengend Snippet: FIGURE 6. miR-185 controls TCR-stimulated intracellular calcium responses. A, intracellular calcium flux was analyzed by flow cytometry in DP thymocytes from the WT (black line), Tg-25 (blue line), and Tg-35 (red line) mice. DP thymocytes were gated by size. Black arrows indicate the time points for each treatment. Fluo-3 AM-loaded thymocytes were treated with biotinylated (Bio) anti-CD3 and anti-CD4 followed by streptavidin (SA), ionomycin (Iono), and MnCl2. B, graph shows relative changes in TCR-triggered peak Ca2-influx over the base line. Each bar represents the mean S.E. of six independent experiments (*, p 0.05, **,p0.01,***,p0.001;two-tailedunpairedStudent’sttest).CandD,Fluo-3AM-loadedthymocytesweretreatedwithionomycin(Iono)(C)andthapsigargin (Thapsi) (D) in the absence of extracellular calcium. DP thymocytes were gated electronically. Experiments were repeated two times, and representative plots are shown from the WT (black line), Tg-25 (blue line), and Tg-35 (red line) mice. E, representative immunoblot shows Mzb1 expression in Jurkat T cells transfected with varying concentrations of miR-185 inhibitor (a-miR-185) when compared with the control (miR negative control inhibitor). -Actin was used as the endogenous control. Band intensities of Mzb1 and -actin were measured using the ImageJ software. The relative amounts of Mzb1 protein were shown as normalized to the control inhibitor, which was set as 1. This was done by dividing the Mzb1/-actin ratio of each sample to that of the control sample. F, graph represents the mean S.E. of relative Mzb1 levels in Jurkat T cells transfected with the control and miR-185 inhibitor from five independent experiments, performed in at least duplicates (*, p 0.05, **, p 0.01, ***, p 0.001; two-tailed unpaired Student’s t test). G, intracellular calcium responses were analyzed byflowcytometryovertimeinJurkatTcellsfollowingtransfectionwitha-miR-185(redline)andthecontrolinhibitor(blackline).Fluo-3AM-loadedJurkatTcells were treated with the mAb C305.2 (C305) (anti-TCR), ionomycin (Iono), and MnCl2. Black arrows indicate the time points for each treatment. H, graph shows relative changes in TCR-triggered peak Ca2 influx over the base line. Each bar represents the mean S.E. of four independent experiments (n.s. nonsig- nificant, *, p 0.05, **, p 0.01, ***, p 0.001; two-tailed unpaired Student’s t test).

Article Snippet: Immunoblotting was performed as described previously with the following antibodies: Mzb1 (11454-1-AP, Proteintech), NFATc3 (SC-8321, Santa Cruz Biotechnology), -actin (4967, Cell Signaling), GFP (632380, Clontech), Myc epitope (2272, Cell Signaling), anti-rabbit HRP-conjugated secondary antibody, and anti-mouse IgGHRP-conjugated secondary antibody (19).

Techniques: Flow Cytometry, Western Blot, Expressing, Transfection, Control, Negative Control, Software, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: The protein interaction of mitochondrial transcription factors A and B2 is associated with 30-day survival in critical COVID-19

doi: 10.3389/fimmu.2025.1445403

Figure Lengend Snippet: mRNA transcripts of MQC-related genes did not differ between 30-day survivors and 30-day non-survivors on day 1. (A) Comparison of relative MFN2 mRNA expression, (B) relative PINK mRNA expression, (C) mtDNA copy-number, (D) relative TFAM mRNA expression and (E) relative TFB2M mRNA expression between 30-day survivors and 30-day non-survivors.

Article Snippet: Slides were re-hydrated, permeabilized and primary antibodies against TFAM (1:50, sc-376672, Santa Cruz Biotechnology, Heidelberg, Germany) and TFB2M (1:100, 46-481, ProSci Incorporated, Poway, CA, USA) were incubated overnight at 4°C.

Techniques: Comparison, Expressing

Journal: Frontiers in Immunology

Article Title: The protein interaction of mitochondrial transcription factors A and B2 is associated with 30-day survival in critical COVID-19

doi: 10.3389/fimmu.2025.1445403

Figure Lengend Snippet: Interactions of TFAM with TFB2M in the human mitochondrial transcription initiation complex increases in 30-day survivors compared to 30-day non-survivors (** p = 0.0017).

Article Snippet: Slides were re-hydrated, permeabilized and primary antibodies against TFAM (1:50, sc-376672, Santa Cruz Biotechnology, Heidelberg, Germany) and TFB2M (1:100, 46-481, ProSci Incorporated, Poway, CA, USA) were incubated overnight at 4°C.

