Journal: Bioengineered

Article Title: Zinc finger protein 521 attenuates osteoarthritis via the histone deacetylases 4 in the nucleus

doi: 10.1080/21655979.2022.2090203

Figure Lengend Snippet: The primary chondrocytes infected with Zfp521 were in good condition. (a) The effect of different doses of Zfp521 on chondrocyte viability was monitored in RTCA experiments. (b–c) Quantitative analysis of the cell index at 28 and 52 h. (d) The chondrocytes were observed using a fluorescence microscope in a pilot Zfp521 dose within an MOI range of 50–400. Scale bar: 100 μm. (e) Comparison of the rate of fluorescent positive cells between MOIs of 50 and 100. (f) The Zfp521 expression increased 8.304-fold at an MOI of 50 in the Ad-Zfp521 group compared with the vehicle group. (g) The Toluidine blue staining for the Ad-Zfp521 group at an MOI of 50 and the vehicle group. *P < 0.05, **P < 0.01, and ***P < 0.001. ns: not significant.

Article Snippet: Both the Ad encoding Zfp521 and mCherry fluorescent protein (Ad-Zfp521) and a negative control (vehicle) were purchased from Genechem (Shanghai, China).

Techniques: Infection, Fluorescence, Microscopy, Comparison, Expressing, Staining

Journal: Bioengineered

Article Title: Zinc finger protein 521 attenuates osteoarthritis via the histone deacetylases 4 in the nucleus

doi: 10.1080/21655979.2022.2090203

Figure Lengend Snippet: Here, Zfp521 affected the cell cycle progression and extracellular matrix–related pathways in the proteomics analysis (a) The hierarchical clustering of differential expression proteins. (b–c) Volcano plots and statistical plots of differential proteins. (d) Differential proteins were enriched in the pathway of the cell cycle phase and extracellular matrix in Biological Process of Gene Ontology analysis. (e) The cellular component analysis showed that differential proteins were enriched in the extracellular-associated region. (f) Network analysis for cyclin-dependent kinase-4 and MMP-13.

Article Snippet: Both the Ad encoding Zfp521 and mCherry fluorescent protein (Ad-Zfp521) and a negative control (vehicle) were purchased from Genechem (Shanghai, China).

Techniques: Quantitative Proteomics

Journal: Bioengineered

Article Title: Zinc finger protein 521 attenuates osteoarthritis via the histone deacetylases 4 in the nucleus

doi: 10.1080/21655979.2022.2090203

Figure Lengend Snippet: Here, Zfp521 promoted chondrocyte proliferation and inhibited apoptosis. (a) The EdU staining for the Zfp521-siRNA and vehicle siRNA chondrocytes was performed. Scale bar: 200 μm. (b) Comparison of the TUNEL staining for the Zfp521-siRNA and vehicle siRNA chondrocytes. Scale bar: 200 μm. (c-d) Quantitative analysis of the EdU and TUNEL assays between the Zfp521-siRNA group and vehicle siRNA group. (e–f) Immunohistochemical staining for PCNA and caspase-3 in the ACLT rats at nine weeks postop. Black scale bar: 50 μm. Blue scale bar: 15 μm. (g–h) Quantitative analysis of the immunohistochemical staining for PCNA and caspase-3 in the ACLT rats. **P < 0.01 and ***P < 0.001. ns: not significant.

Article Snippet: Both the Ad encoding Zfp521 and mCherry fluorescent protein (Ad-Zfp521) and a negative control (vehicle) were purchased from Genechem (Shanghai, China).

Techniques: Staining, Comparison, TUNEL Assay, Immunohistochemical staining

Journal: Bioengineered

Article Title: Zinc finger protein 521 attenuates osteoarthritis via the histone deacetylases 4 in the nucleus

doi: 10.1080/21655979.2022.2090203

Figure Lengend Snippet: Here, Zfp521 suppressed the catabolic responses by downregulating Runx2 in the cartilage. (a) The RT-PCR of the IL1β-induced rat OA chondrocytes for catabolic biomarkers. (b) The total MMP levels in the living rats were monitored using FMT. (c) Quantitative comparison of FMT by the region of interest. (d) Immunohistochemical staining for Runx2, MMP-13, and Col10a1 at the protein level after nine weeks of treatment in the rats. Black scale bar: 50 μm. Blue scale bar: 15 μm. * P < 0.05. ns: not significant.

