Journal: bioRxiv

Article Title: AalpiRNA-18529 regulates vitellogenesis of Aedes albopictus via the Gadd45a -mediated JNK-dependent nurse cell apoptosis pathway

doi: 10.1101/2024.08.20.608798

Figure Lengend Snippet: (A) Schematic diagram showing the relative position of mature Aalpi00018529 in the Ae. albopictus genome. Aalpi18529 originates from piRNA Cluster 215 (NW_021837156.1, 62116929-62144956) in scaffold_11 (NW_021837156.1, length 62,838,808 bp) and is located in exon 15 of the antisense transcript of a lncRNA (TCONS_00030668). (B) Temporal profiles of Aalpi-18529 and TCONS_00030668 at different developmental stages of Aedes albopictus determined by qRT‒PCR. E = n hours postoviposition embryo; 1 st -2 nd L= 1 st -2 nd instar larvae; 3 rd -4 th L = 3 rd -4 th instar larvae; P = pupae; F = adult female; M = adult male. The relative expression levels of Aalpi-18529 and TCONS_00030668 in 0-2 h postoviposition embryos were set as 1. (C) Spatial expression patterns of Aalpi-18529 and TCONS_00030668 in different tissues of Ae. albopictus adult females determined by qRT‒PCR. The relative expression levels of Aalpi-18529 and TCONS_00030668 in the head were set as 1. (D) Dynamic expression levels of Aalpi-18529 and TCONS_00030668 in the ovaries of adult females at different time points after a blood meal determined by qRT‒PCR. PE = hours postemergence; PBM = hours post blood meal. The relative expression levels of Aalpi-18529 and TCONS_00030668 in the 72 h PE groups were set as 1. (E) The relative expression quantities of Aalpi-18529 and TCONS_00030668 in the cell nucleus and cytoplasm fractions of adult females were measured by qRT‒PCR. AalRps7 and β-actin mRNA served as markers of cytoplasmic fractions, whereas U6 served as a marker of nuclear fractions. The X-axis indicates the different sample groups, and the Y-axis shows the relative expression levels. All qRT-PCRs were performed in triplicate with three biological replicates, and the values are presented as the means ± SEMs. (F) Schematics of the previtellogenic stage ovary and its ovarian ovariole (48 PE), and the major cellular types of the primary egg chamber are indicated. (G) Localization of Aalpi-18529 in the ovaries of adult females. The nucleic acid probe targeting Aalpi-18529 was conjugated to the dual fluorophore Cy3 (red). Probes targeting a scrambled nucleotide sequence were used as the control for piRNA. Egg chambers were stained with DAPI to visualize nurse cell nuclear morphology. BC, blank control; NC, negative control. The images were acquired using an All-in-One Fluorescence microimaging system BZ-X800/BZ-X810 (KEYENCE).

Article Snippet: Ovaries were mounted in Antifade Mounting Medium with DAPI (Beyotime Biotechnology, Cat#P0131) and imaged 3– 10 days after mounting.

Techniques: Expressing, Marker, Sequencing, Control, Staining, Negative Control, Fluorescence

Journal: bioRxiv

Article Title: AalpiRNA-18529 regulates vitellogenesis of Aedes albopictus via the Gadd45a -mediated JNK-dependent nurse cell apoptosis pathway

doi: 10.1101/2024.08.20.608798

Figure Lengend Snippet: (A) Ovaries from treatment and control groups were stained with TUNEL assay for apoptotic cells (green), and nuclei were stained with DAPI (blue). Representative pictures of ovaries dissected at 24 h PMB are shown. The right insets show a zoomed image of a typical TUNEL-positive (purpe frame, blue arrow) or TUNEL-negative chamber (yellow frame) and TUNEL-positive germarium (red frame, red arrow). Scale bar: 100 µm. (B) The percentage of TUNEL-positive nurse cells. A total of 15 chambers were randomly selected for TUNEL-positive nurse cell counts in ovaries per adult female, with 8 females per group. All data are shown as the mean ± SEM. Student’s t- test was used to compare the means between two groups, and one-way ANOVA was used to compare the means among different groups. * P < 0.05. ** P < 0.01. *** P <0.001; ns, no significance.

Article Snippet: Ovaries were mounted in Antifade Mounting Medium with DAPI (Beyotime Biotechnology, Cat#P0131) and imaged 3– 10 days after mounting.

Techniques: Control, Staining, TUNEL Assay

Journal: FEBS Open Bio

Article Title: Differential heme release from various hemoglobin redox states and the upregulation of cellular heme oxygenase‐1

doi: 10.1002/2211-5463.12103

Figure Lengend Snippet: Hemoglobin‐mediated induction of heme oxygenase‐1 ( HO ‐1) expressions in mouse E10 cells. E10 cells were exposed to either ferrous, ferric, or ferryl Hb at equimolar concentration (100 µ m ) for 12 h for immunocytochemistry experiment (A) or 24 h for western blotting experiment (B). Laser confocal images showing expression of heme oxygenase‐1 (green, Alexa‐488) in E10 cells (A). E10 cells were also counterstained with Phalloidin Alexa‐647 (red) to show the cellular morphology. Nuclear staining was done with DAPI (blue). Cell lysates were immunoblotted and analyzed for HO ‐1 expression (B, upper panel). Equal loading was confirmed by reprobing the blots against β‐actin (B, lower panel). The ratio of average ( n = 3) band intensity of HO ‐1 with corresponding β‐actin was plotted as relative intensity (C), * P < 0.05 vs. control; # P < 0.05 vs. HbFe 3+ , ‘ t ’ test unpaired.

Article Snippet: Cells were visualized under a Zeiss LSM710 meta confocal microscope (Zeiss, Thornwood, NY, USA) after mounting with Fluoroshield mounting medium with DAPI (Abcam).

Techniques: Concentration Assay, Immunocytochemistry, Western Blot, Expressing, Staining, Control

Journal: Advanced Science

Article Title: Epigenetic Regulation of DAPK1 and Netrin‐1 Drives Diabetic Encephalopathy

