Journal: Scientific Reports

Article Title: Involvement of endothelins in neuroprotection of valosin-containing protein modulators against retinal ganglion cell damage

doi: 10.1038/s41598-022-20497-w

Figure Lengend Snippet: Genes related to the excretion GO term and upregulated by NMDA injection and downregulated by KUS121 and KUS187 treatment.

Article Snippet: The primary antibodies used to probe the blots were mouse anti-EDN1 (1:400; Abcam, Cambridge, UK), rabbit anti-EDNRB (1:400; Alomone Labs, Jerusalem, Israel), rabbit anti-AKT (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-AKT (1:2000, Cell Signaling Technology), rabbit anti-ERK (1:20,000, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phospho-ERK (1:1000, Cell Signaling Technology) and anti-actin (1:5000; Sigma-Aldrich).

Techniques: Injection

Journal: Scientific Reports

Article Title: Involvement of endothelins in neuroprotection of valosin-containing protein modulators against retinal ganglion cell damage

doi: 10.1038/s41598-022-20497-w

Figure Lengend Snippet: mRNA expressions of endothelin-1 ( Edn1 ), endothelin receptor type A ( Ednra ) and endothelin receptor type B ( Ednrb ) in the mouse retina and primary retinal ganglion cells (RGCs). ( a – c ) Relative mRNA expression in neural retina. Neural retina of non-treated wild-type mice (W, n = 7) and NMDA-injected (C, n = 8), NMDA-injected-KUS121-treated (K121, n = 8) and NMDA-injected-KUS187-treated (K187, n = 8) mice were analyzed. The relative expression levels of Edn1 ( a ), Ednrb ( b ) and Ednra ( c ) mRNA were analyzed using qRT-PCR. The ratios of mRNA expression of each gene to that of glyceraldehyde-3-phosphate dehydrogenase were calculated. * p < 0.05 and ** p < 0.01, vs. W, Tukey’s honestly significant difference (HSD). NS no significant difference compared with W, Tukey’s HSD. ( d – f ) Relative mRNA expression in primary RGCs. Primary RGCs isolated from 3-day-old rats by two-step immunopanning were cultured with or without KUS121 (50 µM) for 2 h and then with or without KUS121 and with or without NMDA (500 µM) for another 4 h. The relative expression levels of Edn1 ( d ), Ednrb ( e ) and Ednra ( f ) mRNA were analyzed using qRT-PCR. (-): without KUS121 without NMDA n = 3, C: with NMDA without KUS121, n = 3, K121: with NMDA with KUS121, n = 3, respectively. The ratios of mRNA expression of each gene to that of glyceraldehyde-3-phosphate dehydrogenase were calculated. ** p < 0.01, control vs. KUS121, NS no significant difference compared with (-), Tukey’s HSD.

Article Snippet: The primary antibodies used to probe the blots were mouse anti-EDN1 (1:400; Abcam, Cambridge, UK), rabbit anti-EDNRB (1:400; Alomone Labs, Jerusalem, Israel), rabbit anti-AKT (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-AKT (1:2000, Cell Signaling Technology), rabbit anti-ERK (1:20,000, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phospho-ERK (1:1000, Cell Signaling Technology) and anti-actin (1:5000; Sigma-Aldrich).

Techniques: Expressing, Injection, Quantitative RT-PCR, Isolation, Cell Culture

Journal: Scientific Reports

Article Title: Involvement of endothelins in neuroprotection of valosin-containing protein modulators against retinal ganglion cell damage

doi: 10.1038/s41598-022-20497-w

Figure Lengend Snippet: Protein expression of endothelin-1 (EDN1) and endothelin receptor type B (EDNRB) in retinal tissue. ( a ) The retina of non-treated wild-type (labeled ‘‘W’’), NMDA-injected (control, labeled ‘‘C’’), or NMDA-injected-KUS121-treated mice (labeled ‘‘K’’) was analyzed by western blotting using an anti-EDN1 antibody. Actin was used as a loading control. Complete scans of western blots are shown in Supplementary Fig. a. ( b ) Comparison of EDN1 expression shown as ratios to actin ( n = 5, for all treatments). ** p < 0.01, vs. W, Tukey’s HSD. ( c – f ) Vertical sections of non-treated wild-type, NMDA-injected, NMDA-injected-KUS121-treated and NMDA-injected-KUS187-treated mice retinae were stained with an anti-EDN1 antibody or anti-EDNRB antibody. ( c , d ) Staining intensities of RGC layers with anti-EDN1 ( c ) or anti-EDNRB ( d ) antibody. The staining intensity of the RGC layer at distances of 400–800 μm from the optic nerve head was analyzed using BZ II Analyzer software. W: non-treated wild-type, C: NMDA-injected, K121: NMDA-injected-KUS121-treated, K187: NMDA-injected-KUS187-treated. ( n = 3, for C and K121, n = 2, for W and K187) NS no significant difference compared with W, Tukey’s HSD. ( e,f ) Vertical sections of non-treated wild-type (WT), NMDA-injected (control), NMDA-injected-KUS121-treated (K121) and NMDA-injected-KUS187-treated mice (K187) retinae. The black bar represents 100 µm. GCL ganglion cell layer, IPL inner plexiform layer.

Article Snippet: The primary antibodies used to probe the blots were mouse anti-EDN1 (1:400; Abcam, Cambridge, UK), rabbit anti-EDNRB (1:400; Alomone Labs, Jerusalem, Israel), rabbit anti-AKT (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-AKT (1:2000, Cell Signaling Technology), rabbit anti-ERK (1:20,000, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phospho-ERK (1:1000, Cell Signaling Technology) and anti-actin (1:5000; Sigma-Aldrich).

Techniques: Expressing, Labeling, Injection, Western Blot, Staining, Software

Journal: Scientific Reports

Article Title: Involvement of endothelins in neuroprotection of valosin-containing protein modulators against retinal ganglion cell damage

doi: 10.1038/s41598-022-20497-w

Figure Lengend Snippet: EDN1 and EDNRB protein expression and cell viability in 661W cultured cells under glucose-free conditions. ( a – d ) The relative amount of live cells was measured using WST-8 following 48 h treatment with DMEM/high glucose media or DMEM/glucose-free media with or without KUS121 (100 µM). ( a ) Quantitative analysis of live cells ( n = 3, for all treatments). 661W cells were cultured with high glucose [labeled ‘‘(-)’’] or treated in glucose-free media with (labeled ‘‘K121’’) or without (control, labeled ‘‘C’’) KUS121. ( b – d ) Representative images of 661W cells cultured under each condition. The black bar represents 50 µm. ( e – h ) Protein expression of EDN1 ( e , g ) and EDNRB ( f , h ) in 661W cells was analyzed by western blotting. 661W cells were cultured with high glucose media [labeled ‘‘(-)’’] or with glucose-free media with (labeled ‘‘K’’) or without (control, labeled ‘‘C’’) KUS121 for 24 h before the analysis. Actin was used as a loading control. Complete scans of western blots are shown in Supplementary Fig. b,c. Relative expression of EDN1 ( g ) and EDNRB ( h ) was shown as a ratio to actin ( n = 4, for both treatments). * p < 0.05, ** p < 0.01 and *** p < 0.001, compared with the control (in a and g ) or to the cells cultured in high glucose media (in h ), Tukey’s HSD.