Article Snippet: Both the Ad encoding Zfp521 and mCherry fluorescent protein (Ad-Zfp521) and a negative control (vehicle) were purchased from Genechem (Shanghai, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Comparison, Immunohistochemical staining, Staining

Journal: Bioengineered

Article Title: Zinc finger protein 521 attenuates osteoarthritis via the histone deacetylases 4 in the nucleus

doi: 10.1080/21655979.2022.2090203

Figure Lengend Snippet: Here, Zfp521 enhanced anabolism by upregulating SOX9 in the cartilage. (a) The RT-PCR of the IL1β-induced rat OA chondrocytes for anabolic biomarkers. (b) Immunohistochemical staining for SOX9 and Col2a1 at the protein level after nine weeks of treatment in the rats. Black scale bar: 50 μm. Blue scale bar: 15 μm. (c–d) Safranin O/fast green staining and the OARSI scores for the cartilage evaluation. Scale bar: 50 μm. (e) Sample preparation for the nanoindentation test and labeling of the cartilage areas to be measured. (f–g) Young’s modulus of the cartilage in the medial and lateral tibial plateau. *P < 0.05.

Article Snippet: Both the Ad encoding Zfp521 and mCherry fluorescent protein (Ad-Zfp521) and a negative control (vehicle) were purchased from Genechem (Shanghai, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Staining, Sample Prep, Labeling

Journal: Bioengineered

Article Title: Zinc finger protein 521 attenuates osteoarthritis via the histone deacetylases 4 in the nucleus

doi: 10.1080/21655979.2022.2090203

Figure Lengend Snippet: Here, Zfp521 relieved pain and attenuated knee degeneration. (a) Gait analysis for pain evaluation using the mean intensity/body weight of the right hind limb. (b–c) Quantitative comparison of the mean intensity/body weight and duty cycle. (d) India ink staining for the assessment of the surface erosion of the articular cartilage. (e) The severity of the osteoporosis and subchondral osteosclerosis were evaluated via X-rays. (f) Osteophytosis scores in the X-rays. * P < 0.05. ns: not significant.

Article Snippet: Both the Ad encoding Zfp521 and mCherry fluorescent protein (Ad-Zfp521) and a negative control (vehicle) were purchased from Genechem (Shanghai, China).

Techniques: Comparison, Staining

Journal: Bioengineered

Article Title: Zinc finger protein 521 attenuates osteoarthritis via the histone deacetylases 4 in the nucleus

doi: 10.1080/21655979.2022.2090203

Figure Lengend Snippet: The molecular function of Zfp521 depended on the HDAC4 in the cell nucleus. (a) Domain analysis of proteomics for differentially expressed proteins. (b) A reduction in the phosphorylation metabolism in the KEGG pathway analysis. (c) The Zfp521 upregulation enabled HDAC4 to move into the nucleus at 48 h and onwards, as shown by the enhanced green fluorescence in the nucleus. Scale bar: 100 μm. (d) Here, Zfp521 did not affect the total protein expression of HDAC4. (e) The level of HDAC4 increased in the nucleus and decreased in the cytoplasm after Zfp521 upregulation. (f) The functions of Zfp521 were significantly weakened in the presence of okadaic acid.

Article Snippet: Both the Ad encoding Zfp521 and mCherry fluorescent protein (Ad-Zfp521) and a negative control (vehicle) were purchased from Genechem (Shanghai, China).

Techniques: Phospho-proteomics, Fluorescence, Expressing

Journal: Microbial Cell Factories

Article Title: Generation of diploid Pichia pastoris strains by mating and their application for recombinant protein production

doi: 10.1186/1475-2859-11-91

Figure Lengend Snippet: Vectors for anti-HER2 light-chain and heavy chain expression. ( a ) Plasmid pGLY10969 encodes the light chain of anti-HER2 antibody and mCherry red fluorescent protein in association with the arsenic resistance selection marker and targets the URA6 locus in P. pastoris genome ( b ) Plasmid pGLY10970 encodes the heavy chain of anti-HER2 antibody and yEGFP3 green fluorescent protein in association with nourseothricin resistance selection marker and targets the URA6 locus in P. pastoris genome. ( c ) Plasmid pGLY6830 encodes both light-chain and heavy-chain of anti-HER2 expression cassettes in association with zeocin resistance selection marker and targets to the TRP2 locus in P. pastoris genome. The Spe I restriction enzyme site used to linearize each vector prior to yeast transformation is underlined.