doi: 10.1002/advs.202502535

Figure Lengend Snippet: DAPK1 levels were increased in the brain of diabetic mice with diabetic encephalopathy. A) Schematic overview of diabetic mouse modeling and cognitive‐related behavior assessments. B,C) Representative swimming paths on day 7, B) latency to reach the platform from days 1–7, C) swimming paths on day 9 and number of crossing the platform region on day 9 in the Morris water maze task for diabetic groups (6 month old STZ‐treated mice and db/db mice) and control groups (6 month old SCB‐treated mice and db/m mice). n = 10–12 per group. D) Western blot analysis and E) quantification of DAPK1 protein expression relative to β‐actin in the hippocampus of diabetic and control groups. n = 6 per group. F) Representative images and G) quantitative analysis of immunohistochemical staining for DAPK1 in hippocampal subregions (CA1, DG, and CA3) of diabetic and control mice. n = 6 slices from 3 mice per group. H) Distribution of DAPK1 (red) in excitatory pyramidal neurons (CaMKII, green) and its absence in inhibitory neurons (GAD1, green). Nuclei were stained with DAPI (blue). Higher‐magnification images of the regions marked with white squares in the CA1 area are shown on the right. I) Colocalization analysis of DAPK1 with CaMKII (up) and GAD1 (down), showing overlay levels and Pearson's correlation coefficient. J) Representative images and K) qualification of microglia (IBA1, red) and astrocytic (GFAP, green) activation in the hippocampus of diabetic and control groups. n = 6 slices from three mice per group. L) Representative images and M) quantification of apoptotic cells in the hippocampus of diabetic and control groups, as indicated by TUNEL‐positive staining (green). Nuclei were stained with DAPI (blue). The regions marked with white squares in the CA1 area are shown at higher magnification on the right. The percentage of apoptotic cells was calculated with the number of TUNEL‐positive cells divided by the total number of DAPI‐labeled cells in the same microscopic field. n = 4 slices from three mice per group. N,O) Electrophysiological recordings of N) field excitatory postsynaptic potential (fEPSP) slope to evaluate the LTP at the CA3‐CA1 synapse. n = 9 cells from three mice per group. Data are presented as mean ± S.E.M., and statistical analysis was performed using an unpaired Student's t ‐test or one‐way or two‐way analysis of variance (ANOVA), unless otherwise specified. ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking with 5% nonfat milk for 30 min, the membranes were incubated with primary antibodies overnight at 4 °C to DAPK1 (rabbit, 1:1000, No.25136‐1‐AP, Proteintech), DAPK1 (mouse, 1:1000, No.67815‐1‐Ig, Proteintech), β‐actin (mouse, 1:800, No.66009‐1‐Ig, Proteintech), HNF1A polyclonal antibody (rabbit, 1:1000, No. 22426‐1‐AP, Proteintech), Phospho‐HNF1A (Ser247) Ab (rabbit, 1:1000, AF7045, Affinity), or Netrin‐1 (rabbit, 1:500, DF8579, Affinity), followed by washes with Tris‐buffered saline (PBS)‐Tween 20.

Techniques: Control, Western Blot, Expressing, Immunohistochemical staining, Staining, Activation Assay, TUNEL Assay, Labeling

Journal: Advanced Science

Article Title: Epigenetic Regulation of DAPK1 and Netrin‐1 Drives Diabetic Encephalopathy

doi: 10.1002/advs.202502535

Figure Lengend Snippet: Knockdown hippocampus DAPK1 in excitatory neurons rescued diabetic encephalopathy in diabetes. A) Schematic of the strategy for conditional DAPK1 kinase domain knockout in hippocampal excitatory neurons using CaMKII‐CreERT and tamoxifen (TAM) induction in diabetic mice. Western blot analysis of DAPK1 and β‐actin protein expression in the hippocampus of STZ/KD f/f and STZ/KD −/− groups. n = 2 per group. B) Representative path traces to the hidden platform on day 7 and latency over 7 days in different groups. n = 10 per group. C) Swimming paths on day 9 and number of crossing the platform region on day 9 in the MWM. n = 10 per group. D) Immunofluorescence staining of IBA1 (red) and GFAP (green) in the hippocampus, showing changes in microglial and astrocytic activation across groups. n = 6 slices from three mice per group. E) TUNEL staining of hippocampal sections for apoptosis detection, with DAPI counterstaining; apoptotic cells appear green in images. F) Quantification of TUNEL‐positive cells in the hippocampus, showing reduced apoptosis in DAPK1 −/− mice. n = 6 slices from three mice per group. G) Electrophysiological recordings illustrating normalized field excitatory postsynaptic potential (fEPSP) slopes in CA3–CA1 synapses, with reduced long‐term potentiation (LTP) impairment in DAPK1 −/− mice. H) Summary of fEPSP slope changes among groups, highlighting improved synaptic function in DAPK1 −/− mice. n = 9 cells from three mice per group. I) Representative traces (up) and quantification (down) of paired‐pulse facilitation (PPF) recorded from hippocampal slices. n = 10 cells from three mice per group. J) Representative traces (up) and quantification (down) of AMPA/NMDA current ratio measured in CA1 neurons. n = 10 cells from three mice per group. K) Representative transmission electron microscopy (TEM) images showing hippocampal synapses in the stratum radiatum. L) Quantification of postsynaptic density (PSD) length from TEM images. n = 130–150 synapses from 3 to 5 mice per group. Data are shown as mean ± S.E.M., and statistical analysis was performed using one‐way or two‐way analysis of variance (ANOVA). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking with 5% nonfat milk for 30 min, the membranes were incubated with primary antibodies overnight at 4 °C to DAPK1 (rabbit, 1:1000, No.25136‐1‐AP, Proteintech), DAPK1 (mouse, 1:1000, No.67815‐1‐Ig, Proteintech), β‐actin (mouse, 1:800, No.66009‐1‐Ig, Proteintech), HNF1A polyclonal antibody (rabbit, 1:1000, No. 22426‐1‐AP, Proteintech), Phospho‐HNF1A (Ser247) Ab (rabbit, 1:1000, AF7045, Affinity), or Netrin‐1 (rabbit, 1:500, DF8579, Affinity), followed by washes with Tris‐buffered saline (PBS)‐Tween 20.

Techniques: Knockdown, Knock-Out, Western Blot, Expressing, Immunofluorescence, Staining, Activation Assay, TUNEL Assay, Transmission Assay, Electron Microscopy

Journal: Advanced Science

Article Title: Epigenetic Regulation of DAPK1 and Netrin‐1 Drives Diabetic Encephalopathy

doi: 10.1002/advs.202502535

Figure Lengend Snippet: The elevation of DAPK1 is driven by suppression of miR‐216a‐5p. A) Relative mRNA levels of DAPK1 in hippocampus of control (SCB and db/m) and diabetic (STZ and db/db) mice, showing no significant differences across groups. n = 5. B) Heatmap of miRNA expression profiles in different groups. C) Diagram to display the conserved binding site in DAPK1 3′UTR to the miR‐216a‐5p. The mutant sequence in 3′UTR of DAPK1 for luciferase analysis was provided at the bottom. D) Luciferase reporter assay of wild‐type (WT) and mutant (MUT) DAPK1 3′UTR constructs in cells transfected with miR‐216a‐5p mimic or scramble control. n = 6 per group. E,F) N2a cells were transfected with miR‐216a‐5p agomir or antagomir. E) The cell lysates were collected, and the protein levels of DAPK1 were then detected by western blot (WB) after 48 h, F) with quantification. n = 4 per group. G,H) Immunofluorescent staining showing DAPK1 (green) and miR‐216a‐5p (red) in the hippocampus of db/m and db/db mice, with DAPI counterstaining (blue). Correlation analysis between miR‐216a‐5p and DAPK1 levels, showing a significant negative relationship ( R 2 = 0.7465, p < 0.001). n = 15 slices per group from five mice. Data are presented as mean ± S.E.M., and statistical analysis was performed using an unpaired Student's t ‐test or linear regression. * p < 0.05, ** p < 0.01, and **** p <0.0001.

Article Snippet: After blocking with 5% nonfat milk for 30 min, the membranes were incubated with primary antibodies overnight at 4 °C to DAPK1 (rabbit, 1:1000, No.25136‐1‐AP, Proteintech), DAPK1 (mouse, 1:1000, No.67815‐1‐Ig, Proteintech), β‐actin (mouse, 1:800, No.66009‐1‐Ig, Proteintech), HNF1A polyclonal antibody (rabbit, 1:1000, No. 22426‐1‐AP, Proteintech), Phospho‐HNF1A (Ser247) Ab (rabbit, 1:1000, AF7045, Affinity), or Netrin‐1 (rabbit, 1:500, DF8579, Affinity), followed by washes with Tris‐buffered saline (PBS)‐Tween 20.