Techniques:

Journal: Frontiers in Immunology

Article Title: The protein interaction of mitochondrial transcription factors A and B2 is associated with 30-day survival in critical COVID-19

doi: 10.3389/fimmu.2025.1445403

Figure Lengend Snippet: (A) Proximity ligation assay (PLA) in PBMCs of critical COVID-19 patients on day 1 and healthy control. PLA was conducted to assess the interaction between TFAM and TFB2M within the human mitochondrial transcription initiation complex, serving as an indicator of mitochondrial functionality and viability. Each green dot represents a signal for a TFAM/TFBM interaction. Nuclear staining with DAPI (blue) can be used to determine the average PLA signal per cell. The average PLA signal per cell was then associated with 30-day survival of critical COVID-19. (B) Patients with >10.7 PLA signals experience more ICU-free days over a 60-day period compared to patients with <10.7 PLA signals (*** p = 0.0008).

Article Snippet: Slides were re-hydrated, permeabilized and primary antibodies against TFAM (1:50, sc-376672, Santa Cruz Biotechnology, Heidelberg, Germany) and TFB2M (1:100, 46-481, ProSci Incorporated, Poway, CA, USA) were incubated overnight at 4°C.

Techniques: Proximity Ligation Assay, Control, Staining

Journal: Transfusion medicine and hemotherapy : offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin und Immunhamatologie

Article Title: CRISPR/Cas9-Editing K562 Cell Line as a Potential Tool in Transfusion Applications: Knockout of Vel Antigen Gene.

doi: 10.1159/000534012

Figure Lengend Snippet: Fig. 2. Selection of sgRNA with high efficiency to edit the SMIM1 gene in K562 cells. a GFP expression in different cell lines after transfection. Data are shown in mean ± SD. p values were calculated by t tests (n = 3, **p < 0.01). b A chromatograph shows direct DNA sequencing results in exon 3 of the SMIM1 gene from unedited K562 (K562 WT) and CRISPR/Cas9-mediated K562 cells (K562 KO). Underline and arrow denote the proto-spacer adjacent motif (PAM) sequences and sgRNA sequences, respectively.

Article Snippet: Following transfer to a PVDF membrane (Immobilon-E, Merck Millipore Ltd), the membrane was blocked and shaken in a 3% bovine serum albumin solution for 1 h. The membrane was incubated with the rabbit antibody against SMIM1 (ProteinTech, Cat.: 27849-1-AP) at 4°C overnight.

Techniques: Selection, Expressing, Transfection, DNA Sequencing, CRISPR

Journal: Transfusion medicine and hemotherapy : offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin und Immunhamatologie

Article Title: CRISPR/Cas9-Editing K562 Cell Line as a Potential Tool in Transfusion Applications: Knockout of Vel Antigen Gene.

doi: 10.1159/000534012

Figure Lengend Snippet: Fig. 3. Sequencing results of two single KO (SMIM1 KO) cell clones. a The chromatograph above (KO clone 1) showed deletion of two bases and that below (KO clone 2) showed deletion of four bases. b The deduced amino acids sequences of the SMIM1 gene in wild-type (WT) and KO clones. Bold fonts highlight part of the same amino acids and “*” represents the stop codon.

Article Snippet: Following transfer to a PVDF membrane (Immobilon-E, Merck Millipore Ltd), the membrane was blocked and shaken in a 3% bovine serum albumin solution for 1 h. The membrane was incubated with the rabbit antibody against SMIM1 (ProteinTech, Cat.: 27849-1-AP) at 4°C overnight.

Techniques: Sequencing, Clone Assay

Journal: Cell reports

Article Title: Activation of autophagy depends on Atg1/Ulk1-mediated phosphorylation and inhibition of the Hsp90 chaperone machinery

doi: 10.1016/j.celrep.2023.112807

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-Cdc37 , StressMarq Biosciences , Cat# SPC-142; RRID:AB_2570605.

Techniques: Virus, Recombinant, Proximity Ligation Assay, Software

Journal: BMC Genomics

Article Title: Generation, annotation and analysis of ESTs from Trichoderma harzianum CECT 2413

doi: 10.1186/1471-2164-7-193

Figure Lengend Snippet: The most abundantly represented genes.

Article Snippet: T34C834 , 38 , Hypothetical protein FG05928.1 ( G. zeae ) (contains InterPro Zinc finger domain) , 2,00E-09 , L02, L03, L10, L11.

Techniques: Binding Assay