Journal: PLoS ONE

Article Title: Repeated Small Perturbation Approach Reveals Transcriptomic Steady States

doi: 10.1371/journal.pone.0029241

Figure Lengend Snippet: A and B ) Seven genes were chosen randomly from the set of genes with steady-state characteristics for this comparison. egfl5, cnn3, and bcar3 ( A ) exhibit immediate down-regulated responses, while pmaip1, jun, id2, and gadd45a ( B ) exhibit immediate up-regulated responses followed by relaxation. Solid and dash lines represent real-time PCR data and microarray data, respectively. The error bar of the microarray data indicates standard deviation estimated by the log linear model. The error bar of real-time PCR data is the standard deviation derived from three sets of CTs. C ) Scatter plot of real-time PCR results versus microarray data. The correlation coefficient, R is 0.93. The red line is a linear fit through all points. These results clearly demonstrate that even at the small fold change data points, these two sets of data agree very well. It also demonstrates the high reliability and resolution of our cDNA microarray system, which makes this kind of small perturbation study possible.

Article Snippet: The assay ID of corresponding probe sets and primers in the ABI's database are Hs00182488_m1, Hs00156565_m1, Hs00323519_m1, Hs00169255_m1, Hs00747379_m1, Hs01103582_s1, and Hs00382168_m1, respectively.

Techniques: Real-time Polymerase Chain Reaction, Microarray, Standard Deviation, Derivative Assay

Journal: Journal of Inflammation Research

Article Title: Diltiazem Hydrochloride Protects Against Myocardial Ischemia/Reperfusion Injury in a BNIP3L/NIX-Mediated Mitophagy Manner

doi: 10.2147/jir.s493037

Figure Lengend Snippet: Figure 1 The effects of DIL on histological and immunohistochemical analyses of cardiac muscular tissue in rats with MI/R injury. (A) Hematoxylin and eosin (HE) staining of cardiac muscular tissue in the three groups. Original magnification: ×100, scale bar = 100 μm (upper row); ×200, scale bar = 50 μm (lower row). (B) Representative images of immunohistochemical (IHC) staining of DUSP1, JNK, and BNIP3L/NIX in the three groups. (C–E) Comparison of the average IOD values of DUSP1, JNK, and BNIP3L/ NIX expression in myocardial tissue among the three groups determined by IHC analysis. **P < 0.01 relative to the sham group, ##P < 0.01 relative to the MI/R group.

Article Snippet: Next, the sections were incubated with primary antibodies against DUSP1, JNK, and BNIP3L/NIX (1:200, Proteintech, USA) for 1 h at 37°C.

Techniques: Immunohistochemical staining, Staining, Immunohistochemistry, Comparison, Expressing, Paraffin-embedded Immunohistochemistry

Journal: Journal of Inflammation Research

Article Title: Diltiazem Hydrochloride Protects Against Myocardial Ischemia/Reperfusion Injury in a BNIP3L/NIX-Mediated Mitophagy Manner

doi: 10.2147/jir.s493037

Figure Lengend Snippet: Figure 5 RT‒qPCR analysis of the mRNA levels of Beclin 1, DUSP1, JNK, and BNIP3L/NIX in the control, OGD/R, and OGD/R + DIL groups. Compared to those in the control group, the expression levels of Beclin 1 (A), JNK (C) and BNIP3L/NIX (D) were significantly greater in the OGD/R group, but only DUSP1 expression (B) was significantly lower (all P < 0.01). Remarkably, this effect could be reversed by DIL intervention (all P < 0.01). **P < 0.01 relative to the control group; ##P < 0.01 relative to the OGD/R group.

Article Snippet: Next, the sections were incubated with primary antibodies against DUSP1, JNK, and BNIP3L/NIX (1:200, Proteintech, USA) for 1 h at 37°C.

