Journal: Oncology Reports

Article Title: HSH2D contributes to methotrexate resistance in human T-cell acute lymphoblastic leukaemia

doi: 10.3892/or.2020.7772

Figure Lengend Snippet: Effect of MTX on the biological function of HUT-78 cells. (A) Chemical formula of MTX. (B) Effect of different concentrations of MTX on cell survival. (C) Flow cytometric analysis of (D) the effects of different concentrations of MTX on cells effects of apoptosis. Data are presented as the mean ± SD, n=3, and were analysed using a one-way ANOVA with Tukey's test. *P<0.05, **P<0.01, ***P<0.005 vs. NC. MTX, methotrexate; NC, negative control.

Article Snippet: Cell apoptosis was evaluated using Annexin V FITC/PI apoptosis detection kit (Beyotime Institute of Biotechnology; cat. no. C1062).

Techniques: Negative Control

Journal: Oncology Reports

Article Title: HSH2D contributes to methotrexate resistance in human T-cell acute lymphoblastic leukaemia

doi: 10.3892/or.2020.7772

Figure Lengend Snippet: HSH2D is essential for MTX resistance of HuT-78 cells. (A) Silencing efficacy was evaluated using reverse transcription-quantitative PCR after transfection of two siRNAs and a NC of HSH2D. (B) Knockdown of HSH2D promoted apoptosis induced by MTX treatment in both MTX-resistant cell lines. (C) IC 50 value of MTX was detected for both sensitive and resistant cells using a Cell Counting Kit-8 assay. (D) EdU results of the cell proliferation of drug resistant strains with HSH2D knockdown. (E) Flow cytometry was used to detected (F) the cell cycle in drug resistant strains with HSH2D knockdown. Data are presented as the mean ± SD, n=3, and were analysed using a two-tailed Student's t-test or one-way ANOVA. *P<0.05 and **P<0.01 vs. siNC, siRNA negative control; MTX, methotrexate; HSH2D, haematopoietic SH2 domain containing.

Article Snippet: Cell apoptosis was evaluated using Annexin V FITC/PI apoptosis detection kit (Beyotime Institute of Biotechnology; cat. no. C1062).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, Knockdown, Cell Counting, Flow Cytometry, Two Tailed Test, Negative Control

Journal: Molecular and Cellular Biology

Article Title: Adiponectin Secretion Is Regulated by SIRT1 and the Endoplasmic Reticulum Oxidoreductase Ero1-Lα

doi: 10.1128/mcb.02279-06

Figure Lengend Snippet: FIG. 2. Perturbation of SIRT1 activity affects secretion of HMW forms of adiponectin. 3T3-L1 adipocytes (4 days) were exposed to either resveratrol (50 M) or nicotinamide (5 mM) in standard DMEM containing 10% FBS for 2 days. The cells were then cultured in fresh DMEM overnight, at which time the medium and total cell layer were harvested for Western blot analysis of intra- and extracellular proteins on reducing (A) and nonreducing (B) SDS-PAGE as outlined in Materials and Methods.

Article Snippet: The antibodies employed in the analysis were as follows: mouse polyclonal antiadiponectin antibody (Affinity BioReagents, Golden, CO), polyclonal anti-SIRT1 antibody (Upstate), anti-PPAR and anti-C/EBP (Santa Cruz Biotechnology), anti-Ero1 polyclonal antibody (Abnova Co., Taiwan, China), and polyclonal anti-aP2 serum (David Benlohr, University of Minnesota).

Techniques: Activity Assay, Cell Culture, Western Blot, SDS Page

Journal: Molecular and Cellular Biology

Article Title: Adiponectin Secretion Is Regulated by SIRT1 and the Endoplasmic Reticulum Oxidoreductase Ero1-Lα

doi: 10.1128/mcb.02279-06

Figure Lengend Snippet: FIG. 4. Inhibition of SIRT1 expression enhances secretion of HMW adiponectin. 3T3-L1 preadipocytes expressing a control vector or SIRT1 siRNA were differentiated for the indicated number of days, and medium (extracellular) as well as the total cell layer (intracellular) was harvested for Western blot analysis of proteins on reducing (A) or nonreducing (B) SDS-PAGE employing antibodies to the following proteins: SIRT1, PPAR, C/EBP, adiponectin, adipsin, and aP2/FABP4.

Article Snippet: The antibodies employed in the analysis were as follows: mouse polyclonal antiadiponectin antibody (Affinity BioReagents, Golden, CO), polyclonal anti-SIRT1 antibody (Upstate), anti-PPAR and anti-C/EBP (Santa Cruz Biotechnology), anti-Ero1 polyclonal antibody (Abnova Co., Taiwan, China), and polyclonal anti-aP2 serum (David Benlohr, University of Minnesota).