Techniques: Control, Expressing, Binding Assay, Mutagenesis, Sequencing, Luciferase, Reporter Assay, Construct, Transfection, Western Blot, Staining

Journal: Advanced Science

Article Title: Epigenetic Regulation of DAPK1 and Netrin‐1 Drives Diabetic Encephalopathy

doi: 10.1002/advs.202502535

Figure Lengend Snippet: Knockdown of DAPK1 in excitatory neurons restores Netrin‐1 (Ntn1) expression. A) Heatmap showing the relative mRNA levels of various neurotrophic factors, including Ntn1, in db/db mice compared to controls. n = 3 per group. B) Western blot analysis of Ntn1 protein levels in the hippocampus of db/m and db/db mice, with quantification showing a significant reduction of Ntn1 in db/db mice. n = 5 per group. C) Western blot analysis of Ntn1 in DAPK1‐KD +/− and DAPK1‐KD −/− diabetic mice treated with tamoxifen (TAM), with quantification indicating restoration of Ntn1 levels following DAPK1 knockdown. n = 5 per group. D) Immunofluorescent staining for DAPK1 (green) and Ntn1 (red) in the hippocampus of different groups, with merged images showing colocalization. E) Quantitative analysis of Ntn1 levels in different group mice, demonstrating increased Ntn1 expression following DAPK1 kinase domain knockdown. n = 6 slices per group from three mice. F) Correlation analysis between the relative protein levels of DAPK1 and Ntn1 ( R 2 = 0.4808, p < 0.001). n = 6 per group. G) Schematic of the dual‐luciferase reporter assay construct used to assess Ntn1 promoter activity in response to DAPK1 kinase domain knockdown. H) Relative luciferase activity in cells after cotransfection with the reporter vector and the control or effector vector for 48 h. I) Western blot analysis of HNF1A and Ntn1 levels in control and DAPK1‐overexpressing (oe‐DAPK1) cells, with J) quantification showing a decrease in HNF1A and Ntn1 levels upon DAPK1 overexpression. n = 3 per group. K) Chromatin immunoprecipitation (ChIP) assay was performed to assess the binding of HNF1A to the Ntn1 promoter in HEK293T cells transfected with either DAPK1 vector, wild‐type HNF1A, or phosphorylation‐deficient mutant HNF1A‐S249A. Immunoprecipitated DNA was quantified using qPCR targeting the Ntn1 promoter region. n = 3 per group. L) Luciferase reporter assay showing the transcriptional activity of wild‐type HNF1A and its phosphorylation mutants (S249A and S249D) on the Ntn1 promoter. HEK293T cells were co‐transfected with the Ntn1 promoter‐luciferase construct and the indicated HNF1A variants. Luciferase activity was measured 24 post‐transfection and normalized to Renilla luciferase. n = 5 per group. Data are presented as mean ± S.E.M., and statistical analysis was performed using an unpaired Student's t ‐test or one‐way or two‐way analysis of variance (ANOVA), unless otherwise specified. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After blocking with 5% nonfat milk for 30 min, the membranes were incubated with primary antibodies overnight at 4 °C to DAPK1 (rabbit, 1:1000, No.25136‐1‐AP, Proteintech), DAPK1 (mouse, 1:1000, No.67815‐1‐Ig, Proteintech), β‐actin (mouse, 1:800, No.66009‐1‐Ig, Proteintech), HNF1A polyclonal antibody (rabbit, 1:1000, No. 22426‐1‐AP, Proteintech), Phospho‐HNF1A (Ser247) Ab (rabbit, 1:1000, AF7045, Affinity), or Netrin‐1 (rabbit, 1:500, DF8579, Affinity), followed by washes with Tris‐buffered saline (PBS)‐Tween 20.

Techniques: Knockdown, Expressing, Western Blot, Staining, Luciferase, Reporter Assay, Construct, Activity Assay, Cotransfection, Plasmid Preparation, Control, Over Expression, Chromatin Immunoprecipitation, Binding Assay, Transfection, Phospho-proteomics, Mutagenesis, Immunoprecipitation

Journal: Microbiology Spectrum

Article Title: Leishmania donovani elongator protein Elp3a plays a crucial role in modulating the parasite response to genotoxic stress

doi: 10.1128/spectrum.02439-25

Figure Lengend Snippet: elp3a is not essential for survival of Leishmania promastigotes. ( A ) Conserved domains identified in LdElp3a. Boxes demarcate the relevant domains. Numbers indicate the positions of the starting and ending amino acids of the domains. ( B ) Analysis of subcellular localization of LdElp3a-FLAG. Upper panel: Western blot analysis of whole cell lysates isolated from transfectant promastigotes using anti-FLAG antibodies (1:2,500 dil). Tubulin served as loading control. Arrowhead indicates ~85 kDa Elp3a-FLAG protein. Full-length uncropped blots in . Lower panel: microscopic analysis of transfectant promastigotes using immunofluorescence with anti-FLAG antibodies. DAPI stained both the nuclear (N) and kinetoplast (K) compartments. Kinetoplast morphology and segregation pattern was used as cell cycle stage marker. G1/early S: roundish/short rod-like kinetoplast, single nucleus (1N, 1K). Late S/early G2: one elongated kinetoplast, single nucleus (1N,1K). G2/M: one kinetoplast, two nuclei (2N,1K). Post-mitosis: two kinetoplasts, two nuclei (2N, 2K). Magnification bar: 5 µm. ( C ) Analysis of growth of elp3a −/− promastigotes. Cultures were initiated from stationary phase cultures. The experiment was done thrice with technical replicates in each experiment. Values plotted are average of three experiments, and error bars indicate standard deviation. Raw data excel sheets in . ( D ) Analysis of elp3a −/− cell cycle progression. Cells were synchronized at G1/S boundary with 5 mM HU and then released into fresh drug-free medium. Sampling time-points are indicated above the histogram frames. “R” refers to hours after release. Cells in G1, S, and G2M are gated as M1, M2, and M3, respectively. The experiment was done thrice, with comparable results, and one dataset is shown here. The gating strategy is shown in .

Article Snippet: Briefly, Leishmania promastigotes were fixed in 2% paraformaldehyde, cells spread on poly-lysine coated coverslips, permeabilized with 0.1% Triton X-100, blocked with chicken serum (10%), incubated with primary antibody (1:100 FLAG antibody: Cat. no. F1804, Sigma Aldrich, USA), washed and incubated with Texas Red-labeled secondary antibody (1:100, Jackson ImmunoResearch Laboratories, USA), and washed and mounted in Vectashield antifade mounting medium carrying DAPI (VectorLabs, USA).

Techniques: Western Blot, Isolation, Transfection, Control, Immunofluorescence, Staining, Marker, Standard Deviation, Sampling

Journal: Microbiology Spectrum

Article Title: Leishmania donovani elongator protein Elp3a plays a crucial role in modulating the parasite response to genotoxic stress

doi: 10.1128/spectrum.02439-25

Figure Lengend Snippet: Deletion of elp3a has no impact on survival of the parasite within the mammalian host cell. ( A ) Analysis of murine macrophages (J774A.1) infected with Leishmania metacyclics. ( B ) Analysis of human macrophages (THP1 cells) infected with Leishmania metacyclics. Intracellular parasites were scored by mounting the cells in DAPI-containing medium and capturing Z-stack images using a confocal microscope, followed by image analysis using LAS X software. Both experiments were performed thrice, and average values are plotted in the bar graphs. Error bars denote standard deviation. Statistical significance was determined using the student’s t -test. *: P < 0.05, **: P < 0.005, ns: not significant. Raw data Excel sheets in .

