Journal: Cell death & disease

Article Title: USP11-mediated LSH deubiquitination inhibits ferroptosis in colorectal cancer through epigenetic activation of CYP24A1.

doi: 10.1038/s41419-023-05915-9

Figure Lengend Snippet: Fig. 2 LSH is degraded upon erastin treatment and interacts with USP11. A Protein and mRNA expression of LSH were analyzed by WB (left) or RT-qPCR (right) in HCT116 and SW620 cells that treated with erastin (20 μM) for 24 h. Values are presented as mean ± SD (n = 3). B WB analysis of HCT116 cells that were treated with erastin (20 µM) for 16 h, then were incubated with MG132 (20 μM) for 6 h or Baf-A1 (1 µM) for 16 h. C HCT116 cells incubated with DMSO or erastin (20 µM) for 24 h were subjected to Co-IP with control IgG or LSH antibodies. Ubiquitination of LSH was analyzed by WB. D Liquid chromatography-tandem mass spectrometry analysis of Flag-LSH immunoprecipitated proteins from HCT116 cells (left). Representative peptide fragment counts and peptide coverage of indicated proteins are shown (right). E Co-IP analysis of the interaction between Flag-LSH and MYC-USP11 in HEK293T cells. MYC-IP (left); Flag-IP (right). F Cytoplasmic and nuclear extracts from HCT116 cells were fractionated for Co-IP using USP11 or LSH antibody. USP11-IP (left); LSH-IP (right). G Immunofluorescence analysis of USP11 and LSH in HCT116 and SW620 cells. Scale bar, 25 μm. H Mapping of the region in LSH that interacts with USP11. A schematic diagram of the constructed LSH truncations (top) and purified proteins with Coomassie blue staining (bottom) are shown.

Article Snippet: Antibodies were USP11 (Proteintech, 22340-AP), LSH (Santa Cruz, sc-46665), fluorescent secondary antibodies (Jackson, 111-585-003; 115-545-003).

Techniques: Expressing, Quantitative RT-PCR, Incubation, Co-Immunoprecipitation Assay, Control, Ubiquitin Proteomics, Liquid Chromatography, Mass Spectrometry, Immunoprecipitation, Construct, Staining

Journal: Cell death & disease

Article Title: USP11-mediated LSH deubiquitination inhibits ferroptosis in colorectal cancer through epigenetic activation of CYP24A1.

doi: 10.1038/s41419-023-05915-9

Figure Lengend Snippet: Fig. 3 USP11 stabilizes LSH by deubiquitination. A HCT-8 and HT-29 cells transfected with control or USP11 plasmids were lysed for WB (left) and RT-qPCR (right) analyses. Values are presented as mean ± SD (n = 3). B HCT116 cells transfected with different doses of USP11 plasmids were collected for WB (left) and RT-qPCR (right) analyses. Values are presented as mean ± SD (n = 3). C SW480 and HCT116 cells transfected with control or USP11 siRNAs were collected for WB (left) or RT-qPCR analyses (right). Values are presented as mean ± SD (n = 3). D HCT116 and SW620 cells treated with DMSO or MTX (1 μM) for 12 h were lysed for WB analysis. E Cells transfected with control, USP11, or USP11 mutant (C318A) plasmids were lysed for WB analysis. F Cells were treated with cycloheximide (CHX, 50 μg/ml) for the indicated times and harvested for WB analysis with the indicated antibodies. Quantification was performed and normalized to tubulin levels. Values are presented as mean ± SD (n = 3). G HCT116 cells transfected with control or USP11 siRNAs were treated with DMSO or MG132 (20 μM) for 6 h and lysed for WB analysis. H HEK293T cells transfected with the indicated plasmids were incubated with MG132 (20 µM) for 6 h. Cells were lysed for the Ni-NTA pull-down assay, and ubiquitination of LSH was analyzed by WB. I HEK293T cells transfected with the indicated plasmids were treated with DMSO or MTX (1 μM) for 12 h and MG132 (20 µM) for 6 h. Cell lysates were analyzed as described above. J HCT116 cells were lysed for Co- IP with IgG or LSH antibodies, and ubiquitination of LSH was analyzed by WB. K In vitro deubiquitination (right). Purified GST, GST-USP11, and GST-USP11 (C318A) are shown (left). L, M HEK293T cells transfected with the indicated plasmids were incubated with MG132 (20 µM) for 6 h. Cells were lysed for Flag-IP and ubiquitination of LSH was analyzed by WB.