Techniques: Control, Expressing

Journal: Journal of Inflammation Research

Article Title: Diltiazem Hydrochloride Protects Against Myocardial Ischemia/Reperfusion Injury in a BNIP3L/NIX-Mediated Mitophagy Manner

doi: 10.2147/jir.s493037

Figure Lengend Snippet: Figure 6 Western blot analyses of the Beclin 1, LC3B, DUSP1, JNK, and BNIP3L/NIX proteins in the control, OGD/R, and OGD/R + DIL groups. (A) The electrophoresis results of mitophagy-related proteins from the Western blot. The analyses indicated significantly increased protein expression of Beclin 1 (B), LC3B (C), JNK (E), and BNIP3L/NIX (F) and decreased DUSP1 protein expression (D) in the OGD/R group compared to the control group. DIL intervention reversed these changes (all P < 0.05). *P < 0.05, **P < 0.01 relative to the control group; #P < 0.05, ##P < 0.01 relative to the OGD/R group.

Article Snippet: Next, the sections were incubated with primary antibodies against DUSP1, JNK, and BNIP3L/NIX (1:200, Proteintech, USA) for 1 h at 37°C.

Techniques: Western Blot, Control, Electrophoresis, Expressing

Journal: Journal of Inflammation Research

Article Title: Diltiazem Hydrochloride Protects Against Myocardial Ischemia/Reperfusion Injury in a BNIP3L/NIX-Mediated Mitophagy Manner

doi: 10.2147/jir.s493037

Figure Lengend Snippet: Figure 7 RT‒qPCR analysis of the mRNA levels of the DUSP1, JNK, and BNIP3L/NIX genes after treatment with DUSP1, JNK, and BNIP3L/NIX inhibitors or activators in the different groups. (A) The DUSP1 expression level was significantly decreased in the OGD/R+BCI (an inhibitor of DUSP1) group (P < 0.01), and these changes were reversed by DIL in the OGD/R+BCI+DIL group (P < 0.01). (B) Ani (an activator of JNK) significantly upregulated JNK expression in the OGD/R+Ani group (P < 0.01). However, DIL and SP (an inhibitor of JNK) significantly downregulated JNK expression in the OGD/R+DIL and OGD/R+SP groups compared with that in the OGD/R group (P < 0.01). Unexpectedly, compared with that in the OGD/R+Ani group, the JNK expression level did not increase further under DIL intervention but obviously decreased in the OGD/R+Ani+DIL group (P < 0.01), which indicated that DIL-induced DUSP1 partly inhibited JNK expression. (C) MAC (an activator of BNIP3L/NIX) significantly upregulated BNIP3L/NIX expression in the OGD/R+MAC group. Compared with that in the OGD/R+DIL or OGD/R+BAF (an inhibitor of BNIP3L/NIX) group, BNIP3L/ NIX expression in the OGD/R+DIL or OGD/R+BAF group was significantly lower in the DIL and BAF groups (P < 0.01). Moreover, compared with that in the OGD/R +MAC group, the BNIP3L/NIX expression level did not increase further under DIL intervention but obviously decreased in the OGD/R+ MAC+DIL group (P < 0.01), which indicated that DIL-induced DUSP1 partly inhibited BNIP3L/NIX expression. The data are presented as the means ± SDs of three independent experiments performed in duplicate. *P < 0.05, **P < 0.01.

Article Snippet: Next, the sections were incubated with primary antibodies against DUSP1, JNK, and BNIP3L/NIX (1:200, Proteintech, USA) for 1 h at 37°C.

Techniques: Expressing

Journal: Journal of Inflammation Research

Article Title: Diltiazem Hydrochloride Protects Against Myocardial Ischemia/Reperfusion Injury in a BNIP3L/NIX-Mediated Mitophagy Manner