Article Snippet: Green fluorescent protein yEGFP3 [ ] and red fluorescent protein mCherry [ ] were codon optimized and synthesized from Genscript (Piscataway, NJ).

Techniques: Expressing, Plasmid Preparation, Selection, Marker, Transformation Assay

Journal: Microbial Cell Factories

Article Title: Generation of diploid Pichia pastoris strains by mating and their application for recombinant protein production

doi: 10.1186/1475-2859-11-91

Figure Lengend Snippet: Yeast strains used in this study

Article Snippet: Green fluorescent protein yEGFP3 [ ] and red fluorescent protein mCherry [ ] were codon optimized and synthesized from Genscript (Piscataway, NJ).

Techniques: Plasmid Preparation, Glycoproteomics

Journal: eLife

Article Title: Complex effects of kinase localization revealed by compartment-specific regulation of protein kinase A activity

doi: 10.7554/eLife.66869

Figure Lengend Snippet: ( A ) Expression levels of PKA-RIIβ (top panel) and PKA-RIIα (middle panel). PKAR-FKBP-FP appears as a separate band above endogenous PKA-RIIβ due to its larger size. ( B ) Quantification of total (endogenous plus exogenous) RIIβ expression in cells transiently or stably expressing PKAR-FKBP-FP, normalized to expression in HeLa cells (endogenous only). RIIβ expression is significantly increased in HeLa PFM and PFY cell lines vs. control. ( C ) Quantification of PKA-RIIα expression normalized to expression in HeLa cells. ( D ) PKA-RI expression levels. ( E ) Quantification of RI expression normalized to expression in HeLa cells. ( F ) PKA-C expression levels. ( G ) Quantification of PKA-C expression normalized to expression in HeLa cells. GAPDH used as a loading control for all experiments. Lanes (from left to right) contain lysate from HeLa cells transiently expressing our YFP- or mCherry-tagged PKA-R translocation system (lanes 1 and 2), our HeLa PFM and PFY cell lines (lanes 3 and 4), and untransfected HeLa cells (lane 5). Each blot was repeated twice with representative images shown. Raw immunoblot images are provided in . Quantification, including exact p values, is provided in . (*p<0.05). Figure 1—figure supplement 1—source data 1. Raw immunoblot images, labeled and unlabeled. Images are divided into two folders – one for each technical replicate. Figure 1—figure supplement 1—source data 2. Immunoblot statistical analysis. One-way ANOVAs with multiple comparisons for PKAR-IIb (Sheet 1), PKAR-IIa (Sheet 2), PKA-RI (Sheet 3), and PKA-C (Sheet 4) expression data presented in . Mean, standard deviation, and number of technical replicates also shown for each condition.

Article Snippet: The gel purified PCR product was ligated into a transient expression plasmid containing FKBP and either YFP or mCherry fluorescent protein (gifts from Dr Takanari Inoue, Johns Hopkins University) in the case of PKAR-FKBP-FP, or mCherry alone in the case of mCherry-PKA-C.

Techniques: Expressing, Stable Transfection, Control, Translocation Assay, Western Blot, Labeling, Standard Deviation

Journal: eLife

Article Title: Complex effects of kinase localization revealed by compartment-specific regulation of protein kinase A activity