Techniques: Inhibition, Expressing, Control, Plasmid Preparation, Western Blot, SDS Page

Journal: Molecular and Cellular Biology

Article Title: Adiponectin Secretion Is Regulated by SIRT1 and the Endoplasmic Reticulum Oxidoreductase Ero1-Lα

doi: 10.1128/mcb.02279-06

Figure Lengend Snippet: FIG. 6. SIRT1 regulates expression of the ER oxidoreductase Ero1-L. (A) 3T3-L1 preadipocytes were differentiated for the indicated days, and total cell RNA was extracted for RT-PCR analysis of mRNAs corresponding to PPAR, C/EBP, adiponectin, Ero1-L, FABP4/aP2, and GAPDH. (B and C) 3T3-L1 preadipocytes expressing a control vector (-) or a SIRT1 siRNA () were differentiated for 8 days for RT-PCR analysis of mRNAs (B) or for the indicated days for Western blot analysis of intracellular proteins (C). (D) Control and SIRT1 siRNA-expressing 3T3-L1 preadipocytes were differentiated in the absence (Con) or presence of either a PPAR antagonist (Antag; 10 M T0070907) or PPAR agonist (Trog; 5 M troglitazone), and the medium (extracellular) and total cell layer (intracellular) were harvested for Western blot analysis of the indicated proteins.

Article Snippet: The antibodies employed in the analysis were as follows: mouse polyclonal antiadiponectin antibody (Affinity BioReagents, Golden, CO), polyclonal anti-SIRT1 antibody (Upstate), anti-PPAR and anti-C/EBP (Santa Cruz Biotechnology), anti-Ero1 polyclonal antibody (Abnova Co., Taiwan, China), and polyclonal anti-aP2 serum (David Benlohr, University of Minnesota).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Plasmid Preparation, Western Blot

Journal: Cancer Communications

Article Title: Targeting autophagy overcomes cancer‐intrinsic resistance to CAR‐T immunotherapy in B‐cell malignancies

doi: 10.1002/cac2.12525

Figure Lengend Snippet: Autophagy limits CAR‐T cell‐mediated cytotoxicity by suppressing TNF‐α induced apoptosis. (A) Volcano plot showing varied genes in Nalm6 cells with the addition of vehicle (as control) and autophinib (log 2 FC > 1 and P < 0.05). (B) Volcano plot showing varied genes between sgControl and indicated RB1CC1 KO Nalm6 cells (log 2 FC > 1 and P < 0.05). (C) KEGG pathway enrichment analysis of varied genes identified in RNA sequencing for Nalm6 cells treated with vehicle (as control) and autophinib. (D) Western blotting showing the expression levels of Caspase‐8, Cleaved caspase‐8, Caspase‐9, Cleaved caspase‐9 and p62 proteins after the addition of vehicle (as control), autophinib and SAR405 in Nalm6 and Raji cells when co‐cultured with or without CD19 CAR‐T cells. GAPDH was used as a loading control. (E) Western blotting showing the expression levels of Caspase‐8, Cleaved caspase‐8, Caspase‐9, Cleaved caspase‐9 and p62 proteins in sgControl and indicated gene‐KO (sgBECN1, sgRB1CC1) Nalm6 and Raji cells when co‐cultured with or without CD19 CAR‐T cells. GAPDH was used as a loading control. (F) Expression of TNFRSF1A mRNA by RT‐qPCR and TNFR1 protein by western blotting in Nalm6 and Raji cells after addition of vehicle (as control), autophinib and SAR405 ( n = 3). Values are shown as the mean ± SD. Statistical differences among three groups in each cell line are calculated with one‐way ANOVA with tests. (G‐H) Cytotoxic analysis of sgControl and indicated gene‐KO (sgTNFRSF1A) Nalm6 and Raji cells co‐cultured with CD19 CAR‐T cells (E:T ratio = 1:4) when treated with vehicle (as control), autophinib and SAR405 and then with or without TNF‐block ( n = 3). Values are shown as the mean ± SD. Statistical differences are calculated with two‐way ANOVA with tests. (I) Cytotoxic analysis of sgControl and indicated gene‐KO (sgBECN1, sgRB1CC1, sgTNFRSF1A) Nalm6 and Raji cells co‐cultured with CD19 CAR‐T cells (E: T ratio = 1: 4) when treated with or without TNF block ( n = 3). Values are shown as the mean ± SD. Statistical differences are calculated with two‐way ANOVA with tests. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0,001; ns: not significant. Abbreviations: ANOVA analysis of variance; CAR‐T chimeric antigen receptor T; E:T effector:target; FC fold change; FC fold change; KO knockout; KEGG Kyoto Encyclopedia of Genes and Genomes; RT‐qPCR real‐time quantitative polymerase chain reaction; ns: not significant; SD standard deviation; sg single guide; TNF tumor necrosis factor; TNFR1 tumor necrosis factor receptor 1.