doi: 10.2147/jir.s493037

Figure Lengend Snippet: Figure 8 Western blot analyses of DUSP1, JNK, and BNIP3L/NIX after treatment with DUSP1, JNK and BNIP3L/NIX inhibitors or activators in the different groups. (A and B) Compared with that in the OGD/R group, the DUSP1 gene expression level was significantly upregulated in the OGD/R+DIL and OGD/R+CTB (an activator of DUSP1) groups (P < 0.01). Moreover, the DUSP1 expression level was significantly decreased in the OGD/R+BCI (an inhibitor of DUSP1) group (P < 0.01), and these changes were reversed by DIL in the OGD/R+BCI+DIL group (P < 0.01). (C and D) Ani (an activator of JNK) significantly upregulated JNK expression in the OGD/R+Ani group (P < 0.01). However, DIL and SP (an inhibitor of JNK) significantly downregulated JNK expression in the OGD/R+DIL and OGD/R+SP groups compared with that in the OGD/R group (P < 0.01). Unexpectedly, compared with that in the OGD/R+Ani group, the JNK expression level did not increase further under DIL intervention but obviously decreased in the OGD/R+Ani+DIL group (P < 0.01), which indicated that DIL-induced DUSP1 partly inhibited JNK expression. (E and F) MAC (an activator of BNIP3L/NIX) significantly upregulated BNIP3L/NIX expression in the OGD/R+MAC group. Compared with that in the OGD/R+DIL or OGD/R+BAF (an inhibitor of BNIP3L/NIX) group, BNIP3L/NIX expression in the OGD/R+DIL or OGD/R +BAF group was significantly lower in the DIL and BAF groups (P < 0.01). Moreover, compared with that in the OGD/R+MAC group, the BNIP3L/NIX expression level did not increase further under DIL intervention but obviously decreased in the OGD/R+ MAC+DIL group (P < 0.01), which indicated that DIL-induced DUSP1 partly inhibited BNIP3L/NIX expression. The data are presented as the means ± SDs of three independent experiments performed in duplicate. *P < 0.05, **P < 0.01.

Article Snippet: Next, the sections were incubated with primary antibodies against DUSP1, JNK, and BNIP3L/NIX (1:200, Proteintech, USA) for 1 h at 37°C.

Techniques: Western Blot, Gene Expression, Expressing

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP9X regulates FBW7 stability and suppresses colorectal cancer

doi: 10.1172/jci97325

Figure Lengend Snippet: Figure 1. FBW7 is downregulated in human CRC. (A) Representative images of RNAscope for PPIB and FBW7, and FBW7 IHC on serial sections from human TMAs. u.d., undetected. Scale bars: 10 μm. (B) Quantification of FBW7 mRNA on CRC TMAs in 28 tissue cores positive for a control PPIB mRNA. (C) Quantification of FBW7 IHC in tissue cores from A. (D–F) Endogenous FBW7 interacts with endogenous USP9X and USP9X interacts with endogenous FBW7 in HEK293 cells. Black line in D indicates noncontiguous lanes from the same gel. siFBW7 control in F confirms antibody specificity of FBW7 and represents a negative control for FBW7-USP9X interaction. (G) Endogenous USP9X interacts with epitope-tagged FBW7 isoforms α and β.

Article Snippet: Antibodies against USP9X (Bethyl Laboratories Inc., catalog A301-351A), FBW7α (Bethyl Laboratories Inc., A301-720), c-Jun (BD Biosciences, 610326), active Notch-1 (Abcam, ab8925), c-Myc and cyclin E (Santa Cruz Biotechnology Inc., sc-788 and sc-481), α-tubulin (Abcam, ab7291), Apu2 clone, K48-linkage (MilliporeSigma, 05-1307), and β-actin (MilliporeSigma, A3854) were used.

Techniques: RNAscope, Control, Negative Control

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP9X regulates FBW7 stability and suppresses colorectal cancer

doi: 10.1172/jci97325

Figure Lengend Snippet: Figure 2. FBW7 is a USP9X substrate. (A and B) USP9X knockdown using multiple shRNAs reduced FBW7 protein levels, with no effect on its mRNA. shScr, scrambled control; shNT, nontargeting control. (C) MG132 rescues reduced FBW7 protein levels in USP9X-silenced cells. (D) USP9X knockdown reduced the half-life of FBW7 protein. CHX, cycloheximide. (E) Quantification of 3 independent experiments performed as in D. (F) Increased ubiquityla- tion (Ubi) of FBW7 in USP9X-silenced cells. UM, unmodified. (G) In vitro deubiquitylation of FBW7 by recombinant GST-USP9X. (H and I) Western blots for Flag-FBW7α in HEK293 cells co-overexpressing WT (V5-Usp9x) or catalytically dead (V5-C1566S) mouse Usp9x. (J) Western blots on Ni-NTA pulldown samples from cells cotransfected with Flag-FBW7α and the indicated constructs. All experiments were done in HEK293 cells.