doi: 10.7554/eLife.66869

Figure Lengend Snippet: ( A ) Schematic of PKA-R translocation system. Rapamycin induces heterodimerization of FKBP and FRB, resulting in translocation of PKA-R to the plasma membrane (FKBP = FK506-binding protein, FRB = FKBP-rapamycin-binding domain, R = rapamycin, PKA-R = PKA regulatory subunit, C = PKA catalytic subunit). ( B ) DNA construct design. Two versions of recombinant PKA-R were created with different fluorescent labels. ( C ) Subcellular localization of PKAR-FKBP-YFP (green) within a transiently transfected HeLa cell at 0 and 10 min after addition of 100 nM rapamycin. Scale bar, 10 µm. mCherry protein (red) co-expressed for visualization. ( D ) PKA-R translocation in HeLa PFM cells quantified as cytoplasmic intensity drop in mCherry channel following addition of DMSO or 100 nM rapamycin. p = 0.0039 at t = 24 min post-rapamycin addition; two-tailed Student’s t -test. ( E ) Catalytic subunit of the protein kinase A (PKA-C) translocation in HeLa cells transiently transfected with PKAR-FKBP-YFP, Lyn-FRB, and mCherry-PKA-C, quantified as a cytoplasmic intensity drop in mCherry channel following addition of 100 nM rapamycin. Cells transfected with mCherry protein instead of mCherry-PKA-C were used as a control. p = 0.037 at t = 24 min post-rapamycin addition; two-tailed Student’s t -test. Graphs display the mean of each data set with standard error of the mean (SEM) indicated by shaded region. Number of cells in each data set is as indicated in the figure. Data is the result of one ( D ) and three ( E ) independent experiments, respectively. Mean and SEM values for each condition and time point are provided in . Arrows indicate the timing of drug addition. Figure 1—source data 1. Characterization of regulatory subunit of the protein kinase A (PKA-R) translocation system. ( a ) Sheet 1, Time Course. Change in mCherry cytoplasmic intensity following addition of 0.1% DMSO or 100 nM rapamycin. Mean, standard error of the mean (SEM), and number of cells given for each time point and condition. ( b ) Sheet 2, Time Course. Change in mCherry cytoplasmic intensity following addition of 100 nM rapamycin. Mean, SEM, and number of cells given for each time point and condition.

Article Snippet: The gel purified PCR product was ligated into a transient expression plasmid containing FKBP and either YFP or mCherry fluorescent protein (gifts from Dr Takanari Inoue, Johns Hopkins University) in the case of PKAR-FKBP-FP, or mCherry alone in the case of mCherry-PKA-C.

Techniques: Translocation Assay, Clinical Proteomics, Membrane, Binding Assay, Construct, Recombinant, Transfection, Two Tailed Test, Control

Journal: eLife

Article Title: Complex effects of kinase localization revealed by compartment-specific regulation of protein kinase A activity

doi: 10.7554/eLife.66869

Figure Lengend Snippet: Plasmid maps for ( A ) PKAR-FKBP-YFP and ( B ) PKAR-FKBP-mCherry.

Article Snippet: The gel purified PCR product was ligated into a transient expression plasmid containing FKBP and either YFP or mCherry fluorescent protein (gifts from Dr Takanari Inoue, Johns Hopkins University) in the case of PKAR-FKBP-FP, or mCherry alone in the case of mCherry-PKA-C.

Techniques: Plasmid Preparation

Journal: eLife

Article Title: Complex effects of kinase localization revealed by compartment-specific regulation of protein kinase A activity

doi: 10.7554/eLife.66869

Figure Lengend Snippet: ( A ) Schematic of the microfluidic device used to produce gradients of rapamycin across microchannels housing HeLa PFY or PFM cells. ( B ) Single cell response to 20 nM rapamycin gradient. Numbers show time in minutes. (Green = PKAR-FKBP-YFP [stably expressed], Blue = H2β-mCerulean [transiently expressed nuclear marker], Red = Alexa Fluor 594 dye). ( C ) Average nuclear position (normalized to t = 0) for HeLa PFY cells in 20 nM rapamycin gradient (0.08 nM/µm) vs. no-translocation rapamycin control (HeLa cells stably expressing PKAR-FKBP-YFP but not Lyn-FRB). Standard error of the mean (SEM) indicated by shaded regions. p = 1.34 × 10 -5 at 180 min post-rapamycin addition; two-tailed Student’s t -test. Number of cells in each data set as indicated in the figure. ( D ) Single cell nuclear position data for no-translocation rapamycin control cells in 20 nM rapamycin gradient. Data from one independent experiment. ( E ) Single cell nuclear position data for HeLa PFY cells in 20 nM rapamycin gradient. Data from three independent experiments. Data from ( D ) superimposed in light gray. ( F ) Tracking of intracellular PKA activity gradient in HeLa PFM cells using the transiently expressed FRET probe Lyn-AKAR4. Rapamycin gradient introduced at t = 0. Colorimetric FRET ratio scale as indicated by color bar. ( G ) Mean intracellular plasma membrane (PM) PKA activity tracking along the cell length from the high end of the rapamycin gradient (‘0’ in panel F) to the low end (‘1’ in panel F). Data represent the mean of n = 9 HeLa PFM cells from two independent experiments with SEM indicated by shaded region. Cells were divided into 20 bins with the average FRET ratio value taken for each. Arrows in ( C–E ) indicate addition of rapamycin. Scale bars in ( B ) and ( F ), 10 µm. Mean and SEM values for each time point and condition in ( C ) and each time point and position in ( G ) are provided in . Figure 3—source data 1. Nuclear position and Lyn-AKAR4 data in microfluidic device. ( a ) Sheet 1, and Time Course. Normalized nuclear position data for cells in 20 nM rapamycin gradient (0.08 nM/µm) with or without the membrane subunit of the regulatory subunit of the protein kinase A (PKA-R) translocation system (Lyn-FRB). Results also shown for cells in a volume equivalent DMSO gradient, as displayed in . Mean, standard error of the mean (SEM), and number of cells given for each time point and condition. ( b ) Sheet 2, Intracellular PKA Activity Distribution. Mean intracellular PM PKA activity along the cell length from the high end of the rapamycin gradient (‘0’) to the low end (‘1’) for three time points. Mean, SEM, and number of cells given for all 20 bins and each time point.