Article Snippet: Briefly, Leishmania promastigotes were fixed in 2% paraformaldehyde, cells spread on poly-lysine coated coverslips, permeabilized with 0.1% Triton X-100, blocked with chicken serum (10%), incubated with primary antibody (1:100 FLAG antibody: Cat. no. F1804, Sigma Aldrich, USA), washed and incubated with Texas Red-labeled secondary antibody (1:100, Jackson ImmunoResearch Laboratories, USA), and washed and mounted in Vectashield antifade mounting medium carrying DAPI (VectorLabs, USA).

Techniques: Infection, Microscopy, Software, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Leishmania donovani elongator protein Elp3a plays a crucial role in modulating the parasite response to genotoxic stress

doi: 10.1128/spectrum.02439-25

Figure Lengend Snippet: Analysis of DNA damage in Elp3a-depleted cells subjected to prolonged HU exposure. Microscopic analysis of TUNEL assay reactions carried out on cells that were exposed to 1 mM HU for 24 hours. First row: fluorescein-labeled nuclear and kinetoplast DNA. Second row: DAPI-stained nuclear and kinetoplast DNA. Third row: merged image of fluorescein and DAPI. Log column: untreated cells. 24 h HU column: cells incubated in 1 mM HU for 24 hours. 6.5 hR column: cells incubated in 1 mM HU for 24 hours and then in drug-free medium for a further 6.5 hours. Images were captured by Z stack analysis. Magnification bar: 5 µm. The experiment was performed thrice with comparable results.

Article Snippet: Briefly, Leishmania promastigotes were fixed in 2% paraformaldehyde, cells spread on poly-lysine coated coverslips, permeabilized with 0.1% Triton X-100, blocked with chicken serum (10%), incubated with primary antibody (1:100 FLAG antibody: Cat. no. F1804, Sigma Aldrich, USA), washed and incubated with Texas Red-labeled secondary antibody (1:100, Jackson ImmunoResearch Laboratories, USA), and washed and mounted in Vectashield antifade mounting medium carrying DAPI (VectorLabs, USA).

Techniques: TUNEL Assay, Labeling, Staining, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of PHLPP1 ameliorates cardiac dysfunction via activation of the PI3K/Akt/mTOR signalling pathway in diabetic cardiomyopathy

doi: 10.1111/jcmm.15123

Figure Lengend Snippet: Effects of PHLPP1 on myocardial apoptosis and fibrosis. A, Immunostaining of PHLPP1 (first row, n = 6) and cell apoptosis as determined by TUNEL assay (second‐fourth row, n = 5): apoptosis cell stained red; nuclei stained blue with DAPI. B, Cell apoptosis rate determined by TUNEL assay. C, The levels of cleaved caspase‐3 following PHLPP1 inhibition were measured by Western blot (n = 6). D, The levels of Bax and Bcl‐2 following PHLPP1 inhibition were measured by Western blot (n = 6). E, Representative Masson's trichrome staining (first row) and Sirius red staining (second and third rows) of the myocardium (n = 6). Immunostaining of collagen I (fourth row) and collagen III (fifth row) (n = 6). F, Quantification of Masson's trichrome staining (n = 6). G, Quantification of Sirius red staining (n = 6). H‐K, Western blot analysis of the protein expression of collagen I (F), collagen III (G), MMP2 (H) and MMP9 (I) (n = 6). Control: normal rats. DM: diabetes mellitus. shN.C: negative control shRNA. shPHLPP1: PHLPP1 shRNA. All experiments were performed at least 3 times. Data are expressed as the means ± SD. Statistical analysis was performed using one‐way ANOVA followed by Bonferroni's post hoc test. * P < .05 compared with control, and # P < .05 compared with DM or shN.C in DM

Article Snippet: Finally, cells were sealed in Prolong Gold Anti‐Fade Reagent with DAPI (Invitrogen) after being washed with PBS.

Techniques: Immunostaining, TUNEL Assay, Staining, Inhibition, Western Blot, Expressing, Control, Negative Control, shRNA

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of PHLPP1 ameliorates cardiac dysfunction via activation of the PI3K/Akt/mTOR signalling pathway in diabetic cardiomyopathy

doi: 10.1111/jcmm.15123

Figure Lengend Snippet: The effects of different glucose culture times on PHLPP1 expression. A, Western blot analysis of the expression of PHLPP1 in primary neonatal cardiomyocytes. B, Western blot analysis of the expression of PHLPP1 in H9c2 cells. C, Immunofluorescence of PHLPP1 in primary neonatal cardiomyocytes and H9c2 cells. PHLPP1 stained green in primary neonatal cardiomyocytes and red in H9c2 cells; nuclei stained blue with DAPI. All experiments were performed at least 3 times.NG: 5.5 mmol/L glucose, HG: 33.3 mmol/L glucose. Data are expressed as the means ± SD. Statistical analysis was performed using one‐way ANOVA followed by Dunnett's multiple‐to‐one comparison test. * P < .05 compared with NG

Article Snippet: Finally, cells were sealed in Prolong Gold Anti‐Fade Reagent with DAPI (Invitrogen) after being washed with PBS.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Comparison

Journal: Journal of Nanobiotechnology

Article Title: Exosomes derived from platelet-rich plasma administration in site mediate cartilage protection in subtalar osteoarthritis

doi: 10.1186/s12951-022-01245-8

Figure Lengend Snippet: Exo-Gel enhanced chondrogenic differentiation mBMSCs in vitro. A In vitro estimate the ability of Exo-Gel by coculture in chondrogenic differentiation of mBMSCs. B RT-PCR analysis for COL II, ACAN and SOX9 mRNA in mBMSCs treated as described in (A) at different time-points. Data are represented as means ± SEM (n = 3). C AB staining of mBMSCs in different treatment groups under light microscopy on the 14th day of induction of differentiation (bar = 100 μm). D Immunofluorescent assay of mBMSCs for COL II (red) and DAPI (Blue) in different treatment groups at the same time-point in C (bar = 100 μm). E Western blot assay to detect the protein expression levels of COL II, ACAN and SOX9 at the same time-point in C . F Quantification of protein expression in E . GAPDH was used as loading control. Data are expressed as means ± SEM (n = 3). G Representative Western blot showing the levels of total protein and phosphorylated protein for the indicated molecules (Smad2/3, ERK1/2, and p38) in mBMSCs with or without TGFβ1 neutralizing antibody at the same time-point in C . H Quantification of protein expression and the level of signaling activation in G . GAPDH was used as loading control. Data are expressed as means ± SEM (n = 3). I AB staining of mBMSCs with or without TGFβ1 neutralizing antibody under light microscopy on the 14th day of differentiation (bar = 100 μm). J Immunofluorescent assay of mBMSCs for COL II (red) and DAPI (blue) under the same condition as ( I ) (bar = 100 μm). *p < 0.05

Article Snippet: The primary antibodies used in this study included mouse anti-CD9, CD63, CD81, and SOX9 (Santa Cruz Biotechnology, USA); rabbit anti-COL II (Santa Cruze Biotechnology); rabbit anti-PDGFBB, VEGF, SDF-1, COL X, anti-cleaved caspase-3, Bcl-2, Bax, and GAPDH (Abcam, USA); rabbit anti-TGFβ1, Smad2/3, p-Smad2/3, EKR1/2, p-EKR1/2, p38, and p-p38 (Cell Signaling Technology, USA); rabbit anti- COL X and Ki67 (Novus, USA); rabbit anti-Aggrecan, and MMP13 (Proteintech; China).The secondary antibodies used in this study included Alexa-488 conjugated-goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, USA); horseradishperoxidase–conjugated-goat anti-rabbit IgG (H + L) and horseradish peroxidase–conjugated-goat anti-Mouse IgG (H + L) (Invitrogen, USA); Nuclei was stained with DAPI dihydrochloride (Thermo Fisher Scientific, USA).