Article Snippet: The primary antibodies used to probe the blots were mouse anti-EDN1 (1:400; Abcam, Cambridge, UK), rabbit anti-EDNRB (1:400; Alomone Labs, Jerusalem, Israel), rabbit anti-AKT (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-AKT (1:2000, Cell Signaling Technology), rabbit anti-ERK (1:20,000, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phospho-ERK (1:1000, Cell Signaling Technology) and anti-actin (1:5000; Sigma-Aldrich).

Techniques: Expressing, Cell Culture, Labeling, Western Blot

Journal: Theranostics

Article Title: CDK4/6 inhibition promotes immune infiltration in ovarian cancer and synergizes with PD-1 blockade in a B cell-dependent manner

doi: 10.7150/thno.44871

Figure Lengend Snippet: Abemaciclib treatment induces immune infiltration and T/B cell aggregation. (A) Representative immunohistochemical staining for CD45, CD8, CD19 and PD-L1 (400×), scale bars: 50 μm. (B) TIL was quantified by counting positive signals in four randomly selected fields (400×) in each tumor section using ImageJ, n=6 mice. Statistical comparisons were performed using an unpaired T test. (C) Representative immunofluorescence images of CD3 (green), CD19 (red) and nuclear staining (DAPI, blue) showing different types of cell aggregates (400×). Both CD3+ T cells and CD19+ B cells in the control group were scattered, while the numerous infiltrated T cells and B cells formed ectopic lymphoid-like structures with different shapes in the abemaciclib-treated group, scale bars: 50 μm. (D) Quantification of T cell/B cell aggregates in 3 randomly selected 100× fields in each tumor section.

Article Snippet: The primary antibodies used included rabbit anti-mouse CD45 (1:200, CST, 70257), rabbit anti-mouse CD8α (1:400, CST, 98941), rabbit anti-mouse CD19 (1:800, CST, 90176), and rabbit anti-mouse PD-L1 (1:200, CST, 64988).

Techniques: Immunohistochemical staining, Staining, Immunofluorescence, Control

Journal: Theranostics

Article Title: CDK4/6 inhibition promotes immune infiltration in ovarian cancer and synergizes with PD-1 blockade in a B cell-dependent manner

doi: 10.7150/thno.44871

Figure Lengend Snippet: Proportion of TILs from PBS- or abemaciclib-treated ID8 tumors and phenotypic analysis of T cells by flow cytometry, n=6 mice. (A) The proportions of CD8+ T cells, CD4+ T cells, B220+ B cells, CD11b+F4/80+ tumor-associated macrophages (TAMs) and CD11b+Gr-1+ MDSCs in the CD45+ TIL population were determined by flow cytometry. (B) Representative zebra plots of IFN-γ and CD107a expression in CD8+T cell subsets. (C-D) Representative zebra plots of CD4+foxp3- Tregs and CD4+foxp3+ T cells, histogram overlays show CD73 expression in each subset.

Article Snippet: The primary antibodies used included rabbit anti-mouse CD45 (1:200, CST, 70257), rabbit anti-mouse CD8α (1:400, CST, 98941), rabbit anti-mouse CD19 (1:800, CST, 90176), and rabbit anti-mouse PD-L1 (1:200, CST, 64988).

Techniques: Flow Cytometry, Expressing

Journal: Theranostics

Article Title: CDK4/6 inhibition promotes immune infiltration in ovarian cancer and synergizes with PD-1 blockade in a B cell-dependent manner

doi: 10.7150/thno.44871

Figure Lengend Snippet: CXCL10 or CXCL13 neutralization blocked CD8+T cell or B cell migration in vitro and abrogated the therapeutic effect of abemacilib in vivo. (A) Transwell assays were performed to detect the chemotactic effect of ID8 cell supernatants after PBS or abemaciclib treatment, and serum-free DMEM was used as the culture medium. Transwell assays were also tested in the presence of neutralizing anti-CXCL10 or anti-CXCL13 antibody. Each sample was analyzed in triplicate. (B) Representative images of ID8-luc models at day 21 post first treatment are shown. (C) The data were quantified with an IVIS, n=6 mice.

Article Snippet: The primary antibodies used included rabbit anti-mouse CD45 (1:200, CST, 70257), rabbit anti-mouse CD8α (1:400, CST, 98941), rabbit anti-mouse CD19 (1:800, CST, 90176), and rabbit anti-mouse PD-L1 (1:200, CST, 64988).

Techniques: Neutralization, Migration, In Vitro, In Vivo

Journal: Theranostics

Article Title: CDK4/6 inhibition promotes immune infiltration in ovarian cancer and synergizes with PD-1 blockade in a B cell-dependent manner

doi: 10.7150/thno.44871

Figure Lengend Snippet: Abemaciclib synergizes with anti-PD-1 therapy in ID8 tumor control, and combination therapy achieves longer survival than monotherapy. (A) Tumor progression was monitored every 7 days during treatment, and representative images of 3 mice in each group are shown; n=8 mice. (B) The tumor burden was evaluated by quantification of total flux with Living Image software. Data presented are the group mean of 8 mice ± standard error. (C) The long-term survival of ID8 tumor-bearing mice treated with PBS, abemaciclib or an anti-PD-1 mAb alone or in combination is shown. (D) Bioluminescence images of mice on day 14 after tumor cell re-challenge, naïve C57 mice were used as controls(left). The tumor burden was evaluated by quantification of total flux (right). (E-F) Flow cytometry analysis of functional and activation markers expressed by CD8+ T cells (left) or CD4+ T cells (right) in ID8 tumors at day 10 post treatment initiation, n=6 mice. Error bars, SD.

Article Snippet: The primary antibodies used included rabbit anti-mouse CD45 (1:200, CST, 70257), rabbit anti-mouse CD8α (1:400, CST, 98941), rabbit anti-mouse CD19 (1:800, CST, 90176), and rabbit anti-mouse PD-L1 (1:200, CST, 64988).

Techniques: Control, Software, Flow Cytometry, Functional Assay, Activation Assay

Journal: Theranostics

Article Title: CDK4/6 inhibition promotes immune infiltration in ovarian cancer and synergizes with PD-1 blockade in a B cell-dependent manner

doi: 10.7150/thno.44871

Figure Lengend Snippet: The antitumor activity of abemaciclib plus an anti-PD-1 mAb is abrogated by CD8+ T cell depletion or B cell depletion. (A) Flow cytometry analysis confirmed an obvious decrease in the CD8+ T cell or CD19+ B cell proportion in the mouse spleen. (B) Representative live animal images of ID8-luc models at day 21 post first treatment are shown. (C) The data were quantified with living image software, n=8 mice. (D) The long-term survival of ID8 tumor-bearing mice in the control, combination, combination plus CD8+ T cell depletion, and combination plus B cell depletion groups was evaluated. (E) The expression of CD69, IFN-γ and CD107a in the CD8+ T cell population and (F) CD4+ T cell populations was analyzed by flow cytometry on day 10 after treatment initiation, n=6 mice.