Article Snippet: The gel purified PCR product was ligated into a transient expression plasmid containing FKBP and either YFP or mCherry fluorescent protein (gifts from Dr Takanari Inoue, Johns Hopkins University) in the case of PKAR-FKBP-FP, or mCherry alone in the case of mCherry-PKA-C.

Techniques: Stable Transfection, Marker, Translocation Assay, Control, Expressing, Two Tailed Test, Activity Assay, Clinical Proteomics, Membrane

Journal: eLife

Article Title: Complex effects of kinase localization revealed by compartment-specific regulation of protein kinase A activity

doi: 10.7554/eLife.66869

Figure Lengend Snippet: ( A ) Single cell response to DMSO gradient (0–0.1% from ‘sink’ to ‘source’ as labeled in ). Numbers show time in minutes. Scale bar, 10 µm. (Green = PKAR-FKBP-YFP [stably expressed], Blue = H2β-mCerulean [transiently expressed nuclear marker], Red = Alexa Fluor 594 dye). ( B ) Nuclear position normalized to t = 0 min post-DMSO addition. A positive nuclear position indicates net movement of the nuclear centroid toward the DMSO source as described in Materials and methods. Arrow indicates DMSO addition. Data represent nuclear position for n = 18 cells from two independent experiments. Mean and standard error of the mean (SEM) values for each time point are provided in .

Article Snippet: The gel purified PCR product was ligated into a transient expression plasmid containing FKBP and either YFP or mCherry fluorescent protein (gifts from Dr Takanari Inoue, Johns Hopkins University) in the case of PKAR-FKBP-FP, or mCherry alone in the case of mCherry-PKA-C.

Techniques: Labeling, Stable Transfection, Marker

Journal: bioRxiv

Article Title: Modulating gene expression and protein secretion in the bacterial predator Bdellovibrio bacteriovorus

doi: 10.1101/2025.05.17.654431

Figure Lengend Snippet: ( a ) Schematic representation of B. bacteriovorus AP cell expressing cytosolic mScarletI3 under the control of P merRNA promoter on a pCAT.000-derived plasmid. ( b ) Comparison of fluorescence intensities of different fluorescent reporter proteins (mCherry, mNeonGreen, mScarletI3) expressed in B. bacteriovorus AP cells, measured at their respective emission wavelengths (610 nm, 592 nm, 517 nm respectively). The control is plasmid pCAT:P merRNA -opt.RBS lacking a fluorescent protein; its fluorescence was measured at 610 nm, 592 nm, and 517 nm, while a mean of all three measurements is shown here. White dots indicate median fluorescence. A second independent biological replicate produced highly similar results (see online source data).

Article Snippet: Coding regions for fluorescent proteins mCherry, mScarletI3 [ ], mNeonGreen [ ] were codon optimized for B. bacteriovorus (Twist Biosciences).

Techniques: Expressing, Control, Derivative Assay, Plasmid Preparation, Comparison, Fluorescence, Produced