Techniques: In Vitro, Reverse Transcription Polymerase Chain Reaction, Staining, Light Microscopy, Western Blot, Expressing, Control, Activation Assay

Journal: Journal of Nanobiotechnology

Article Title: Exosomes derived from platelet-rich plasma administration in site mediate cartilage protection in subtalar osteoarthritis

doi: 10.1186/s12951-022-01245-8

Figure Lengend Snippet: Exo-Gel suppressed IL-1β-triggered chondrocyte apoptosis in vitro. A In vitro investigate the effect of Exo-Gel on apoptosis of chondrocytes treated with IL-1β (10 ng/ml) for 24 h. TUNEL (green) staining to analyze chondrocyte apoptosis, DAPI (blue) staining for cell nuclei (bar = 200 μm). B Quantitative assessment of the proportion of apoptotic cells in A . Data are expressed as means ± SEM (n = 6). C Annexin V/FITC/PI double-staining flow cytometry to detect the effect of Exo-Gel coincubated medium on IL-1β-triggered chondrocyte apoptosis. D Quantification of apoptotic cells (Annexin V-positive) in C . Data are expressed as means ± SEM (n = 4). E Representative Western blots of cleaved-Cas3, Bcl-2, and Bax expression in chondrocytes in different groups. F Quantification of cleaved-Cas3 protein expression and the ratio of Bcl-2 to Bax in chondrocyte in E . GAPDH was used as loading control. Data are expressed as means ± SEM (n = 3). *p < 0.05

Article Snippet: The primary antibodies used in this study included mouse anti-CD9, CD63, CD81, and SOX9 (Santa Cruz Biotechnology, USA); rabbit anti-COL II (Santa Cruze Biotechnology); rabbit anti-PDGFBB, VEGF, SDF-1, COL X, anti-cleaved caspase-3, Bcl-2, Bax, and GAPDH (Abcam, USA); rabbit anti-TGFβ1, Smad2/3, p-Smad2/3, EKR1/2, p-EKR1/2, p38, and p-p38 (Cell Signaling Technology, USA); rabbit anti- COL X and Ki67 (Novus, USA); rabbit anti-Aggrecan, and MMP13 (Proteintech; China).The secondary antibodies used in this study included Alexa-488 conjugated-goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, USA); horseradishperoxidase–conjugated-goat anti-rabbit IgG (H + L) and horseradish peroxidase–conjugated-goat anti-Mouse IgG (H + L) (Invitrogen, USA); Nuclei was stained with DAPI dihydrochloride (Thermo Fisher Scientific, USA).

Techniques: In Vitro, TUNEL Assay, Staining, Double Staining, Flow Cytometry, Western Blot, Expressing, Control

Journal: Journal of Nanobiotechnology

Article Title: Exosomes derived from platelet-rich plasma administration in site mediate cartilage protection in subtalar osteoarthritis

doi: 10.1186/s12951-022-01245-8

Figure Lengend Snippet: Exo-Gel suppressed IL-1β-triggered chondrocyte degeneration and enhanced anabolism in vitro. A Immunofluorescent assay to detect the expression of COL II (Gree), COL X (Green), and MMP13 (Green) in chondrocytes in different treatment groups, DAPI (blue) staining for cell nuclei (bar = 200 μm). B Quantification of COL II, COL X, and MMP13 fluorescence intensity in A . Data are expressed as means ± SEM (n = 6). C Representative Western blots of COL II, COL X, and MMP13 expression in chondrocytes in different groups. D Quantification of COL II, COL X, and MMP13 protein expression in chondrocytes in C . GAPDH was used as loading control. Data are expressed as means ± SEM (n = 3). E Representative Western blot showing the levels of total protein and phosphorylated protein for the indicated molecules (p65 and STAT3) in chondrocytes in different treatment groups. F Quantification of protein expression and the level of signaling activation in E . GAPDH was used as loading control. Data are expressed as means ± SEM (n = 3) *p < 0.05

Article Snippet: The primary antibodies used in this study included mouse anti-CD9, CD63, CD81, and SOX9 (Santa Cruz Biotechnology, USA); rabbit anti-COL II (Santa Cruze Biotechnology); rabbit anti-PDGFBB, VEGF, SDF-1, COL X, anti-cleaved caspase-3, Bcl-2, Bax, and GAPDH (Abcam, USA); rabbit anti-TGFβ1, Smad2/3, p-Smad2/3, EKR1/2, p-EKR1/2, p38, and p-p38 (Cell Signaling Technology, USA); rabbit anti- COL X and Ki67 (Novus, USA); rabbit anti-Aggrecan, and MMP13 (Proteintech; China).The secondary antibodies used in this study included Alexa-488 conjugated-goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, USA); horseradishperoxidase–conjugated-goat anti-rabbit IgG (H + L) and horseradish peroxidase–conjugated-goat anti-Mouse IgG (H + L) (Invitrogen, USA); Nuclei was stained with DAPI dihydrochloride (Thermo Fisher Scientific, USA).

Techniques: In Vitro, Expressing, Staining, Fluorescence, Western Blot, Control, Activation Assay

Journal: Journal of Nanobiotechnology

Article Title: Exosomes derived from platelet-rich plasma administration in site mediate cartilage protection in subtalar osteoarthritis

doi: 10.1186/s12951-022-01245-8

Figure Lengend Snippet: Exo-Gel inhibited cartilage degeneration of subtalar joint. A TUNEL (Green) staining in the subtalar joint section to analyze chondrocyte apoptosis at 4 weeks postoperatively, DAPI (blue) staining for cell nuclei (bar = 100 μm). B , C immumohistochemical staining of COL X ( B ) and Ki67 ( C ) in the subtalar joint section (bar = 100 μm). (D) Quantitative assessment of number of apoptotic cells in A . Data are expressed as means ± SEM (n = 4). (E, F) quantitative analysis of COL X and Ki67 positive cells in cartilage tissue in B and C , respectively. Data are expressed as means ± SEM (n = 4). *p < 0.05

Article Snippet: The primary antibodies used in this study included mouse anti-CD9, CD63, CD81, and SOX9 (Santa Cruz Biotechnology, USA); rabbit anti-COL II (Santa Cruze Biotechnology); rabbit anti-PDGFBB, VEGF, SDF-1, COL X, anti-cleaved caspase-3, Bcl-2, Bax, and GAPDH (Abcam, USA); rabbit anti-TGFβ1, Smad2/3, p-Smad2/3, EKR1/2, p-EKR1/2, p38, and p-p38 (Cell Signaling Technology, USA); rabbit anti- COL X and Ki67 (Novus, USA); rabbit anti-Aggrecan, and MMP13 (Proteintech; China).The secondary antibodies used in this study included Alexa-488 conjugated-goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, USA); horseradishperoxidase–conjugated-goat anti-rabbit IgG (H + L) and horseradish peroxidase–conjugated-goat anti-Mouse IgG (H + L) (Invitrogen, USA); Nuclei was stained with DAPI dihydrochloride (Thermo Fisher Scientific, USA).