Article Snippet: The following antibodies were used for Western blotting: ATG3 (Abcam, ab108282, 1:500, Oxford, England), RB1CC1 (Proteintech, 17250‐1‐AP, 1:2,000, Rosemont, USA), BECN1 (Proteintech, 11306‐1‐AP, 1:1,000, Rosemont, USA), p62 (Cell Signaling, #5114, 1:1,000, Boston, IL, USA), LC3B (Cell Signaling, #43566, 1:1,000, Boston, IL, USA), TNFR1 (Proteintech, 21574‐1‐AP, 1:1,000, Rosemont, USA), pSTAT1 (Cell Signaling, #9167, 1:1,000, Boston, IL, USA), STAT1 (Cell Signaling, #14994, 1:1,000, Boston, IL, USA), IRF1 (Proteintech, 11335‐1‐AP, 1:1,000, Rosemont, USA), IRF4 (Proteintech, 11247‐1‐AP, 1:1,000, Rosemont, USA), IRF7 (Abcam, ab238137, 1:1,000, Oxford, England), Caspase‐8 (Proteintech, 66093‐1‐Ig, 1:1,000, Rosemont, USA), Caspase‐9 (Proteintech, 66169‐1‐Ig, 1:1,000, Rosemont, USA), cleaved Caspase‐8 (Cell Signaling, # 9748, 1:1,000, Boston, IL, USA), cleaved Caspase‐9 (Abcam, ab2324, 1:1,000, Oxford, England) and GAPDH (Proteintech, 60004‐1‐Ig, 1:8,000, Rosemont, USA).

Techniques: Control, RNA Sequencing, Western Blot, Expressing, Cell Culture, Quantitative RT-PCR, Blocking Assay, Knock-Out, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Cancer Communications

Article Title: Targeting autophagy overcomes cancer‐intrinsic resistance to CAR‐T immunotherapy in B‐cell malignancies

doi: 10.1002/cac2.12525

Figure Lengend Snippet: STAT1/IRF1 axis mediates the upregulation of CXCL10 and CXCL11 induced by autophagy targeting. (A‐B) Western blotting showing the expression levels of STAT1, pSTAT1, and IRF1 proteins in Nalm6 and Raji cells after the addition of autophagy inhibitors or the knockout of RB1CC1 ( n = 3). GAPDH was used as a loading control. The histograms showing the expression of STAT1 and IRF1 mRNA by RT‐qPCR quantification. Values are shown as the mean ± SD. Statistical differences are calculated with one‐way ANOVA tests. (C‐D) Western blotting showing the expression levels of STAT1 and IRF1 proteins in Nalm6 and Raji cells after the addition of autophagy inhibitors and the silencing of STAT1 ( n = 3). GAPDH was used as a loading control. The histograms showing the expression of STAT1 and IRF1 mRNA by RT‐qPCR quantification. Values are shown as the mean ± SD. Statistical differences are calculated with one‐way ANOVA tests. (E‐F) Expression of CXCL10 and CXCL11 mRNA by RT‐qPCR and ELISA quantification of CXCL10 and CXCL11 protein levels in the supernatants of shControl, shSTAT1 and shIRF1 Nalm6 and Raji cells ( n = 3). Values are shown as the mean ± SD. Statistical differences are calculated with two‐way ANOVA tests. (G‐H) ChIP‐qPCR showing the binding of STAT1 and IRF1 to the promoter region of CXCL10 and CXCL11 ( n = 3). Values are shown as the mean ± SD. Statistical differences for each cell line are calculated with unpaired Student's t tests. (I) Graphic abstract: in the proposed model, inhibition of cancer cell‐autonomous autophagy leads to accumulation of cytosolic DNA, which thereby not only suppresses cancer cell survival by inducing TNFR1‐TNF‐α mediated apoptosis but also promotes the CAR‐T cell recruitment in tumor microenvironment via STAT1/IRF1‐dependent activation of chemokine signaling. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0,001; ns: not significant. Abbreviations: ANOVA analysis of variance; CAR‐T, chimeric antigen receptor T; ChIP, chromatin immunoprecipitation; CXCL CXC, chemokine ligand; ELISA, enzyme‐linked immunosorbent assay; FC, fold change; IRF, interferon regulatory factor; ns: not significant; RT‐qPCR, real‐time quantitative polymerase chain reaction; SD, standard deviation; sh short hairpin; STAT, signal transducers and activators of transcription.

Article Snippet: The following antibodies were used for Western blotting: ATG3 (Abcam, ab108282, 1:500, Oxford, England), RB1CC1 (Proteintech, 17250‐1‐AP, 1:2,000, Rosemont, USA), BECN1 (Proteintech, 11306‐1‐AP, 1:1,000, Rosemont, USA), p62 (Cell Signaling, #5114, 1:1,000, Boston, IL, USA), LC3B (Cell Signaling, #43566, 1:1,000, Boston, IL, USA), TNFR1 (Proteintech, 21574‐1‐AP, 1:1,000, Rosemont, USA), pSTAT1 (Cell Signaling, #9167, 1:1,000, Boston, IL, USA), STAT1 (Cell Signaling, #14994, 1:1,000, Boston, IL, USA), IRF1 (Proteintech, 11335‐1‐AP, 1:1,000, Rosemont, USA), IRF4 (Proteintech, 11247‐1‐AP, 1:1,000, Rosemont, USA), IRF7 (Abcam, ab238137, 1:1,000, Oxford, England), Caspase‐8 (Proteintech, 66093‐1‐Ig, 1:1,000, Rosemont, USA), Caspase‐9 (Proteintech, 66169‐1‐Ig, 1:1,000, Rosemont, USA), cleaved Caspase‐8 (Cell Signaling, # 9748, 1:1,000, Boston, IL, USA), cleaved Caspase‐9 (Abcam, ab2324, 1:1,000, Oxford, England) and GAPDH (Proteintech, 60004‐1‐Ig, 1:8,000, Rosemont, USA).