Article Snippet: Antibodies against USP9X (Bethyl Laboratories Inc., catalog A301-351A), FBW7α (Bethyl Laboratories Inc., A301-720), c-Jun (BD Biosciences, 610326), active Notch-1 (Abcam, ab8925), c-Myc and cyclin E (Santa Cruz Biotechnology Inc., sc-788 and sc-481), α-tubulin (Abcam, ab7291), Apu2 clone, K48-linkage (MilliporeSigma, 05-1307), and β-actin (MilliporeSigma, A3854) were used.

Techniques: Knockdown, Control, In Vitro, Recombinant, Western Blot, Construct

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP9X regulates FBW7 stability and suppresses colorectal cancer

doi: 10.1172/jci97325

Figure Lengend Snippet: Figure 3. USP9X negatively regulates SCF(FBW7) substrates. (A) Accumulation of SCF(FBW7) substrates in USP9X-silenced HEK293 cells. (B) Accumu- lation of SCF(Fbw7) substrates in Usp9x-knockout murine adult fibroblasts. (C) Western blots showing levels of SCF(FBW7) substrates in USP9X- and ITCH-silenced HEK293 cells. (D) Accumulation of SCF(FBW7) substrates with USP9X silencing was abolished in the HCT116-FBW7Δ/Δ CRC cell line. (E and F) Western blots for ubiquitylated c-Myc on Ni-NTA pulldown from HEK293 cells overexpressing Flag–c-Myc and 6x-His–tagged ubiquitin and cotransfected with the indicated shRNAs. c-MYC mRNA levels in the same cells are shown in F. (G) c-MYC luciferase activity in HCT116 cells with the indicated genotypes and siRNA treatments. Mean of 2 independent experiments is shown.

Article Snippet: Antibodies against USP9X (Bethyl Laboratories Inc., catalog A301-351A), FBW7α (Bethyl Laboratories Inc., A301-720), c-Jun (BD Biosciences, 610326), active Notch-1 (Abcam, ab8925), c-Myc and cyclin E (Santa Cruz Biotechnology Inc., sc-788 and sc-481), α-tubulin (Abcam, ab7291), Apu2 clone, K48-linkage (MilliporeSigma, 05-1307), and β-actin (MilliporeSigma, A3854) were used.

Techniques: Knock-Out, Western Blot, Ubiquitin Proteomics, Luciferase, Activity Assay

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP9X regulates FBW7 stability and suppresses colorectal cancer

doi: 10.1172/jci97325

Figure Lengend Snippet: Figure 5. Usp9x is required for tissue regen- eration during acute colitis. (A) Schematic of acute colitis protocol. (B) Western blots for the indicated proteins in 6 independent WT mice fed with (day 7, “peak” and day 21, “recov- ery”) or without (day 0, “normal”) 2.5% DSS in water. (C) qRT-PCR analysis for Usp9x and Fbw7 mRNA in different phases of colitis from the experiment in B. Statistical significance calculated by 1-way ANOVA. (D) Weight curves from DSS-induced colitis experiment in the indicated mice; n = 7–8/group. *P < 0.05. (E) Representative IHC sections for H&E, BrdU, AB/PAS, and NICD1 from Usp9x+/+ and Usp9xΔG mice. Scale bars: 100 μm. (F) Quantification of BrdU+ and AB/PAS+ cells from the experiment in D. Data are presented as mean; statistical significance was calculated by Student’s t test in D and F.

Article Snippet: Antibodies against USP9X (Bethyl Laboratories Inc., catalog A301-351A), FBW7α (Bethyl Laboratories Inc., A301-720), c-Jun (BD Biosciences, 610326), active Notch-1 (Abcam, ab8925), c-Myc and cyclin E (Santa Cruz Biotechnology Inc., sc-788 and sc-481), α-tubulin (Abcam, ab7291), Apu2 clone, K48-linkage (MilliporeSigma, 05-1307), and β-actin (MilliporeSigma, A3854) were used.