Article Snippet: Antibodies were USP11 (Proteintech, 22340-AP), LSH (Santa Cruz, sc-46665), fluorescent secondary antibodies (Jackson, 111-585-003; 115-545-003).

Techniques: Transfection, Control, Quantitative RT-PCR, Mutagenesis, Incubation, Pull Down Assay, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, In Vitro

Journal: Cell death & disease

Article Title: USP11-mediated LSH deubiquitination inhibits ferroptosis in colorectal cancer through epigenetic activation of CYP24A1.

doi: 10.1038/s41419-023-05915-9

Figure Lengend Snippet: Fig. 4 USP11 alleviates erastin-mediated degradation of LSH. A, C WB (top) and RT-qPCR (bottom) were analyzed in USP11 overexpressing stable cell line or USP11-KO cells treated with erastin for 24 h at the indicated concentrations. Values are presented as mean ± SD (n = 3). B, D WB (top) and RT-qPCR (bottom) were analyzed in USP11 overexpressing stable cell line or USP11- KO cells treated with erastin (20 µM) at the indicated time. Values are presented as mean ± SD (n = 3). E Co-IP with LSH antibody in HCT116 cells treated with DMSO or erastin (20 µM) for 24 h (top) and with USP11 antibody in HCT116 cells treated with erastin (20 µM) for 0, 4, 8, and 12 h respectively (bottom). F USP11-KO cells transfected with the indicated plasmids were incubated with DMSO or erastin (20 µM) for 24 h and MG132 (20 µM) for 6 h. Cell lysates were collected for Ni-NTA pull-down, and ubiquitination of LSH was analyzed by WB.

Article Snippet: Antibodies were USP11 (Proteintech, 22340-AP), LSH (Santa Cruz, sc-46665), fluorescent secondary antibodies (Jackson, 111-585-003; 115-545-003).

Techniques: Quantitative RT-PCR, Stable Transfection, Co-Immunoprecipitation Assay, Transfection, Incubation, Ubiquitin Proteomics

Journal: Cell death & disease

Article Title: USP11-mediated LSH deubiquitination inhibits ferroptosis in colorectal cancer through epigenetic activation of CYP24A1.

doi: 10.1038/s41419-023-05915-9

Figure Lengend Snippet: Fig. 7 The USP11/LSH/CYP24A1 axis is aberrantly activated in CRC. A Immunohistochemical analysis of USP11, LSH, and CYP24A1 expression in human CRC and adjacent normal tissues. Scale bar, 50 μm. B Correlations between USP11 and LSH and between LSH and CYP24A1 were analyzed by Linear regression analysis (n = 94). C Analysis of CRC patient prognosis stratified by USP11, LSH, and CYP24A1 expression levels. D Tumor formation in the xenografted mice (n = 6 per group) (left). WB analysis of the indicated proteins (right). E Tumor growth rate of xenografts in BALB/c nude mice. Tumor weight and volumes were measured and plotted. Values are presented as mean ± SD (n = 6). F Immunohistochemical analysis of tumor xenografts. Scale bar, 50 μm. G Schematic model of USP11/LSH/CYP24A1 axis-mediated resistance to ferroptosis in CRC cells.

Article Snippet: Antibodies were USP11 (Proteintech, 22340-AP), LSH (Santa Cruz, sc-46665), fluorescent secondary antibodies (Jackson, 111-585-003; 115-545-003).