Techniques: TUNEL Assay, Staining

Journal: Cancer Communications

Article Title: Unfolded protein response kinase PERK supports survival and metastasis of circulating tumor cell clusters via SAM synthesis and H3K4me3‐dependent PDGFB signaling

doi: 10.1002/cac2.70072

Figure Lengend Snippet: Activated UPR in CTC clusters protects cells from cell death. (A) Schematic diagram of the experimental design for isolating CTCs from mice for RNA sequencing at week 4 following fat pad inoculation with MDA‐MB‐231/LM2 cells. (B) GO pathway enrichment analysis of upregulated gene sets in CTC clusters compared to single CTCs ( n = 3). (C) GSVA enrichment scores of single CTCs and CTC clusters from breast cancer patients and PDX‐Br16 mouse model. The single‐cell RNA sequencing data were obtained from the GEO database ( GSE111065 ). (D) Relative mRNA expression levels of UPR‐related genes in single CTCs and CTC clusters from the NCG‐MDA‐MB‐231/LM2 mouse model ( n = 3). (E) Representative flow cytometry plots showing the gating strategy for identifying ER tracker intensity in single CTCs and CTC clusters from the blood of NCG‐MDA‐MB‐231/LM2 mice. (F) MFI of TPE‐MI in single CTCs and CTC clusters, as determined by flow cytometry ( n = 5). G. Percentage of Annexin V + single and clustered tumor cells pretreated with thapsigargin ( n = 3). (H) Cell viability of single and clustered tumor cells under thapsigargin treatment ( n = 3). (I) Schematic diagram of the experimental design. Clustered tumor cells were pretreated with vehicle or azoramide for 6 h prior to tail vein injection into NCG mice, followed by assessment of disseminated tumor cells in the lung at 24 h. (J‐K) Bioluminescence images (left) and quantification of fluorescence signal intensity (right) of mice (J) and the lungs (K) to assess lung metastasis at 24 h after tail vein injection of clustered MDA‐MB‐231/LM2‐CTC cells ( n = 5). Results represent mean ± SD. Student's t‐test in C, D, F, J, and K, two‐way ANOVA test in G and H. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Abbreviations: CTCs, circulating tumor cells; RNA‐seq, RNA sequencing; GO, gene ontology; GSVA, gene set variation analysis; PDX‐Br16, patient‐derived xenograft‐Br16; GEO, ​gene expression omnibus​; NCG, NOD/ShiLtJGpt‐Prkdc em26Cd52 Il2rg em26Cd22 /Gpt; TPE‐MI, tetraphenylethene‐maleimide; MDA‐MB‐231/LM2​, ​MDA‐MB‐231 lung metastasis 2​; UPR, unfolded protein response; HSPA5, heat shock protein family A member 5; XBP1, x‐box binding protein 1; DDIT3, DNA damage inducible transcript 3; DNAJB1, dnaJ heat shock protein family member B1; ATF3, activating transcription factor 3; ATF4, activating transcription factor 4; PPP1R15A, protein phosphatase 1 regulatory subunit 15A; ER Tracker, endoplasmic reticulum tracker; MFI, mean fluorescence intensity; Azo, azoramide; IV, intravenous; ANOVA, analysis of variance.

Article Snippet: For annexin V detection analysis, Annexin V‐Elab Fluor 647/propidium iodide (PI) Apoptosis Kit (E‐CK‐A213, Elabscience, Wuhan, Hubei, China) or Annexin V‐allophycocyanin (APC)/4',6‐diamidino‐2‐phenylindole (DAPI) Apoptosis Kit (E‐CK‐A258, Elabscience) was employed to detect Annexin V + cells.

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Injection, Fluorescence, Derivative Assay, Binding Assay

Journal: Cancer Communications

Article Title: Unfolded protein response kinase PERK supports survival and metastasis of circulating tumor cell clusters via SAM synthesis and H3K4me3‐dependent PDGFB signaling

doi: 10.1002/cac2.70072

Figure Lengend Snippet: PERK is a key factor in promoting CTC cluster survival. (A) Relative mRNA expression levels of UPR‐related genes in single CTCs and CTC clusters isolated from NCG‐MDA‐MB‐231/LM2 (left) and C57BL/6‐B16F10 (right) mouse models ( n = 3). (B) Percentage of Annexin V + single and clustered MDA‐MB‐231/LM2‐CTC (left) and B16F10 (right) cells transfected with siRNAs against PERK and IRE1α and treated with or without thapsigargin ( n = 3). (C) Percentage of Annexin V + clustered MDA‐MB‐231/LM2‐CTC (left) and B16F10 (right) cells treated with AMG44 prior to thapsigargin ( n = 3). (D) Relative PERK expression in single CTCs and CTC clusters isolated from BC patients and PDX mouse models (PDX‐1, PDX‐2, and PDX‐3) ( n = 15 for patients; n = 10 for PDX‐1; n = 8 for PDX‐2 and PDX‐3). (E) Schematic design of the experimental design. MDA‐MB‐231/LM2 cells were orthotopically implanted into mice, and lung metastases were analyzed via IHC at week 4. Representative IHC images of pPERK in lung metastases. The arrow indicates single CTCs; the dashed circle indicates CTC cluster. (F‐I) Representative immunofluorescence images of single CTCs and CTC clusters from a patient and the NCG‐MDA‐MB‐231/LM2 mouse model. Cells were stained for pPERK (red), EGFR (green) (F) or TUNEL (purple) (H), GFP (green) (G) or TUNEL (purple) (I), and DAPI (blue). (J) Schematic design of the experiment (left). MDA‐MB‐231/LM2‐CTC cells were pretreated with Vehicle, MK‐28 or AMG44 and then cultured in suspension for 24 h. Percentage of Annexin V + tumor cell clusters was analyzed by flow cytometry (right) ( n = 5). Results represent mean ± SD. Student's t‐test in A and D, one‐way ANOVA test in B, C, and J. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Abbreviations: UPR, unfolded protein response; CTCs, circulating tumor cells; NCG, NOD/ShiLtJGpt‐Prkdc em26Cd52 Il2rg em26Cd22 /Gpt; MDA‐MB‐231/LM2​, ​MDA‐MB‐231 lung metastasis 2​; ATF6, activating transcription factor 6; PERK, protein kinase R (PKR)‐like endoplasmic reticulum kinase; IRE1α, inositol‐requiring enzyme 1 alpha; BC, breast cancer; PDX, patient‐derived xenograft; IHC, immunohistochemistry; pPERK, phosphorylated PERK; DAPI, 4′,6‐diamidino‐2‐phenylindole; EGFR, epidermal growth factor receptor; GFP, green fluorescent protein; TUNEL, terminal deoxynucleotidyl transferase dUTP nick‐end labeling; siRNA, small interfering RNA;ANOVA, analysis of variance.

Article Snippet: For annexin V detection analysis, Annexin V‐Elab Fluor 647/propidium iodide (PI) Apoptosis Kit (E‐CK‐A213, Elabscience, Wuhan, Hubei, China) or Annexin V‐allophycocyanin (APC)/4',6‐diamidino‐2‐phenylindole (DAPI) Apoptosis Kit (E‐CK‐A258, Elabscience) was employed to detect Annexin V + cells.