Article Snippet: The primary antibodies used included rabbit anti-mouse CD45 (1:200, CST, 70257), rabbit anti-mouse CD8α (1:400, CST, 98941), rabbit anti-mouse CD19 (1:800, CST, 90176), and rabbit anti-mouse PD-L1 (1:200, CST, 64988).

Techniques: Activity Assay, Flow Cytometry, Software, Control, Expressing

Journal: PLOS One

Article Title: Enhancement of YTHDF2 plays a protective role in acute IRI models through downregulation of TUG1 expression

doi: 10.1371/journal.pone.0319605

Figure Lengend Snippet: Primer sequences used in qRT-PCR analysis.

Article Snippet: YTHDF2 (24744-1-AP, 1:400, Proteintech, Wuhan, China) and Alexa Fluor ® 647 AffiniPure (711-605-152,1:1000, Jackson, China) were adopted as primary and secondary antibody in PBS dilution, respectively.

Techniques: cDNA Synthesis

Journal: PLOS One

Article Title: Enhancement of YTHDF2 plays a protective role in acute IRI models through downregulation of TUG1 expression

doi: 10.1371/journal.pone.0319605

Figure Lengend Snippet: (A) Microscopic images of HK-2 cells of OGD/R model. Left, control cells cultured in normoxia condition; middle, cells cultured in 12 hours of hypoxia and 12 hours reoxygenation; right, cells cultured in 24 hours of hypoxia but no reoxygenation. (scale bar = 200μm) (B) Viability of cells in (A) detected via CCK8. (C) Western Blot of apoptosis related proteins BCL2, Caspase-3 and BAX. (D-F) Quantification of the protein level in (C) by ImageJ software for (D) BCL2, (E) Caspase-3, and (F) BAX. (G) mRNA level of YTHDF2 in HK-2 cells of OGD/R detected by RT-PCR. (H, I) YTHDF2 protein detected in HK-2 cells of OGD/R by (H)Western Blot and (I) quantified base on the gray value of bands. Data was expressed as the mean ± SD of three repeated experiments. **P < 0.01, ***P < 0.001 and ****P < 0.0001 vs. the control group. (n = 3 for cell experiments).

Article Snippet: YTHDF2 (24744-1-AP, 1:400, Proteintech, Wuhan, China) and Alexa Fluor ® 647 AffiniPure (711-605-152,1:1000, Jackson, China) were adopted as primary and secondary antibody in PBS dilution, respectively.

Techniques: Control, Cell Culture, Western Blot, Software, Reverse Transcription Polymerase Chain Reaction

Journal: PLOS One

Article Title: Enhancement of YTHDF2 plays a protective role in acute IRI models through downregulation of TUG1 expression

doi: 10.1371/journal.pone.0319605

Figure Lengend Snippet: (A) Diagram showing the procedure of IRI model in mouse kidney. (B, C) Morphological changes were visualized in kidney with (B) H&E staining and then (C) quantified. (B) Representative images of control (upper left) and modeled tissues (lower left) were compared in 40 × magnification (Bar = 200μm). Renal tubules with regular morphology (upper right) or lumen expansion (lower right) were further magnified from left images (Bar = 50μm). (C) Frequency of enlarged lumens was counted within control and modeled renal cortex. For either group, 5 fields of vision were picked randomly from 5 kidneys. The result was presented as percentage of area occupied by tubules with lumen expansion in fields of view. (D, E) Microscopic imaging of basal membrane of renal tubules via (D) PASM staining and (E) quantified within cortex area. (D) Representative images of control (upper left) and modeled tissues (upper right) were compared in 40 × magnification (Bar = 200μm). Renal tubules with (lower left) or without (lower right) PASM staining were further magnified from upper images (Bar = 50μm). (E) Frequency of lumens absent of PASM was counted within renal cortex under 40 × magnification. For either group, 5 fields of vision were picked randomly from 5 kidneys. The result was presented as percentage of area occupied by tubules negative of PASM. (F) mRNA level of YTHDF2 in control and IRI kidneys by RT-PCR. (G, H) YTHDF2 protein level in control and IRI kidneys detected by (G) Western Blot and (H) quantified results based the gray value of bands. (I, J) Immunofluorescent imaging of YTHDF2 protein in (I) normal or IRI renal tissue under 60 × magnification (Bar = 20μm), while the staining intensity of was (J) quantified in 5 random fields of view for both groups. Data was expressed as the mean ± SD of three repeated experiments. * P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (n = 5 for mouse experiments).

Article Snippet: YTHDF2 (24744-1-AP, 1:400, Proteintech, Wuhan, China) and Alexa Fluor ® 647 AffiniPure (711-605-152,1:1000, Jackson, China) were adopted as primary and secondary antibody in PBS dilution, respectively.

Techniques: Staining, Control, Imaging, Membrane, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: PLOS One

Article Title: Enhancement of YTHDF2 plays a protective role in acute IRI models through downregulation of TUG1 expression

doi: 10.1371/journal.pone.0319605

Figure Lengend Snippet: (A) Microscopic images of HK-2 cells infected with lentivirus for the ectopic expression of YTHDF2 in bright field (left) and fluorescent field (right). The fluorescence signal was generated by the GFP protein in the lentiviral vector. (scale bar = 200μm) (B-C) Confirmation of enhanced YTHDF2 protein level with (B) Western Blot and (C) the quantified results. (D) Morphological characterization of YTHDF2 overexpressed and control cells in OGD/R model. (scale bar = 200μm) (E) Viability of cells with/without YTHDF2 overexpression in OGD/R model. (F-I) The trend of apoptosis related proteins indicted with (F) Western Blot upon YTHDF2 overexpression in OGD/R model and quantified respectively for (G) BCL2, (H) BAX and (I) Caspase-3. Data was expressed as the mean ± SD of three repeated experiments. * P < 0.05, **P < 0.01 and ****P < 0.0001.

Article Snippet: YTHDF2 (24744-1-AP, 1:400, Proteintech, Wuhan, China) and Alexa Fluor ® 647 AffiniPure (711-605-152,1:1000, Jackson, China) were adopted as primary and secondary antibody in PBS dilution, respectively.

Techniques: Infection, Expressing, Fluorescence, Generated, Plasmid Preparation, Western Blot, Control, Over Expression

Journal: PLOS One

Article Title: Enhancement of YTHDF2 plays a protective role in acute IRI models through downregulation of TUG1 expression

doi: 10.1371/journal.pone.0319605

Figure Lengend Snippet: (A) Microscopic images of HK-2 cells infected with lentivirus for the knockdown of YTHDF2 in bright field (left) and fluorescent field (right). The fluorescence signal was generated by the GFP protein in the lentiviral vector. (scale bar = 200μm) (B-C) Confirmation of impaired YTHDF2 protein level with (B) Western Blot and (C) the quantified results. (D) Morphological characterization of YTHDF2 knockdown and control cells in OGD/R model. (scale bar = 200μm) (E) Viability of cells with/without YTHDF2 knockdown in OGD/R model. Data was expressed as the mean ± SD of three repeated experiments. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: YTHDF2 (24744-1-AP, 1:400, Proteintech, Wuhan, China) and Alexa Fluor ® 647 AffiniPure (711-605-152,1:1000, Jackson, China) were adopted as primary and secondary antibody in PBS dilution, respectively.