Techniques: Immunohistochemical staining, Expressing

Journal: Nucleic Acids Research

Article Title: The chromatin regulator HELLS mediates SSB repair and responses to DNA alkylation damage

doi: 10.1093/nar/gkaf1201

Figure Lengend Snippet: Loss of HELLS results in deficient repair of alkylation-derived DNA single SSBs in specific cell types. (A) HELLS and key BER DNA repair protein expression levels in three cell lines as visualized by Western blot in WCE. SE = short exposure, LE = long exposure. GAPDH was used as a loading control. (B) Alkaline comet (AC) and neutral comet (NC) images of HAP1 and HAP1-HELLS KO cells following treatment with 500 μM MMS for 1 h. Bar = 200 µm. (C) DNA strand breaks quantified by alkaline and neutral assays. Data are the average of the comet tail moments of 50 cells per sample. n = 3 independent biological experiments. (D) Double-strand breaks were quantified based on the NC assay in HAP1 cells. Graph is plotted as in C. (E) DNA strand breaks quantified by AC assay in HeLa cells following treatment with 1 mM MMS for 1 h. The data represent individual comet tail moments of 200 cells per sample per experiment, plotted vertically. Two independent experiments are displayed side by side. (F) DNA strand breaks quantified by AC assay in CHON-002 cells at the indicated time points following treatment with 500 individual comet tail moments of 200 cells per sample per experiment, plotted vertically. Two independent experiments are displayed side by side. (G) Western blot of WCE in HeLa-HELLS KO cells transiently transfected with pCMV empty or pCMV-HELLS (HELLS overexpression) vectors, probed for HELLS. GAPDH was used as a loading control. (H) DNA strand breaks quantified by AC assay in HeLa HELLS KO cells transfected with pCMV-HELLS following treatment with 1 mM MMS for 1 h. Data represent mean tail moments of 200 cells per sample. n = 2 independent biological replicates. (I) HeLa HELLS KO cells transiently transfected with pCMV-empty vector and pCMV-HELLS were exposed to MMS for 48 h, and cell survival was determined with CCK8 reagent. Data are mean ± SEM. n = 3 independent biological replicates. (J) Western blot of WCE in HAP1 HELLS KO cells containing the stable genomic integration of HELLS WT, and HELLS K254R (catalytically inactive). GAPDH was used as a loading control. DNA strand breaks were quantified by the AC assay in the cell lines. Data are the average of comet tail moments of 100 cells per sample of two independent biological experiments. (K) HAP1 and HAP1-HELLS KO cells were treated with the indicated doses of MMS for 1 h. The media was changed, and cells were allowed to recover for 0, 1, or 2 h. BER pathway members were visualized on chromatin by Western blot. MacroH2A was utilized as a loading control. SE = short exposure, LE = long exposure. (L) Western blot of WCE in MCF7 cells probed for HELLS, indicating downregulation of HELLS by siRNA for 72 h. GAPDH was used as a loading control. DNA strand breaks quantified by AC assay in MCF7 cells followed by HELLS downregulation. Cells transfected with 3 nM siRNA for 72 h were dosed with 1 mM MMS for 1 h. Data are the average of the comet tail moments of 100 cells per sample. (M) Western blot of WCE in U2OS cells probed for HELLS, indicating downregulation of HELLS by siRNA for 48 h. GAPDH was used as a loading control. DNA strand breaks quantified by AC assay in U2OS cells followed by HELLS downregulation. Cells transfected with 3 nM siRNA for 48 h were dosed with 1 mM MMS for 1 h. Data are the average of comet tail moments of at least 200 cells per sample. n = 2 independent biological experiments. Statistical significance for all comet assays was determined by two-way ANOVA of the mean tail moments with Tukey’s multiple comparisons test. (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: A custom mutation of the Myc-DDK HELLS construct that encodes HELLS K254R was created by Origene. pLenti V HELLS WT or Mutant plasmids were constructed using In-Fusion cloning (Takara Bio) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Expressing, Western Blot, Control, Transfection, Over Expression, Plasmid Preparation