Techniques: Western Blot, Expressing, Knock-Out, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, ChIP-qPCR, Binding Assay, Inhibition, Activation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Experimental & Molecular Medicine

Article Title: GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes

doi: 10.1038/emm.2016.28

Figure Lengend Snippet: Detection of glucocorticoid-induced transcript 1 (GLCCI1) and podocyte-specific marker gene expression by reverse transcription-PCR (RT-PCR). ( a ) Expression of the genes encoding GLCCI1, nephrin, podocin, synaptopodin and podocalyxin showed similar patterns in the podocytes. The expression levels differed significantly between the high glucose-induced and wortmannin-treated diabetic groups of podocytes. ( b ) The expression of the genes encoding GLCCI1, nephrin, podocin, synaptopodin and podocalyxin showed similar patterns in the kidneys of diabetic rats. In addition, wortmannin ameliorated the expression of all podocyte-specific proteins including GLCCI1. ( c , d ) The relative band intensity of proteins in podocytes and the kidneys of diabetic rats was observed by RT-PCR. The band intensity was measured using the Multi Gauge V3.0 software (Fuji Film). *** P <0.001, 5 m M (control) and wortmannin-treated diabetic groups versus the high glucose–induced group by analysis of variance (ANOVA; means±s.e.m., n =3).

Article Snippet: The membranes were incubated with blocking solution containing a 1:1000 dilution of the rabbit anti-GLCCI1 antibody (ab107491, Abcam, Cambridge, UK), a 1:500 dilution of the goat anti-nephrin antibody (sc-19000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), a 1:500 dilution of the rabbit anti-podocin antibody (sc-21009, Santa Cruz Biotechnology), a 1:1000 dilution of the rabbit anti-SGK1 antibody (ab59337, Abcam) or a 1:1000 dilution of the rabbit anti-FOXO3A antibody (ab47285, Abcam).The membranes were then incubated with blocking solution containing 1:1000 (for GLCCI1, podocin, SGK1 and FOXO3A) dilutions of a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1858415, Pierce Biotechnology, Waltham, MA, USA) or a 1:1000 (for nephrin) dilution of a horseradish peroxidase-conjugated donkey anti-goat IgG secondary antibody (ab6885, Abcam).

Techniques: Marker, Gene Expression, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Expressing, Software, Control

Journal: Experimental & Molecular Medicine

Article Title: GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes

doi: 10.1038/emm.2016.28

Figure Lengend Snippet: Analysis of glucocorticoid-induced transcript 1 (GLCCI1) protein expression and involvement in the phosphoinositide 3-kinase (PI3K) pathway by western blotting. ( a ) GLCCI1, nephrin and podocin were expressed only in the 5 m M and the wortmannin-treated diabetic groups. Serum/glucocorticoid-regulated kinase 1 (SGK1) was expressed only in the high glucose-induced group. Forkhead box O3 (FOXO3A) was regulated by wortmannin. ( b ) GLCCI1 was expressed only in the control and wortmannin-treated diabetic groups. GLCCI1 was significantly decreased in the diabetic group. ( c , d ) The relative band intensities of proteins in the podocytes and the kidneys of diabetic rats were observed by western blot analysis. The band intensities were measured using the Multi Gauge V3.0 software (Fuji Film). *** P <0.001, 5 m M (control) and wortmannin-treated diabetic groups versus the 25 m M (diabetes) group by analysis of variance (ANOVA; means±s.e.m., n =3).

Article Snippet: The membranes were incubated with blocking solution containing a 1:1000 dilution of the rabbit anti-GLCCI1 antibody (ab107491, Abcam, Cambridge, UK), a 1:500 dilution of the goat anti-nephrin antibody (sc-19000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), a 1:500 dilution of the rabbit anti-podocin antibody (sc-21009, Santa Cruz Biotechnology), a 1:1000 dilution of the rabbit anti-SGK1 antibody (ab59337, Abcam) or a 1:1000 dilution of the rabbit anti-FOXO3A antibody (ab47285, Abcam).The membranes were then incubated with blocking solution containing 1:1000 (for GLCCI1, podocin, SGK1 and FOXO3A) dilutions of a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1858415, Pierce Biotechnology, Waltham, MA, USA) or a 1:1000 (for nephrin) dilution of a horseradish peroxidase-conjugated donkey anti-goat IgG secondary antibody (ab6885, Abcam).