Techniques: Infection, Knockdown, Fluorescence, Generated, Plasmid Preparation, Western Blot, Control

Journal: PLOS One

Article Title: Enhancement of YTHDF2 plays a protective role in acute IRI models through downregulation of TUG1 expression

doi: 10.1371/journal.pone.0319605

Figure Lengend Snippet: (A-B) The mRNA level of TUG1 detected by RT-PCR in HK-2 cells with (A) YTHDF2 overexpression and (B) YTHDF2 knockdown under hypoxia/reoxygenation condition. (C) RNA immunoprecipitation assay demonstrated YTHDF2 binding to TUG1 (****P < 0.0001). (D) The candidate m 6 A sites on TUG1 RNA predicted with SRAMP. (E) groups of m 6 A sites defined with their confidence level scored by SRAMP. (F) Potential binding sites of YTHDF2 recognized on TUG1 RNA chain based on the defined motif (A/G/U)GAC(A/U) (G) Number of candidate sites matching both criteria, potential of m 6 A modification and allowance for YTHDF2 binding. (H)The expression of miR-449b-5p in OGD/R-induced HK-2 cells with YTHDF2 overexpression were tested using qRT-PCR. (I-J) MMP2 proteins indicted with (I) Western Blot upon YTHDF2 overexpression in OGD/R model and (J) quantified respectively for MMP2. Data was expressed as the mean ± SD of three repeated experiments. * P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: YTHDF2 (24744-1-AP, 1:400, Proteintech, Wuhan, China) and Alexa Fluor ® 647 AffiniPure (711-605-152,1:1000, Jackson, China) were adopted as primary and secondary antibody in PBS dilution, respectively.

Techniques: Reverse Transcription Polymerase Chain Reaction, Over Expression, Knockdown, RNA Immunoprecipitation, Binding Assay, Modification, Expressing, Quantitative RT-PCR, Western Blot

Journal: PLOS One

Article Title: Enhancement of YTHDF2 plays a protective role in acute IRI models through downregulation of TUG1 expression

doi: 10.1371/journal.pone.0319605

Figure Lengend Snippet: 21 potential binding sites of YTHDF2 protein on TUG1 RNA.

Article Snippet: YTHDF2 (24744-1-AP, 1:400, Proteintech, Wuhan, China) and Alexa Fluor ® 647 AffiniPure (711-605-152,1:1000, Jackson, China) were adopted as primary and secondary antibody in PBS dilution, respectively.

Techniques: Binding Assay, Sequencing

Journal: eFood

Article Title: Docosahexaenoic Acid and Nervonic Acid Synergically Enhance Cognitive Memory in Normal Mice via Brain Fatty Acids Remodeling

doi: 10.1002/efd2.70059

Figure Lengend Snippet: FIGURE 3 | DHA and NA enhanced c‐Fos expression in the hippocampus of normal mice. (A) Representative c‐Fos immunopanned photos in the hippocampus. (B) The number of c‐Fos+ neurons in the hippocampal DG area. (C) The expression of PSD95 in the hippocampus. (D) The expression of synaptophysin (SYP) in the hippocampus. *p < 0.05, **p < 0.01 vs control group. One‐way ANOVA post‐hoc Dunnett's test (mean ± SEM, n = 4).

Article Snippet: Immunostaining was performed using monoclonal antibodies against c‐Fos (1:1000; Cell Signaling Technology, cat#2250S) and synaptophysin (SYP, 1:400; Santa Cruz, sc‐17750), polyclonal antibody against PSD95 (1:200; Invitrogen, cat#51‐6900), followed by incubation at 4°C for 24 h. Goat anti‐ mouse (Alexa Fluor 594, 1:500, ab150116) and goat anti‐rabbit (Alexa Fluor 488, 1:500, ab150077) IgG were used as secondary antibodies and incubated in the dark at room temperature for 2 h. Fluorescent images were captured using an ECHO Revlove fluorescence microscope (ECHO, San Diego, California, USA) and quantitatively analyzed using ImageJ software (NIH, Bethesda, Maryland, USA).

Techniques: Expressing, Control

Journal: Leukemia

Article Title: RECQ1 helicase is involved in replication stress survival and drug resistance in multiple myeloma

doi: 10.1038/leu.2017.54

Figure Lengend Snippet: RECQ1 depletion induces DDR in MM cells. ( a ) Alkaline comet assay was used to analyze DNA breaks formation after RECQ1 depletion in XG19 cell line. Tail length and tail moment (fraction of total DNA in the tail × tail length) were analyzed with ImageJ software. Data are calculated from measurement of 50 comets for each sample. Statistical difference was tested using a Student t -test. ( b ) 53BP1 staining was used as marker for DNA damage. The number of 53BP1 foci found in each cell was enumerated 3 days after doxycycline treatment. At least 300 cells were counted for each group. The percentage of cells with more than 10 53BP1 foci per cell is displayed in the histograms. Statistical difference was tested using a Student t -test. ( c ) HMCL-TR-shRECQ1 cells were cultured for 7 days with or without doxycycline (dox) and protein detection was assayed using western blot analysis. Membranes were stained with anti-phospho-ATM, anti-ATM, anti-phospho-p53 (Ser15 or Ser20) and anti-p53, anti-Phospho-Chk2, anti-Chk2 and anti-γH2AX. A mouse monoclonal anti-β-actin antibody was used as control. ( d ) XG19-TR-shRECQ1 cell line was treated or not with doxycycline for 5 days and replication fork progression was determined by DNA fiber spreading after double labeling with IdU and CldU. Statistical difference was tested using a Student t -test.

Article Snippet: The rabbit anti-53BP1 antibody (clone NB100-304, Novus Biologicals, Cambridge, UK) and the mouse anti-γH2AX (Ser139) antibody (clone JBW301, Merck Millipore, Darmstadt, Germany) were diluted 1/300 and 1/100, respectively, in 5% bovine milk in PBS, and deposited on cytospins for 90 min at room temperature.

Techniques: Alkaline Single Cell Gel Electrophoresis, Software, Staining, Marker, Cell Culture, Western Blot, Control, Labeling