Techniques: Expressing, Isolation, Transfection, Immunofluorescence, Staining, TUNEL Assay, Cell Culture, Suspension, Flow Cytometry, Derivative Assay, Immunohistochemistry, Small Interfering RNA

Journal: Cancer Communications

Article Title: Unfolded protein response kinase PERK supports survival and metastasis of circulating tumor cell clusters via SAM synthesis and H3K4me3‐dependent PDGFB signaling

doi: 10.1002/cac2.70072

Figure Lengend Snippet: PERK signaling regulates MAT2A via ATF4 to support enhanced methionine metabolism. (A) KEGG enrichment analysis of upregulated genes in scramble control tumor cell clusters compared to PERK‐KO clusters ( n = 3). (B) Volcano plot showing PERK‐related metabolites identified through targeted metabolomics ( n = 6). (C) Heatmap showing metabolites involved in the methionine cycle in scramble and PERK‐KO tumor cell clusters ( n = 6). (D) Intracellular metabolites in NC‐oe and PERK‐oe tumor cell clusters ( n = 6). (E) The schematic diagram for the conversion of [ 13 C 5 ]‐methionine into various metabolites (left) and LC‐MS quantification of M+5 methionine, M+5 SAM, and M+4 SAH following a 16 h incubation with [ 13 C 5 ]‐methionine in scramble and PERK‐KO tumor cell clusters (right) ( n = 4). (F) Percentage of Annexin V + MDA‐MB‐231/LM2‐CTC cells cultured in CM or medium lacking Ser, Gly, Met, or Cys for 24 h ( n = 5). (G‐I) Western blot analysis of metabolic enzyme expression in MDA‐MB‐231/LM2‐CTC and B16F10 tumor cell clusters with or without PERK (G), ATF4 expression in MDA‐MB‐231/LM2‐CTC with or without PERK (H), and ATF4 and MAT2A expression in MDA‐MB‐231/LM2‐CTC with or without ATF4 (I). (J) Relative MAT2A expression in MDA‐MB‐231/LM2‐CTC transfected with shNC or shATF4 #2 ( n = 3). (K) Percentage of Annexin V + MDA‐MB‐231/LM2‐CTC transduced with shNC or shMAT2A #3 and treated with or without SAM for 24 h (left) or pretreated with PF9366 prior to SAM (right) ( n = 3). (L) Schematic of the in vivo experiment design. (M) Bioluminescence imaging (left) and fluorescence intensity quantification (right) of lung metastases at 24 h after tail vein injection ( n = 5). (N) Representative H&E staining images (left) and quantification of pulmonary nodules (right) in NCG mice at 2 weeks after tail vein injection of MDA‐MB‐231/LM2‐CTC clusters (shNC and shMAT2A #3) ( n = 5). (O) Schematic representation of the experimental design. MDA‐MB‐231/LM2‐CTC clusters were cultured in CM or MRM for 24 h before tail vein injection into NCG mice, with lung metastases assessed at 24 h. (P) Bioluminescence imaging (left) and fluorescence intensity quantification (right) of lung metastases at 24 h after tail vein injection ( n = 5). Results represent mean ± SD. Student's t‐test in D, E, J, M, and P; one‐way ANOVA test in F and K. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Abbreviations: KEGG, Kyoto Encyclopedia of Genes and Genomes; FC, fold change; Sc, scramble; PERK, Protein kinase RNA‐like endoplasmic reticulum kinase; PERK‐KO, PRKR‐knockout; sgPERK, single‐guide RNA targeting PERK; LC‐MS, liquid chromatography‐mass spectrometry; Met, methionine; SAM; S‐adenosylmethionine; SAH, S‐adenosylhomocysteine; CM, control medium; Ser, serine; Gly, glycine; Cys, cysteine; PSAT1, phosphoserine aminotransferase 1; PHGDH, phosphoglycerate dehydrogenase; MTHFR, methylenetetrahydrofolate reductase; MAT2A, methionine adenosyltransferase 2A; ATF4, activating transcription factor 4; shNC, negative control short hairpin RNA; NC‐oe, negative control‐overexpression; PERK‐oe, PERK‐overexpression; shATF4, short hairpin RNA of ATF4; shMAT2A, short hairpin RNA of MAT2A; MRM, methionine‐restricted medium; NCG, NOD/ShiLtJGpt‐Prkdc em26Cd52 Il2rg em26Cd22 /Gpt; MDA‐MB‐231/LM2​, ​MDA‐MB‐231 lung metastasis 2​; CTCs, circulating tumor cells; BLI, lung Bioluminescence imaging; H&E, hematoxylin and eosin; ANOVA, analysis of variance.

Article Snippet: For annexin V detection analysis, Annexin V‐Elab Fluor 647/propidium iodide (PI) Apoptosis Kit (E‐CK‐A213, Elabscience, Wuhan, Hubei, China) or Annexin V‐allophycocyanin (APC)/4',6‐diamidino‐2‐phenylindole (DAPI) Apoptosis Kit (E‐CK‐A258, Elabscience) was employed to detect Annexin V + cells.

Techniques: Control, Liquid Chromatography with Mass Spectroscopy, Incubation, Cell Culture, Western Blot, Expressing, Transfection, Transduction, In Vivo, Imaging, Fluorescence, Injection, Staining, Knock-Out, Liquid Chromatography, Mass Spectrometry, Negative Control, shRNA, Over Expression

Journal: Cancer Communications

Article Title: Unfolded protein response kinase PERK supports survival and metastasis of circulating tumor cell clusters via SAM synthesis and H3K4me3‐dependent PDGFB signaling

doi: 10.1002/cac2.70072

Figure Lengend Snippet: Methionine metabolism promotes H3K4me3 methylation modification to upregulate PDGFB expression in CTC clusters. (A) Western blot analysis of PERK, MAT2A, and H3K4me3 levels in control and PERK‐KO MDA‐MB‐231/LM2‐CTC clusters treated with or without PF9366 or SAM. (B) The volcano plot depicting differentially expressed genes affected by methionine ( n = 3). (C) Metaplot comparing H3K4me3 enrichment profiles in MDA‐MB‐231/LM2‐CTC clusters cultured in CM or MRM. The plot is centered on TSS (±3.0 Kb) ( n = 3). (D) Bioinformatics analysis filtered out 10 survival‐related genes as downstream targets of H3K4me3. (E) Heatmap showing survival‐associated gene expression in MDA‐MB‐231/LM2‐CTC clusters under CM or MRM conditions ( n = 3). (F) Genome browser tracks of H3K4me3 at the PDGFB gene locus by ChIP‐seq ( n = 3). (G) TEM images of MDA‐MB‐231/LM2‐CTC cluster. (H‐J) Representative immunofluorescence images (left) and quantification (right) of PDGFB in CTC clusters from NCG‐MDA‐MB‐231/LM2 mouse model (H, n = 20); or in the complete or dispersed CTC clusters (I, n = 15); or in control or PDGFB‐knockdown CTC clusters (J, n = 15). Arrows indicate cell‐cell border; circle indicates intracellular regions. (K) Representative images of flow cytometry (left) and quantification of AnnexinV + MDA‐MB‐231/LM2‐CTC clusters (right) in control (shNC) and PDGFB‐knockdown (shP#1/2/3) groups ( n = 5). (L) Schematic representation of the experimental design. MDA‐MB‐231/LM2‐CTC clusters (shNC and shPDGFB #3) were injected into NCG mice via the tail vein, and metastatic burden was assessed by bioluminescence imaging at 24 h. (M‐N) Bioluminescence images (left) and quantification of fluorescence intensity (right) showing lung metastasis after tail vein injection at 24 h ( n = 5). Results represent mean ± SD. One‐way ANOVA test in A and K; student's t‐test in H, I, J, M, and N. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Abbreviations: MDA‐MB‐231/LM2​, ​MDA‐MB‐231 lung metastasis 2​; CTCs, circulating tumor cells; FC, fold change; PERK, PRKR‐like endoplasmic reticulum kinase; ERK‐KO, PRKR‐knockout; sgPERK, single‐guide RNA targeting PERK; MAT2A, methionine adenosyltransferase 2A; H3K4me3, trimethylation of histone H3 at lysine 4; SAM, S‐adenosylmethionine; CM, control medium; MRM, methionine‐restricted medium; No., number; TSS, transcription start site; ChIP‐seq, chromatin immunoprecipitation sequencing; RNA‐seq, RNA sequencing; PDGFB, platelet‐derived growth factor subunit B; IGF2BP3, insulin‐like growth factor 2 mRNA‐binding protein 3; PHGDH, phosphoglycerate dehydrogenase; ANXA11, annexin A11; ITGA6, integrin subunit alpha 6; ITGB4, integrin subunit beta 4; LOX, lysyl oxidase; PIK3C2B, phosphatidylinositol‐4‐phosphate 3‐kinase catalytic subunit type 2 beta; IRF9, interferon regulatory factor 9; ATP2A3, ATPase sarcoplasmic/endoplasmic reticulum Ca 2+ transporting 3; TEM, transmission electron microscopy; DAPI, 4′,6‐diamidino‐2‐phenylindole; shNC, negative control short hairpin RNA; shP#, short hairpin RNA targeting PDGFB; NCG, NOD/ShiLtJGpt‐Prkdc em26Cd52 Il2rg em26Cd22 /Gpt; BLI, lung Bioluminescence imaging; ANOVA, analysis of variance.