Techniques: Expressing, Western Blot, Control, Software

Journal: Experimental & Molecular Medicine

Article Title: GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes

doi: 10.1038/emm.2016.28

Figure Lengend Snippet: The localization of glucocorticoid-induced transcript 1 (GLCCI1), podocyte-specific markers and proteins involved in the phosphoinositide 3-kinase (PI3K) signaling pathway in podocytes. ( a ) In podocytes, GLCCI1 (red, Alexa 568 conjugated) was localized in the 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei. No GLCCI1 localization was observed in the 25 m M D -glucose-treated podocytes. However, GLCCI1 was regulated by wortmannin treatment. ( b – d ) The podocyte-specific proteins nephrin (green, Alexa 488 conjugated), podocin (red) and synaptopodin (green) showed a reactivity pattern similar to GLCCI1 in podocytes treated with wortmannin. ( e ) Serum/glucocorticoid-regulated kinase 1 (SGK1; red) was observed only in the 25 m M group. We confirmed that treatment with wortmannin decreased SGK1 expression. ( f ) Forkhead box O3 (FOXO3A; red) was observed in the 5 m M group. A lower FOXO3A signal was accompanied by high reactivity of SGK1 in the 25 m M group. Localization of FOXO3A was regulated by treatment with wortmannin. Original magnification: × 400. Scale bar, 50 μm.

Article Snippet: The membranes were incubated with blocking solution containing a 1:1000 dilution of the rabbit anti-GLCCI1 antibody (ab107491, Abcam, Cambridge, UK), a 1:500 dilution of the goat anti-nephrin antibody (sc-19000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), a 1:500 dilution of the rabbit anti-podocin antibody (sc-21009, Santa Cruz Biotechnology), a 1:1000 dilution of the rabbit anti-SGK1 antibody (ab59337, Abcam) or a 1:1000 dilution of the rabbit anti-FOXO3A antibody (ab47285, Abcam).The membranes were then incubated with blocking solution containing 1:1000 (for GLCCI1, podocin, SGK1 and FOXO3A) dilutions of a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1858415, Pierce Biotechnology, Waltham, MA, USA) or a 1:1000 (for nephrin) dilution of a horseradish peroxidase-conjugated donkey anti-goat IgG secondary antibody (ab6885, Abcam).

Techniques: Staining, Expressing

Journal: Experimental & Molecular Medicine

Article Title: GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes

doi: 10.1038/emm.2016.28

Figure Lengend Snippet: The localization of glucocorticoid-induced transcript 1 (GLCCI1), podocyte-specific markers and proteins associated with the phosphoinositide 3-kinase (PI3K) signaling pathway in the rat glomerulus. ( a ) Staining of rat kidney sections with an antibody against GLCCI1 (red) is shown. The diabetic group showed low reactivity. However, GLCCI1 was regulated by wortmannin treatment. ( b – d ) The podocyte-specific proteins nephrin (green), podocin (red) and synaptopodin (green) showed a reactivity pattern similar to GLCCI1 following wortmannin treatment. ( e ) Serum/glucocorticoid-regulated kinase 1 (SGK1; red) was observed only in the diabetic group. We confirmed that SGK1 expression was decreased by wortmannin. ( f ) Forkhead box O3 (FOXO3A; red) was observed in the control group. The lower reactivity of FOXO3A was accompanied by the overexpression of SGK1 in the diabetic group. The localization of FOXO3A was regulated by wortmannin treatment. Original magnification: × 400. Scale bar, 50 μm.

Article Snippet: The membranes were incubated with blocking solution containing a 1:1000 dilution of the rabbit anti-GLCCI1 antibody (ab107491, Abcam, Cambridge, UK), a 1:500 dilution of the goat anti-nephrin antibody (sc-19000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), a 1:500 dilution of the rabbit anti-podocin antibody (sc-21009, Santa Cruz Biotechnology), a 1:1000 dilution of the rabbit anti-SGK1 antibody (ab59337, Abcam) or a 1:1000 dilution of the rabbit anti-FOXO3A antibody (ab47285, Abcam).The membranes were then incubated with blocking solution containing 1:1000 (for GLCCI1, podocin, SGK1 and FOXO3A) dilutions of a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1858415, Pierce Biotechnology, Waltham, MA, USA) or a 1:1000 (for nephrin) dilution of a horseradish peroxidase-conjugated donkey anti-goat IgG secondary antibody (ab6885, Abcam).

Techniques: Staining, Expressing, Control, Over Expression

Journal: Experimental & Molecular Medicine

Article Title: GLCCI1 is a novel component associated with the PI3K signaling pathway in podocyte foot processes

doi: 10.1038/emm.2016.28

Figure Lengend Snippet: Colocalization of glucocorticoid-induced transcript 1 (GLCCI1) with the podocyte-specific proteins nephrin and synaptopodin. ( a ) Double labeling with the podocyte-specific marker nephrin (green) showed a complete overlap (merge, yellow) with the distribution of GLCCI1 (red) in the 5 m M and wortmannin-treated diabetic groups of cultured podocytes. ( b ) GLCCI1 (red) also colocalized with synaptopodin (green). We observed overlapping reactivity (yellow indicates red plus green) in the 5 m M and wortmannin-treated diabetic groups of cultured podocytes. ( c , d ) In rat glomeruli, we observed overlapping reactivity (yellow) via the colocalization of GLCCI1 (red) with nephrin and synaptopodin (green). Wortmannin ameliorated the lower reactivity in glomeruli from rats in the diabetic group. Original magnification: × 400. Scale bar, 50 μm.