Techniques: Western Blot, Quantitative RT-PCR

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP9X regulates FBW7 stability and suppresses colorectal cancer

doi: 10.1172/jci97325

Figure Lengend Snippet: Figure 7. Reduced USP9X is associated with poor prognosis in human CRC. (A) IHC for the indicated proteins on human CRC TMAs, including associated normal tissue. Scale bars: 50 μm. Insets show x2 magnifications of areas in the main image. (B) Quantification of staining intensities from sections in A, and their Spearman’s rank correlation. (C) Western blots showing positive correlation of USP9X and FBW7 protein levels in 8 different CRC cell lines. Red: USP9X mutant, pink: FBW7 truncation mutant (homozygous) cell lines. Black line indicates noncontiguous lanes from the same gel. (D) Kaplan-Meier plot showing comparison of survival between USP9X-low and -high expression groups of CRC patients. Data are from TCGA. (E) Mutational status of USP9X and FBW7 in CRC patients. Data are from the TCGA dataset of 212 patients and the DFCI Colorectal Adenocarcinoma dataset of 619 patients (published in refs. 6, 45 [Nature 2012 and Cell Reports 2016, respectively]). Data viewed with cBioPortal. Only samples with alterations in either USP9X or FBW7 are shown. Fisher’s exact test P = 0.01 for females and P = 1 for males. WCL, whole cell lysate.

Article Snippet: Antibodies against USP9X (Bethyl Laboratories Inc., catalog A301-351A), FBW7α (Bethyl Laboratories Inc., A301-720), c-Jun (BD Biosciences, 610326), active Notch-1 (Abcam, ab8925), c-Myc and cyclin E (Santa Cruz Biotechnology Inc., sc-788 and sc-481), α-tubulin (Abcam, ab7291), Apu2 clone, K48-linkage (MilliporeSigma, 05-1307), and β-actin (MilliporeSigma, A3854) were used.

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

Journal: Genome Biology

Article Title: Deciphering cellular states of innate tumor drug responses

doi: 10.1186/gb-2006-7-3-r19

Figure Lengend Snippet: Selection of 30 genes considered for further Q-PCR analysis

Article Snippet: JUN , JUN-Hs00277190_s1 , 22.8 , 22.1 , -1.6 , 1.86E-02 , 22.1 , 22.3 , -2.5 , 9.00E-06.

Techniques: Selection

Journal: Genome Biology

Article Title: Deciphering cellular states of innate tumor drug responses

doi: 10.1186/gb-2006-7-3-r19

Figure Lengend Snippet: Validation of microarray gene expression data

Article Snippet: JUN , JUN-Hs00277190_s1 , 22.8 , 22.1 , -1.6 , 1.86E-02 , 22.1 , 22.3 , -2.5 , 9.00E-06.

Techniques: Biomarker Discovery, Microarray, Gene Expression

Journal: BIOCELL

Article Title: Nicotinic acid induces apoptosis of glioma cells via the calcium-dependent endoplasmic reticulum stress pathway

doi: 10.32604/biocell.2022.017383

Figure Lengend Snippet: FIGURE 4. NA induces ER stress and apoptosis. The levels of activated caspase-9, GRP94, calreticulin, PDI and CHOP (A), and phospho-c- Jun (B) were analyzed by western blotting after treatment with 14 mM NA for the indicated length of time. The relative expression of indicated proteins was quantified densitometrically and subsequently normalized against β-actin.

Article Snippet: Membranes were blocked at room temperature (RT, 25°C) with 5% bovine serum albumin (BSA; Sigma-Aldrich) for 2 h and incubated with one of the following primary antibodies: caspase-3 (#14220, 1:2,000), cleaved caspase-3 (#9664, 1:2,000), cleaved caspase-9 (#52873, 1:2,000), GRP94 (#20292, 1:2,000), calreticulin (#12238, 1:2,000), protein disulfide isomerase (PDI) (#3501, 1:2,000), CHOP (#2895, 1:2,000), phospho-c-Jun (#91952, 1:2,000,) and GAPDH (#5174, 1:5,000; all from Cell Signaling Technology, Burlington, MA, USA), as well as β-actin (1:5,000; SigmaAldrich A5316), at 4°C overnight.

Techniques: Western Blot, Expressing