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a Domain structures of short and long Ank2 protein variants (220 and 440 kDa, respectively). Note that exon 4 deletion (red star) affects both short and long variants. MBD membrane-binding domain, SBD spectrin-binding domain; linker, a linker for the MBD and SBD domains; giant exon, a domain uniquely present in the long Ank2 variant; DD death domain, CT C-terminal region. b Immunoblot analysis of WT and Ank2-cKO cortical and hippocampal lysates (P21). ( n = 4 mice [WT, cKO], one sample t -test). c Hyperactivity of Ank2-cKO mice (P19–21) in the open-field test (100 lux), as shown by distance moved. The reduced time spent in the center region suggests anxiety-like behavior. ( n = 25 [WT; males and females mixed here and in all other behavioral experiments], 19 [cKO], Mann-Whitney test [total distance moved, center time], two-way repeated-measures/RM-ANOVA with Sidak’s test [distance moved]). d Anxiolytic-like behavior in Ank2-cKO mice (P19–21) in the light-dark test (600 lux), as shown by light-chamber time/entry. ( n = 20 [WT], 17 [cKO], Student’s t -test). e Anxiolytic-like behavior in Ank2-cKO mice (P24–25) in the elevated plus-maze test, as shown by open-arm time/entry. ( n = 18 [WT], 10 [cKO], Student’s t -test). f Normal social interaction and impaired social novelty recognition in Ank2-cKO mice (P19–20) in the three-chamber test, as shown by sniffing time. S1/S2, novel/familiar stranger; O, object. ( n = 18 [WT], 14 [cKO], Mann–Whitney test [WT-S1/O, WT-S1/S2, and cKO-S1/O], Student’s t-test [cKO-S1/S2]). g Abnormally increased social interaction in Ank2-cKO mice (P23–25) in a direct social-interaction test. ( n = 9 [WT], 7 [cKO], Student’s t -test). h Decreased self-grooming and digging, without a change in jumping, in Ank2-cKO mice (P25–27). Note that hyperactivity is observed here, similar to the open-field hyperactivity. ( n = 24 [WT], 17 [cKO], Mann-Whitney test [digging/self-grooming]). Source data for uncropped immunoblot images are provided as a Source Data file. The statistical tests involved two-sided analyses, and adjustments were made for multiple comparisons. Data are presented as mean values +/- SEM. P values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Membrane, Binding Assay, Variant Assay, Western Blot, MANN-WHITNEY

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a Examples of epileptiform spike discharges in Ank2-cKO mice (P35; parietal right lobe) monitored until death, as shown by electroencephalography (EEG). b Epileptiform discharges in EEG observed in an Ank2-cKO mouse until death. c Juvenile seizure-related death occurring in Ank2-cKO mice during ~P20–50. ( n = 23 [WT], 23 [cKO], Log-rank test). d – h Increased pentylenetetrazole (PTZ)-induced seizures in Ank2-cKO mice (P25–27), as shown by final seizure stage (1–4) reached, latency to stage 2 seizure, animals reached stage 2, and frequency and total duration of stage 2. The stage 2 was mainly compared because of its predominance in the mutant mice. ( n = 17 [WT], 10 [cKO], chi-square test [final seizure stage], Mann-Whitney test [stage 2 latency/duration]). The statistical tests involved two-sided analyses. Data are presented as mean values +/− SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Mutagenesis, MANN-WHITNEY

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a Examples of neuronal firing in the somatosensory cortex (SSC) in brain slices from Ank2-cKO mice, as measured by multielectrode array (MEA; 64 electrodes in 8 × 8 arrays; cutoff for significant firing > 4.5 x standard deviation). b , c Increased mean firing rate and mean burst rate (inter-spike-interval <10 ms) in the Ank2-cKO SSC (P19–22), as measured by MEA. ( n = 9 slices/3 mice [WT], 10, 3 [cKO], Student’s t -test [total], Welch’s test [layer 2/3], Mann–Whitney test [layer 5]). d Unaltered number of spikes in a single burst in the Ank2-cKO SSC (P19–22) measured by MEA. ( n = 9, 3 [WT], 10, 3 [cKO], Student’s t-test [total], Mann–Whitney test [layer 2/3 and 5]). e Unaltered percentage of spikes in bursts in the Ank2-cKO SSC (P19–22) measured by MEA. ( n = 9, 3 [WT], 10, 3 [cKO], Welch’s test [total], Mann-Whitney test [layer 2/3 and 5]). f Diagram showing examples of local field potentials (LFPs) and depicting parameters of LFP (length and amplitude). g – i Increased duration but normal frequency and amplitude of LFPs in layer 2/3 but not layer 5 in the Ank2-cKO SSC (P19–22) measured by MEA. ( n = 9, 3 [WT], 10, 3 [cKO], Student’s t -test [LFP frequency-layer 2/3, amplitude-total and layer 2/3, duration-total and layer 5], Mann–Whitney test [frequency/amplitude-layer 5], Welch’s test [duration-layer 2/3]). The statistical tests involved two-sided analyses. Data are presented as mean values +/- SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Standard Deviation, MANN-WHITNEY

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a Normal resting membrane potential in Ank2-cKO SSC layer 2/3 pyramidal neurons (P19–22). ( n = 12 neurons/8 mice [WT], 22,8 [cKO], Mann–Whitney test). b , c Increased input resistance in Ank2-cKO SSC layer 2/3 neurons (P19–22). ( n = 10, 3 [WT], 12, 3 [cKO], two-way RM-ANOVA with Sidak’s test and Student’s t -test). d Increased current-firing curve slope in Ank2-cKO SSC layer 2/3 neurons (P19–22). ( n = 7, 3 [WT], 9, 3 [cKO], two-way RM-ANOVA with Sidak’s test). e Diagram explaining mAHP. f Decreased mAHP amplitude in Ank2-cKO SSC layer 2/3 neurons (P19–22). ( n = 16, 5 [WT], 21, 5 [cKO], Welch’s test). g Diagram explaining AP threshold and AP shape-related parameters. h – l Normal AP threshold and AP shape-related parameters in Ank2-cKO SSC layer 2/3 neurons (P19–22), as indicated by AP-threshold voltage and the height, width, and time-dependent voltage changes (dV/dt; maximal/depolarizing and minimal/repolarizing) of APs. ( n = 13, 9 [WT], 18, 11 [cKO], Student’s t -test [AP threshold and Max dV/dt and min dV/dt], Mann-Whitney test [FWHM and amplitude]). The statistical tests involved two-sided analyses, and adjustments were made for multiple comparisons. Data are presented as mean values +/− SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Membrane, MANN-WHITNEY

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a Diagram depicting procedures of PTM (posttranslational modification) analyses for samples from WT and Ank2-cKO cortex and hippocampus; (P19–22) ( n = 3 groups from 9 mice [3 mice/group] [WT, cKO]). b , c DAVID gene ontology (GO) analyses of biological functions for the proteins with up- and downregulated PTM levels (PTM proteins). d – f Volcano plots highlighting the PTM proteins with significant fold changes (>1.5) and p -values (<0.05, Welch’s t-test) and the presence of potassium channels in the down-PTM proteins (fold change > 1.5 and p < 0.05 [Welch’s t -test]). g Diagram depicting procedures of proteomic analysis of total (whole-lysate) proteins in Ank2-cKO mice (cortex + hippocampus; P19–22). h , i Volcano plots showing the differentially expressed proteins (DEPs) derived from the analysis of total (whole-lysate) proteins in Ank2-cKO mice (cortex and hippocampus; P19–22) with significant fold changes (>1.5) and p -values (<0.05) and the presence of Kv7.2 and Kv7.3 in the downregulated total DEPs ( p < 0.05 [Welch’s t -test] but not fold change > 1.5). ( n = 3 mice [WT, cKO]). j Decreased total and crude synaptosomal levels of Kv7.2 and Kv7.3 in Ank2-cKO brains (cortex + hippocampus; P19–23). Note that the levels of Kv2.1, Kv3.1, and Kv1.2 (unrelated control) were not changed. ( n = 4 mice [WT, cKO] except for Kv7.3 in total lysates ( n = 5 mice [WT, cKO]), one sample t -test). k Decreased surface levels of Kv7.2 and Kv7.3 in cultured cortical neurons (DIV 17). ( n = 15 dishes [5 + 5 + 5] from 3 biologically independent experiments [Kv7.2] and 14 dishes [5 + 5 + 4] from 3 biologically independent experiments [Kv7.3], one sample t-test). Source data for uncropped immunoblot images are provided as a Source Data file. The statistical tests involved two-sided analyses. Data are presented as mean values +/− SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Modification, Derivative Assay, Control, Cell Culture, Western Blot