Article Snippet: The membranes were incubated with blocking solution containing a 1:1000 dilution of the rabbit anti-GLCCI1 antibody (ab107491, Abcam, Cambridge, UK), a 1:500 dilution of the goat anti-nephrin antibody (sc-19000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), a 1:500 dilution of the rabbit anti-podocin antibody (sc-21009, Santa Cruz Biotechnology), a 1:1000 dilution of the rabbit anti-SGK1 antibody (ab59337, Abcam) or a 1:1000 dilution of the rabbit anti-FOXO3A antibody (ab47285, Abcam).The membranes were then incubated with blocking solution containing 1:1000 (for GLCCI1, podocin, SGK1 and FOXO3A) dilutions of a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1858415, Pierce Biotechnology, Waltham, MA, USA) or a 1:1000 (for nephrin) dilution of a horseradish peroxidase-conjugated donkey anti-goat IgG secondary antibody (ab6885, Abcam).

Techniques: Labeling, Marker, Cell Culture

Journal: Aging (Albany NY)

Article Title: ASIC1 and ASIC3 mediate cellular senescence of human nucleus pulposus mesenchymal stem cells during intervertebral disc degeneration

doi: 10.18632/aging.202850

Figure Lengend Snippet: The effect of acidic culture conditions on senescence-related protein expression in primary cultured human NP-MSCs. Western blotting analyses were performed against Rb1, p53, p27, p21, p16, Aggrecan and Collagen II, MMP9 and MMP3 expression in Pfirrmann grade III NP-MSCs exposed to pH 6.6 culture conditions with or without treatment using the inhibitors Amiloride, PcTx1 or APETx2, respectively. The samples were from case 5, 9 and 12, respectively.

Article Snippet: Membranes were blocked with 5% skim milk in Tris-Buffered Saline-Tween-20 (TBST) for 1.5 h before incubating with diluted primary antibodies at 4° C overnight as follows: ASIC1 (Abcam ab240896, 1:1000), ASIC2 (Proteintech 17851-1-AP, 1:1000), ASIC3 (Alomone ASC018AN0702, 1:200), ASIC4 (Proteintech 12003-1-AP, 1:1000), p16 (Santa Cruz sc-166760, 1:500), p21 (Proteintech 10355-1-AP, 1:500), p53 (Proteintech 10442-1-AP, 1:1000), pRb1 (Proteintech 10048-2-Ig, 1:500), MMP3 (Proteintech 6338-1-Ig, 1:500), MMP9 (Proteintech 10375-2-AP, 1:500), Aggrecan (Proteintech 13880-1-AP, 1:500), Collagen II (Proteintech 15943-1-AP, 1:500) and β-actin (Proteintech 66009-1-lg, 1:10000).

Techniques: Expressing, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: A coregulatory influence map of glioblastoma heterogeneity and plasticity – From cells in vitro to tumors and back again

doi: 10.1101/2024.08.23.609303

Figure Lengend Snippet: (A) Analysis of PAX8 Protein Expression in U87MG, U87MG-R50, and U87MG-OFF Cells Representative Western blot detection of PAX8 protein and its predicted activity based on the regulatory network (colored dots under the WB), along with quantitative analysis (n>3) of its relative protein level (Top panel). Bottom panel: detection of the PAX8 protein by immunofluorescence in the same samples analyzed by WB. (B) Top panel shows the mRNA expression levels of NKX2.5 in primary and recurrent gliomas, as analyzed using the CGGA (Chinese Glioma Genome Atlas, http://www.cgga.org.cn/index.jsp ) cohort. NKX2.5 expression is significantly higher in grade IV gliomas compared to grade III and grade II gliomas. Bottom panel illustrates the survival analysis of recurrent GBM patients (grade IV) based on NKX2.5 expression levels. (C) NKX2.5 Mediated Transcriptional Repression of Glycolytic Genes. Top Panel: Functional enrichment analysis of the repressed and activated genes of NKX2.5 using clusterProfiler. The plot visualizes the enriched Gene Ontology Biological Processes (GO-BP) terms, with the size of each dot representing the gene count and the color indicating the adjusted p-value, reflecting the significance of the enrichment. Middle Panel: RT-qPCR analysis showing the reduced expression of glycolytic genes (HK2, GPI, GAPDH, LDHA) in U87MG cells upon overexpression of NKX2.5. Bottom Panel: Western Blot Analysis of NKX2.5 and HK2 protein expression in U87MG cells in 4 conditions: control (untreated cells), lipofectamine treated cells, negative control plasmid and NKX2.5 plasmid transfected cells. (D) Metabolic signatures of assigned classes for each cell line (U87MG, U87MG-R50, and U87MG-OFF) and energy metabolism characterization using Seahorse XFp extracellular flux analysis. This includes ATP production rates and the contribution of glycolysis and OXPHOS, as well as an energy map, depicting both Oxygen Consumption Rates (OCR) and Extracellular Acidification Rates (ECAR). All Seahorse data are represented as means from three independent experiments ± SEM.