Article Snippet: For annexin V detection analysis, Annexin V‐Elab Fluor 647/propidium iodide (PI) Apoptosis Kit (E‐CK‐A213, Elabscience, Wuhan, Hubei, China) or Annexin V‐allophycocyanin (APC)/4',6‐diamidino‐2‐phenylindole (DAPI) Apoptosis Kit (E‐CK‐A258, Elabscience) was employed to detect Annexin V + cells.

Techniques: Methylation, Modification, Expressing, Western Blot, Control, Cell Culture, Gene Expression, ChIP-sequencing, Immunofluorescence, Knockdown, Flow Cytometry, Injection, Imaging, Fluorescence, Knock-Out, RNA Sequencing, Derivative Assay, Binding Assay, Transmission Assay, Electron Microscopy, Negative Control, shRNA

Journal: Frontiers in Physiology

Article Title: Protective Effect of Unacylated Ghrelin on Compression-Induced Skeletal Muscle Injury Mediated by SIRT1-Signaling

doi: 10.3389/fphys.2017.00962

Figure Lengend Snippet: TUNEL and dystrophin staining. Apoptotic nuclear DNA breaks in muscle tissue were measured using the technique of TUNEL staining (A) and were expressed as TUNEL index (B) . Representative images of TUNEL and dystrophin staining of compressed and uncompressed muscles (A) . Immunofluorescence labeling of dystrophin (red) was performed to identify the localization of the TUNEL-positive nuclei (green) with regard to muscle sarcolemmal membrane and nuclei were labeled with DAPI (blue) (A) . UnAG opposed the increase of TUNEL positive nuclei induced by moderate compression but this protective effect was absent when co-treated with EX527 (A) . TUNEL index was higher in compressed muscles of saline and UnAG+EX527 groups compared to compressed muscle of UnAG group (B) . Arrows indicate TUNEL-positive nuclei. * p < 0.05, compressed muscle compared to uncompressed control muscle; # p < 0.05, UnAG compared to Saline; + p < 0.05, UnAG+EX527 compared to UnAG; Cont, control muscle; Comp, compressed muscle; DAPI, 4′,6-diamidino-2-phenyl-lindole.

Article Snippet: The sections were then mounted with 4', 6-diamidino-2-phenylindole (DAPI, H-1200, Vector Laboratories) to visualize the nuclei.

Techniques: TUNEL Assay, Staining, Immunofluorescence, Labeling

Journal: Investigative ophthalmology & visual science

Article Title: Safety and Feasibility of Intrastromal Injection of Cultivated Human Corneal Stromal Keratocytes as Cell-Based Therapy for Corneal Opacities.

doi: 10.1167/iovs.17-23575

Figure Lengend Snippet: FIGURE 4. Expression of CSK, SF, and inflammatory markers in rat stroma at 2 and 4 weeks after cell injection. (A) CSK markers (Kera, keratocan; Lum, lumican; Col1, collagen I) were expressed predominantly along the CSK-injected region (marked by white arrowhead) as indicated by human- specific HuNu labelling. The staining was absent for SF- and PBS-injected corneas. (B) SF markers (Thy1; tenascin C, TNC; fibronectin, FN) were expressed in SF-injected corneas and the intensity of staining appeared to be more diffuse at 4 weeks compared to 2 weeks after injection. The myofibroblast marker (aSMA) became detectable at the fourth week post injection (region of dense haze illustrated under slit-lamp biomicroscopy marked by white bracket). In contrast, SF and myofibroblast markers were absent in CSK- and PBS-injected corneas. (C) The expression of inflammatory markers (MMP2, CD45) was stronger in SF-injected corneas when compared to the other two groups. The human cells were distinguished by human-specific HuNu staining. PBS-injected corneas were used as control. Nuclei were counterstained using DAPI (blue). Scale bar: 100 lm.

Article Snippet: After washes, samples were mounted in Fluoroshield with DAPI (Santa Cruz Biotech) and viewed under fluorescence microscopy (Zeiss Axioplan 2).

Techniques: Expressing, Injection, Staining, Marker, Control

Journal: Investigative ophthalmology & visual science

Article Title: Safety and Feasibility of Intrastromal Injection of Cultivated Human Corneal Stromal Keratocytes as Cell-Based Therapy for Corneal Opacities.

doi: 10.1167/iovs.17-23575

Figure Lengend Snippet: FIGURE 5. Time-lapse cell intensity changes after injection. (A) Intrastromal injection of Molday ION–labelled cells (2 3 104 per injection) and weekly imaging using Spectralis HRA with excitation under FA model. Representative images with good cell focusing were selected from a minimum of 50 consecutive frames taken along the entire stromal depth at each examination time point. (B) Graph showing a time-dependent reduction of label intensities (mean percentages of time 0 level) for both injection dosages (23104 and 53104 cells) until 6 weeks post injection. Approximately ~50% label intensity remained at around 3 weeks post injection. *P < 0.05, compared to label intensity measured immediately after injection (week 0) (Mann-Whitney U test). (C) TUNEL signal (red) was detected to colocalize with Molday ION EverGreen–labelled cells (green). Sections pretreated with DNase I and incubated with complete TUNEL reagent mix served as positive control whereas similar sections incubated with TUNEL reagent mix without TdT enzyme served as negative control. Nuclei were counterstained using DAPI (blue). Scale bar: 100 lm.

Article Snippet: After washes, samples were mounted in Fluoroshield with DAPI (Santa Cruz Biotech) and viewed under fluorescence microscopy (Zeiss Axioplan 2).

Techniques: Injection, Imaging, MANN-WHITNEY, TUNEL Assay, Incubation, Positive Control, Negative Control