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a – c Increased AIS area (marked by Ank3; arrows) in cultured hippocampal Ank2 fl/fl neurons infected with AAV-Cre (DIV 4 ~ 10–14 ~ 28), and decreased mean intensities of Kv7.3 and Ank3 in the AIS area; these changes together yield normal total intensities of Kv7.3 and Ank3. Note that the width of Ank3 was not changed. ( n = 36 neurons from three independent experiments [WT], 36 [cKO], Welch’s test [Ank3 mean intensity], Mann–Whitney test [Ank3 area/total intensity; Kv7.3 mean/total intensity in AIS]). Scale bar, 10 µm. d – g Increased AIS length and area (marked by Ank3) in the Ank2-cKO somatosensory cortex (P19–23), as determined by three-dimensional imaging and quantification of expanded brain tissues. ( n = 60 neurons from 4 mice [WT], 60, 4 [cKO], Student’s t-test [Ank3 length], Welch’s t -test [Ank3 area]). Scale bar, 4 µm. h – j The Kv7.2/3 antagonist, XE991, increases the slope of the current-spike curve in WT, but not Ank2-cKO, SSC layer 2/3 pyramidal neurons (P19–23). ( n = 7 neurons from 3 mice [WT], 14,4 [WT + XE991], 9,3 [cKO], 13,5 [cKO+XE991], two-way RM-ANOVA with Sidak’s test [i] and with Tukey’s test [j]). The statistical tests involved two-sided analyses, and adjustments were made for multiple comparisons. Data are presented as mean values +/− SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Cell Culture, Infection, MANN-WHITNEY, Imaging

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a , b Retigabine treatment (from P16–17) rescues the increased neuronal excitability and firing in Ank2-cKO SSC layer 2/3 pyramidal neurons (P19–23) without affecting WT neurons, as shown by current-firing curves. ( n = 8 neurons from 4 mice [WT-Veh], 8,4 [WT-RTG], 8,4 [cKO-Veh], 8,4 [cKO-RTG], two-way RM-ANOVA with Tukey’s test [a], two-way RM-ANOVA with Sidak’s test [b]). c and d Retigabine treatment (from P16–17) rescues the decreased mAHP (medium afterhyperpolarization) amplitude in Ank2-cKO SSC layer 2/3 neurons (P19–23) without affecting that of WT neurons. ( n = 8, 4 [WT], 8,4 [WT-RTG], 8,4 [cKO], 8,4 [cKO-RTG], two-way RM-ANOVA with Tukey’s test [c], two-way RM-ANOVA with Sidak’s test [d]). e , f Retigabine treatment (from P16–17) does not affect input resistance in Ank2-cKO or WT SSC layer 2/3 neurons (P19–23). ( n = 7,4 [WT], 7,4 [WT-RTG], 7,4 [cKO], 7,4 [cKO-RTG], two-way RM-ANOVA with Tukey’s test [e], two-way RM ANOVA with Sidak’s test [f]). The statistical tests involved two-sided analyses, and adjustments were made for multiple comparisons. Data are presented as mean values +/- SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques:

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a , b Chronic retigabine treatment (from P16–17) improves juvenile seizure-related death of Ank2-cKO mice without affecting WT mice. ( n = 11 mice [WT-Veh/vehicle], 12 [WT-RTG/retigabine], 16 [cKO-Veh], 10 [cKO-RTG], Log-rank test). c , d Cessation of chronic retigabine treatment at around ~P42 (~26 days after the initiation of retigabine treatment) eliminates the treatment effect of retigabine on juvenile seizure-related death in Ank2-cKO mice. ( n = 14 mice [cKO-Veh], 13 [cKO-RTG], Log-rank test). e – g Chronic retigabine treatment (from P16–17) does not improve hyperactivity or anxiety-like behavior (center time) of Ank2-cKO mice in the open-field test. ( n = 11 mice [WT-Veh], 12 [WT-RTG], 11 [cKO-Veh], 10 [cKO-RTG], two-way ANOVA with Tukey test). h – k Acute retigabine treatment (5 mg/kg) at P21/22 improves hyperactivity but not anxiety-like behavior (center time) of Ank2-cKO mice in the open-field test. Drug treatment groups were divided into two (retigabine/saline first) to minimize cross-treatment effects. ( n = 13 mice [WT-Veh], 15 [WT-RTG], 16 [cKO-Veh], 17 [cKO-RTG], two-way ANOVA with Tukey’s test [distance moved_4min], Sidak’s test [distance moved_total]). The statistical tests involved two-sided analyses, and adjustments were made for multiple comparisons. Data are presented as mean values +/− SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Saline

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet:

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Virus, Recombinant, Software

Journal: iScience

Article Title: Tumor stage-driven disruption of NK cell maturation in human and murine tumors

doi: 10.1016/j.isci.2024.111233

Figure Lengend Snippet:

Article Snippet: As soon as the total flux reached 2.105 p/s, mice were injected intraperitoneally with either 400 μg of anti-NK1.1 mAb (PK136 - BioXcell, BX-BE0036) or the control isotype (IgG2a - BioXcell, BX-BE0085) in 200 μL of PBS with insulin syringe (Dutscher, 324891).

Techniques: Staining, Control, Recombinant, Modification, Luciferase, Software

Journal: Oncotarget

Article Title: Next-generation multimodality of nutrigenomic cancer therapy: sulforaphane in combination with acetazolamide actively target bronchial carcinoid cancer in disabling the PI3K/Akt/mTOR survival pathway and inducing apoptosis

doi: 10.18632/oncotarget.28011

Figure Lengend Snippet: Cleaved caspase-3, cleaved caspase -7, and cleaved PARP expressions in typical H727 ( A – D ) and atypical H720 ( E – H ) BC cells after AZ (40 μM), SFN (40 μM), and the combination treatment for 72 hours using immunohistochemistry (IHC) and immunofluorescence labeling assays, and Western blot. (D, H) The percent positive cells were quantified using Image J software. ( I – O ) Western blot analyses of cell lysates for the expression of cleaved caspase-3, caspase-7, or cleaved PARP. The blots were stripped and reprobed for β actin as a loading control. The data represent one out of three independent experiments showing similar results. The results show a progressive increase in apoptotic proteins from AZ, SFN to AZ+SFN. The expression of cleaved caspase-3, -7, and PARP was compared to the untreated control for each treatment group by ANOVA using the unpaired t -test with 95% confidence with indicated asterisks for statistical significance. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, n = 3 independent experiments. Abbreviations: AZ: acetazolamide; SFN: sulforaphane; BC: bronchial carcinoma; PARP: poly [ADP-ribose] polymerase; IF: immunofluorescence.

Article Snippet: Cells were fixed and permeabilized in 80% cold methanol for 10 minutes on ice, washed three times in cold-PBS and incubated with primary antibodies against cleaved caspase-3 (Asp175, 1:400), cleaved caspase-7 (Asp198, 1:1600), and cleaved PARP (Asp214, 1:400) (Cell Signaling Technology, New England Biolabs, Ltd., Whitby, Ontario, Canada).