Article Snippet: The primary antibodies used were antibodies for NKX2-5 (Cell Signaling, cat #8792, 1:500), HK2 (Proteintech, cat #22029-1-AP, 1:5000) and α-Tubulin (cat #T9026, Sigma-Aldrich, 1:1000 dilution).

Techniques: Expressing, Western Blot, Activity Assay, Immunofluorescence, Functional Assay, Quantitative RT-PCR, Over Expression, Control, Negative Control, Plasmid Preparation, Transfection

Journal: International Journal of Molecular Medicine

Article Title: circNRIP1 facilitates keloid progression via FXR1-mediated upregulation of miR-503-3p and miR-503-5p

doi: 10.3892/ijmm.2021.4903

Figure Lengend Snippet: circNRIP1 is upregulated in keloid tissue and is associated with keloid progression. RT-qPCR assay was performed to detect the abundance of circNRIP1 in (A) keloid and adjacent normal skin tissue and (B) keloid-derived and normal human fibroblasts. (C) Knockdown efficiency of circNRIP1 was evaluated in keloid-derived fibroblasts by RT-qPCR. (D) Cell Counting Kit-8 assay was used to determine the viability of keloid-derived fibroblasts in the presence or absence circNRIP1 knockdown. Colony-forming ability of keloid-derived fibroblasts was (E) assessed and (F) quantified. Flow cytometric assays were conducted to evaluate the effect of circNRIP1 on (G and H) apoptosis and (I and J) cell cycle progression of keloid-derived fibroblasts. (K and L) Changes in the expression of extracellular matrix-associated regulatory proteins in keloid-derived fibroblasts were determined by western blot assay. ** P<0.01 vs. Ctrl or human normal fibroblasts. circNRIP1, circular nuclear receptor interacting protein 1; RT-q, reverse transcription-quantitative; Ctrl, control; si, small interfering; OD, optical density.

Article Snippet: For cell apoptosis, keloid-derived fibroblasts underwent double staining with 20 ng/l FITC-Annexin V (10 μ l, 30 min, 25°C) then 50 ng/l propidium iodide (5 μ l, 5 min, 25°C) using the Annexin V-FITC cell apoptosis detection kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions.

Techniques: Quantitative RT-PCR, Derivative Assay, Knockdown, Cell Counting, Expressing, Western Blot, Reverse Transcription, Control

Journal: International Journal of Molecular Medicine

Article Title: circNRIP1 facilitates keloid progression via FXR1-mediated upregulation of miR-503-3p and miR-503-5p

doi: 10.3892/ijmm.2021.4903

Figure Lengend Snippet: FXR1 promotes keloid-derived fibroblast proliferation and accumulation of extracellular matrix but inhibits apoptosis. (A and B) Knockdown efficiency of FXR1 was investigated in keloid-derived fibroblasts via western blot analysis. (C) Viability of keloid-derived fibroblasts in the presence or absence of FXR1 knockdown was detected by Cell Counting Kit-8 assay. (D and E) Proliferation of keloid-derived fibroblasts was determined by colony formation assay. (F and G) Cell apoptosis and (H and I) cell cycle analysis were determined via flow cytometric assays in siFXR1-transfected keloid-derived fibroblasts. (J and K) Expression levels of extracellular matrix-associated proteins were determined in siFXR1-transfected keloid-derived fibroblasts by western blot assay. ** P<0.01 vs. Ctrl. FXR1, Fbxo4-mediated FMR1 autosomal homolog 1; si, small interfering; Ctrl, control; OD, optical density; SMA, smooth muscle actin.

Article Snippet: For cell apoptosis, keloid-derived fibroblasts underwent double staining with 20 ng/l FITC-Annexin V (10 μ l, 30 min, 25°C) then 50 ng/l propidium iodide (5 μ l, 5 min, 25°C) using the Annexin V-FITC cell apoptosis detection kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions.

Techniques: Derivative Assay, Knockdown, Western Blot, Cell Counting, Colony Assay, Cell Cycle Assay, Transfection, Expressing, Control

Journal: International Journal of Molecular Medicine

Article Title: circNRIP1 facilitates keloid progression via FXR1-mediated upregulation of miR-503-3p and miR-503-5p

doi: 10.3892/ijmm.2021.4903

Figure Lengend Snippet: Mature miR-503 induces keloid-derived fibroblast proliferation and accumulation of extracellular matrix but inhibits apoptosis. (A) Knockdown efficiency of the miR-503-3p and miR-503-5p specific inhibitors was investigated in keloid-derived fibroblasts via reverse transcription-quantitative PCR. Viability of keloid-derived fibroblasts transfected with (B) miR-503-3p or (C) miR-503-5p inhibitor was determined by Cell Counting Kit-8 assay. (D and E) Proliferation of miR-503-3p or miR-503-5p inhibitor-transfected keloid-derived fibroblasts was measured by colony formation assay. (F and G) Apoptosis and (H) cell cycle progression in keloid-derived fibroblasts transfected with (I) miR-503-3p or (J) miR-503-5p inhibitor was assessed by flow cytometry. Western blot assays were used to determine the expression levels of extracellular matrix-associated proteins in keloid-derived fibroblasts transfected with (K and L) miR-503-3p or (M and N) miR-503-5p inhibitor. ** P<0.01 vs. Ctrl. miR, microRNA; Ctrl, control; OD, optical density; SMA, smooth muscle actin.