Techniques: Immunohistochemistry, Immunofluorescence, Labeling, Western Blot, Software, Expressing, Control

Journal: Journal of biological engineering

Article Title: A facile in vitro platform to study cancer cell dormancy under hypoxic microenvironments using CoCl 2 .

doi: 10.1186/s13036-018-0106-7

Figure Lengend Snippet: Fig. 1 CoCl2-induced hypoxia-mimicking conditions can lead to prolonged growth inhibition in MCF-7 cells. Cell growth analysis (a) and representative fluorescence images of cell cycling marker Ki67 (red) expression (b) in MCF-7 cells treated with 100 and 300 μM CoCl2 compared to untreated control cells (day 6 of culture). Immunofluorescence analysis of Ki67 was performed after 6 days of CoCl2 treatment. Nuclei were stained with DAPI. Scale bars indicate 200 μm. c Flow cytometric analysis of MCF-7 cell cycle distribution through propidium iodide (PI) staining intensity in cells treated with 300 μM CoCl2 for 6 days compared to untreated cells (day 6 of culture). Data were analyzed by Modfit LT software (* P < 0.001 compared to untreated control)

Article Snippet: Blocking and permeabilization were performed in blocking buffer, phosphate buffered saline (PBS) containing 10% Normal Goat Serum (Thermo Fisher Scientific) and 0.3% Triton X-100 (Sigma Aldrich), for 1 h. Then, the cells were stained with rabbit anti-human Ki67 (D3B5; 1:400 dilution, Cell Signaling Technology), mouse anti-human HIF1α (54/HIF1α, 1:200, BD Biosciences), or mouse anti-human GLUT1 (SPM498, 1:200, Thermo Fisher Scientific) in antibody dilution buffer, PBS containing 1% bovine serum albumin (Sigma Aldrich) and 0.3% Triton X-100, overnight at 4 °C.

Techniques: Inhibition, Fluorescence, Marker, Expressing, Control, Immunofluorescence, Staining, Software

Journal: Journal of biological engineering

Article Title: A facile in vitro platform to study cancer cell dormancy under hypoxic microenvironments using CoCl 2 .

doi: 10.1186/s13036-018-0106-7

Figure Lengend Snippet: Fig. 2 Quiescent MCF-7 cells can resume growth upon removal of CoCl2. a Cell growth analysis of MCF-7 cells treated with 300 μM CoCl2 for 6 days (from day 4 to day 10) compared to untreated control cells. Cells recovered in normal growth media after the 6-day CoCl2 treatment. b Representative fluorescence images of cycling marker Ki67 (red) and nuclei (blue) in MCF-7 cells after 6 days of treatment with 300 μM CoCl2, 12 days of treatment with 300 μM CoCl2, or 6 days of treatment with 300 μM CoCl2 followed by 6 days of recovery. The untreated control consists of cells at day 4, prior to CoCl2 treatment. Scale bars indicate 200 μm

Article Snippet: Blocking and permeabilization were performed in blocking buffer, phosphate buffered saline (PBS) containing 10% Normal Goat Serum (Thermo Fisher Scientific) and 0.3% Triton X-100 (Sigma Aldrich), for 1 h. Then, the cells were stained with rabbit anti-human Ki67 (D3B5; 1:400 dilution, Cell Signaling Technology), mouse anti-human HIF1α (54/HIF1α, 1:200, BD Biosciences), or mouse anti-human GLUT1 (SPM498, 1:200, Thermo Fisher Scientific) in antibody dilution buffer, PBS containing 1% bovine serum albumin (Sigma Aldrich) and 0.3% Triton X-100, overnight at 4 °C.

Techniques: Control, Fluorescence, Marker

Journal: Journal of biological engineering

Article Title: A facile in vitro platform to study cancer cell dormancy under hypoxic microenvironments using CoCl 2 .

doi: 10.1186/s13036-018-0106-7

Figure Lengend Snippet: Fig. 4 Induction of dormancy by CoCl2 is dependent on HIF1α. a Cell growth analysis of HIF1α-suppressed MCF-7 cells treated with 300 μM CoCl2 for 6 days (from day 4 to day 10) compared to untreated HIF1α-suppressed MCF-7 cells. Treated and untreated MCF-7 cells transduced with shRNA containing a scrambled sequence served as additional control groups. b Representative fluorescence images of Ki67 (red) and nuclei (blue) in HIF1α-suppressed MCF-7 cells (top) and MCF-7 cells transduced with scrambled shRNA (bottom) after 4-day treatment with 300 μM CoCl2. Scale bars indicate 200 μm

Article Snippet: Blocking and permeabilization were performed in blocking buffer, phosphate buffered saline (PBS) containing 10% Normal Goat Serum (Thermo Fisher Scientific) and 0.3% Triton X-100 (Sigma Aldrich), for 1 h. Then, the cells were stained with rabbit anti-human Ki67 (D3B5; 1:400 dilution, Cell Signaling Technology), mouse anti-human HIF1α (54/HIF1α, 1:200, BD Biosciences), or mouse anti-human GLUT1 (SPM498, 1:200, Thermo Fisher Scientific) in antibody dilution buffer, PBS containing 1% bovine serum albumin (Sigma Aldrich) and 0.3% Triton X-100, overnight at 4 °C.

Techniques: Transduction, shRNA, Sequencing, Control, Fluorescence

Journal: Journal of biological engineering

Article Title: A facile in vitro platform to study cancer cell dormancy under hypoxic microenvironments using CoCl 2 .

doi: 10.1186/s13036-018-0106-7

Figure Lengend Snippet: Fig. 5 CoCl2 treatment also induces dormancy in OVCAR-3 cells. a Representative fluorescence images of HIF1α and GLUT1 expression in OVCAR- 3 cells after 72 h of 100 μM CoCl2 treatment compared to untreated control cells. Scale bars indicate 200 μm. b Cell growth analysis of OVCAR-3 cells treated with 100 μM CoCl2 for 6 days (from day 6 to day 12) compared to untreated cells. Cells recovered in normal growth media after the 6-day CoCl2 treatment. c Percentages of Ki67-positive cells in untreated control, 6-day 100 μM CoCl2 treatment, and 6-day 100 μM CoCl2 treatment followed by 6-day recovery in normal growth media (* P < 0.001 compared to untreated control; # P < 0.001 compared to 6-day CoCl2 treatment). Quantification was performed with ImageJ software. d Representative fluorescence images of Ki67 (red), GLUT1 (green) and nuclei (blue) in OVCAR-3 cells in untreated control, 6-day 100 μM CoCl2 treatment, and 6-day 100 μM CoCl2 treatment followed by 6-day recovery in normal growth media. Scale bars indicate 200 μm

Article Snippet: Blocking and permeabilization were performed in blocking buffer, phosphate buffered saline (PBS) containing 10% Normal Goat Serum (Thermo Fisher Scientific) and 0.3% Triton X-100 (Sigma Aldrich), for 1 h. Then, the cells were stained with rabbit anti-human Ki67 (D3B5; 1:400 dilution, Cell Signaling Technology), mouse anti-human HIF1α (54/HIF1α, 1:200, BD Biosciences), or mouse anti-human GLUT1 (SPM498, 1:200, Thermo Fisher Scientific) in antibody dilution buffer, PBS containing 1% bovine serum albumin (Sigma Aldrich) and 0.3% Triton X-100, overnight at 4 °C.

Techniques: Fluorescence, Expressing, Control, Software

Journal: Journal of biological engineering

Article Title: A facile in vitro platform to study cancer cell dormancy under hypoxic microenvironments using CoCl 2 .

doi: 10.1186/s13036-018-0106-7

Figure Lengend Snippet: Fig. 6 In vitro cellular responses to hypoxia in ER-negative MDA-MB-231 cells can be mimicked by CoCl2. a Cell growth analysis of MDA-MB-231 cells treated with CoCl2 (100, 300 and 500 μM) or true hypoxic conditions (0.1% O2) for 6 days (from day 2 to day 8) compared to untreated cells. b Representative fluorescence images of Ki67 (red) and nuclei (blue) in MDA-MB-231 cells after 4 days of treatment with 300 μM CoCl2 or 0.1% O2, compared to untreated control cells (day 2 of culture). c Representative fluorescence images of Ki67 (red) and nuclei (blue) in MDA-MB-231 cells at day 6 (after 4-day CoCl2 treatment) compared to untreated control cells (day 2 of culture). Scale bars indicate 200 μm

Article Snippet: Blocking and permeabilization were performed in blocking buffer, phosphate buffered saline (PBS) containing 10% Normal Goat Serum (Thermo Fisher Scientific) and 0.3% Triton X-100 (Sigma Aldrich), for 1 h. Then, the cells were stained with rabbit anti-human Ki67 (D3B5; 1:400 dilution, Cell Signaling Technology), mouse anti-human HIF1α (54/HIF1α, 1:200, BD Biosciences), or mouse anti-human GLUT1 (SPM498, 1:200, Thermo Fisher Scientific) in antibody dilution buffer, PBS containing 1% bovine serum albumin (Sigma Aldrich) and 0.3% Triton X-100, overnight at 4 °C.

Techniques: In Vitro, Fluorescence, Control

Journal: Journal of biological engineering

Article Title: A facile in vitro platform to study cancer cell dormancy under hypoxic microenvironments using CoCl 2 .

doi: 10.1186/s13036-018-0106-7

Figure Lengend Snippet: Fig. 7 Differential hypoxic regulation of dormancy in MCF-7 and MDA-MB-231 cells can be recapitulated by CoCl2 in 3D culture models. a-c Representative fluorescence images of cycling marker Ki67 (red) and nuclei (blue) in MCF-7 cells embedded in collagen gels (a) or grown in pHEMA-coated plates (b), and quantification of the percentage of Ki67-positive cells (c) in each condition: untreated, 3-day 300 μM CoCl2 treatment, and 3-day 300 μM CoCl2 treatment followed by 3-day recovery in normal growth media (* P < 0.001 compared to untreated control; # P < 0.005 compared to 3-day CoCl2 treatment). d-f Representative fluorescence images of cycling marker Ki67 (red) and nuclei (blue) in MDA-MB-231 cells embedded in collagen gels (d) or grown in pHEMA-coated plates (e), and quantification of the percentage of Ki67-positive cells (f) in each condition: untreated, 3-day 300 μM CoCl2 treatment, and 6-day 300 μM CoCl2 treatment. Quantification was performed with ImageJ software. Scale bars indicate 200 μm

Article Snippet: Blocking and permeabilization were performed in blocking buffer, phosphate buffered saline (PBS) containing 10% Normal Goat Serum (Thermo Fisher Scientific) and 0.3% Triton X-100 (Sigma Aldrich), for 1 h. Then, the cells were stained with rabbit anti-human Ki67 (D3B5; 1:400 dilution, Cell Signaling Technology), mouse anti-human HIF1α (54/HIF1α, 1:200, BD Biosciences), or mouse anti-human GLUT1 (SPM498, 1:200, Thermo Fisher Scientific) in antibody dilution buffer, PBS containing 1% bovine serum albumin (Sigma Aldrich) and 0.3% Triton X-100, overnight at 4 °C.

Techniques: Fluorescence, Marker, Control, Software

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Indomethacin Treatment Post-irradiation Improves Mouse Parotid Salivary Gland Function via Modulation of Prostaglandin E 2 Signaling

doi: 10.3389/fbioe.2021.697671

Figure Lengend Snippet: Indomethacin treatment enhances amylase levels and reduces compensatory proliferation in whole parotid glands at day 30 post-IR. (A,B) C57BL/6J or (C-F) FVB mice were untreated (UT) or received 5 Gy ionizing radiation (IR) with intraperitoneal injections of vehicle (Veh, saline with 10% ethanol) or indomethacin (Indo, 1 mg/kg body weight) at days 3, 5, and 7 post-IR. Parotid glands were extracted at day 30 post-IR. (A,B) Immunoblots were generated from tissue lysates and an anti-amylase antibody, which were stripped and re-probed for ERK1/2 as a loading control. (A) Representative Western blot images of changes in amylase levels following radiation and indomethacin treatments. (B) Densitometry was performed using ImageJ software and protein content was normalized to the average of the UT + Veh group. (C–F) Parotid glands were fixed, sectioned and immunohistochemistry was performed with (C,D) an anti-amylase antibody or (E,F) an anti-Ki67 antibody, as described in section “Materials and Methods.” (C) Representative images of amylase positive area (40× magnification, scale bar: 50 μm). (D) Percent positive amylase area was determined using ImageJ software. The graph represents the amylase positive area as a percentage of the total area. (E) Representative images of Ki67 positive area (white arrows indicate Ki67+, DAPI+ cells; 40× magnification, scale bar: 50 μm). (F) Ki67 positive and total cell numbers were manually counted from 5 fields of view/mouse. The graph represents the Ki67 positive cell number as a percentage of the total cell number. (B,D,F) Graphs represent the mean ± SEM. Each closed circle represents an independent sample. Significant differences were determined via a one-way ANOVA followed by Bonferroni’s post hoc comparisons (** p < 0.01, **** p < 0.0001; ns = not significant).

Article Snippet: Sections were washed with PBS three times for 5 min each, blocked in 0.5% New England nuclear blocking agent (Perkin Elmer, no. 2346249) for 1 h at room temperature and then incubated in anti-amylase antibody (1:1,000 in 1% BSA; Sigma, no. A8273) or anti-Ki67 antibody [1:400 in 1% BSA, Cell Signaling, no. 9129] overnight at 4°C.

Techniques: Saline, Western Blot, Generated, Control, Software, Immunohistochemistry

Journal: STAR Protocols

Article Title: Generation of glioblastoma patient-derived organoids and mouse brain orthotopic xenografts for drug screening

doi: 10.1016/j.xpro.2021.100345

Figure Lengend Snippet:

Article Snippet: SOX2 antibody (1:400) , Cell Signaling Technology , 23064.

Techniques: Recombinant, Membrane, Saline, Cell Recovery, SYBR Green Assay, Microscopy, Imaging, Adhesive, Control, Ointment, Cream, Software