Article Snippet: For cell apoptosis, keloid-derived fibroblasts underwent double staining with 20 ng/l FITC-Annexin V (10 μ l, 30 min, 25°C) then 50 ng/l propidium iodide (5 μ l, 5 min, 25°C) using the Annexin V-FITC cell apoptosis detection kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions.

Techniques: Derivative Assay, Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, Cell Counting, Colony Assay, Flow Cytometry, Western Blot, Expressing, Control

Journal: International Journal of Molecular Medicine

Article Title: circNRIP1 facilitates keloid progression via FXR1-mediated upregulation of miR-503-3p and miR-503-5p

doi: 10.3892/ijmm.2021.4903

Figure Lengend Snippet: Schematic representation of the mechanism by which circNRIP1 promotes keloid progression. circNRIP1 induced pre-miR-503 maturation via protecting FXR1 from Fbxo4-mediated ubiquitination and degradation, which facilitated proliferation and extracellular matrix accumulation, but inhibited apoptosis, in keloid-derived fibroblasts. circNRIP1, circular nuclear receptor interacting protein 1; miR, microRNA; FXR1, Fbxo4-mediated FMR1 autosomal homolog 1; AGO2, argonaute RISC catalytic component 2.

Article Snippet: For cell apoptosis, keloid-derived fibroblasts underwent double staining with 20 ng/l FITC-Annexin V (10 μ l, 30 min, 25°C) then 50 ng/l propidium iodide (5 μ l, 5 min, 25°C) using the Annexin V-FITC cell apoptosis detection kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions.

Techniques: Ubiquitin Proteomics, Derivative Assay

Journal: Radiation Oncology

Article Title: Association of anti-apoptotic Mcl-1L isoform expression with radioresistance of oral squamous carcinoma cells

doi: 10.1186/1748-717x-7-135

Figure Lengend Snippet: Figure 2 Time course profile of Mcl-1 splice variants & apoptosis related proteins post-IR. (a) Expression of Mcl-1L, Mcl-1S transcripts and proteins at different time points post-IR in FBM & AW8507. Post-IR cells were harvested at different time points, used for RT-PCR and Western blotting. (b) Western blot illustrates expression of Bax, Bak, Bcl-xl & Bcl-2 proteins at different time points post-IR in oral cell lines, using β-actin as loading control. A representative blot for three independent experiments is shown.

Article Snippet: Apoptosis detection by flow cytometry The Annexin V-FITC apoptosis detection kit (Santa Cruz Biotechnology, CA) was used for the detection of apoptotic cells in the three oral cell lines, as per the manufacturer’s specifications.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Radiation Oncology

Article Title: Association of anti-apoptotic Mcl-1L isoform expression with radioresistance of oral squamous carcinoma cells

doi: 10.1186/1748-717x-7-135

Figure Lengend Snippet: Figure 4 Apoptosis induction and localization of Mcl-1 post-IR. (a) Percentage of apoptosis induction at different time points post-IR by Annexin-V & PI staining analyzed by FACS. The flow cytometry data shown is representative of three independent experiments (*P < 0.05 & **P < 0.01) (b) Immunofluorescence staining of Mcl-1 protein counterstained with DAPI (blue) shows peri-nuclear accumulation (inset) and additional nuclear localization 4 hrs post-IR in AW8507 cells.

Article Snippet: Apoptosis detection by flow cytometry The Annexin V-FITC apoptosis detection kit (Santa Cruz Biotechnology, CA) was used for the detection of apoptotic cells in the three oral cell lines, as per the manufacturer’s specifications.

Techniques: Staining, Flow Cytometry, Immunofluorescence

Journal: Radiation Oncology

Article Title: Association of anti-apoptotic Mcl-1L isoform expression with radioresistance of oral squamous carcinoma cells

doi: 10.1186/1748-717x-7-135

Figure Lengend Snippet: Figure 6 Microscopic analyses of cell proliferation and apoptosis after different treatment combinations. (a & b) After different treatment combinations as described, cell growth of AW8507 & AW13516 cells were determined by counting cell numbers using Trypan blue dye exclusion assay. Cell numbers are given in percent of untreated controls. Data are means ± SD of three independent experiments. (c & d) Apoptosis was determined by DAPI staining, cells either untreated (Control) or treated with UC siRNA, Mcl-1L siRNA alone or in combination with IR. Apoptotic cells were counted in different fields and shown as percent of apoptosis relative to control. Data are means ± SD of three independent experiments. *indicates P < 0.05, ** P < 0.01.

Article Snippet: Apoptosis detection by flow cytometry The Annexin V-FITC apoptosis detection kit (Santa Cruz Biotechnology, CA) was used for the detection of apoptotic cells in the three oral cell lines, as per the manufacturer’s specifications.

Techniques: Exclusion Assay, Staining, Control