Journal: mBio

Article Title: Nrf2 Regulates Granuloma Formation and Macrophage Activation during Mycobacterium avium Infection via Mediating Nramp1 and HO-1 Expressions

doi: 10.1128/mBio.01947-20

Figure Lengend Snippet: Genes related to oxidative stress and heme metabolism, such as Nrf2 ( Nfe2l2 ), HO-1 ( Hmox1 ), and Nramp1 ( Slc11a1 ), are highly altered in the lungs of Nrf2 −/− mice following MAC infection. At 2 months after MAC infection, hierarchical clustering based on the 79 differentially expressed genes between the lungs of wild-type mice infected with MAC bacteria and those of Nrf2 −/− mice infected with MAC bacteria was done, with statistically significant differences (FDR-adjusted P < 0.01) and with more than 2-fold changes. WTC, uninfected lungs from wild-type mice; KOC, uninfected lungs from Nrf2 −/− mice; WTI, infected lungs from wild-type mice; KOI, infected lungs from Nrf2 −/− mice.

Article Snippet: Mouse Nramp1 cDNA open reading frame (ORF) clones (MR227222) were purchased from OriGene.

Techniques: Infection, Bacteria

Journal: mBio

Article Title: Nrf2 Regulates Granuloma Formation and Macrophage Activation during Mycobacterium avium Infection via Mediating Nramp1 and HO-1 Expressions

doi: 10.1128/mBio.01947-20

Figure Lengend Snippet: Alveolar macrophages from infected lungs are responsible for the expression of Nramp1 regulated by Nrf2. Alveolar macrophages were obtained from BAL fluids of wild-type and Nrf2 −/− mice 2 months after intranasal inoculation of 1 × 10 7 CFU of MAC bacteria or saline (Cont). (A) Expression of Nrf2 , Nramp1 , and HO-1 in alveolar macrophages from infected lungs of wild-type and Nrf2 −/− mice. (B and C) Representative Western blots of Nrf2 expression (B) and its semiquantitative analysis (C) in lung nuclear extracts from wild-type mice that were infected with MAC bacteria or uninfected. Values were normalized to lamin B. (D) Representative fluorescence images of Nrf2 immunoreactivity (green) and nuclear staining with DAPI (blue) in BAL-recovered cells of MAC infected wild-type mice (magnification, ×200). (E) Semiquantitative analysis of the mean fluorescence density of Nrf2 staining in the nuclei of alveolar macrophages from each group of mice. The fold changes between MAC-infected alveolar macrophages and control alveolar macrophages (saline) are shown. Experiments were performed in duplicate with four mice in each group. Significant differences between infected mice and noninfected mice (*, P < 0.05) and between Nrf2 −/− mice and wild-type mice (#, P < 0.05) are shown. Data are means and SEM.

Article Snippet: Mouse Nramp1 cDNA open reading frame (ORF) clones (MR227222) were purchased from OriGene.

Techniques: Infection, Expressing, Bacteria, Saline, Western Blot, Fluorescence, Staining, Control

Journal: mBio

Article Title: Nrf2 Regulates Granuloma Formation and Macrophage Activation during Mycobacterium avium Infection via Mediating Nramp1 and HO-1 Expressions

doi: 10.1128/mBio.01947-20

Figure Lengend Snippet: The Nrf2-Nramp1 pathway promotes MAC bacterial killing in alveolar macrophages by enhancing phagolysosome formation. (A) Representative images of intracellular MAC bacteria in alveolar macrophages from infected lungs of wild-type (WT) and Nrf2 −/− mice. Kinyoun staining was used. Magnification, ×200. (B) Number of bacteria per alveolar macrophage. (C) Electron micrographs of alveolar macrophages from wild-type and Nrf2 −/− mice 2 months after MAC infection. Phagosomes, lysosomes, and phagolysosomes are indicated by open arrowheads, closed arrowheads, and arrows, respectively. Bar = 2.0 μm. (D) Fluorescent staining to detect the formation of phagolysosomes in alveolar macrophages obtained from MAC-infected wild-type mice (top) and Nrf2 −/− mice (bottom). MAC bacteria were labeled with auramine (green), and lysosomes were stained with Lysotracker Red. Magnification, ×200. (E) Percentage of vesicles costained with MAC bacteria and lysosomes in macrophages obtained from MAC-infected wild-type mice (WT) and Nrf2 −/− mice. A minimum of 100 cells were analyzed. (F) Electron micrographs of MAC-infected alveolar macrophages transfected with Nramp1 or mock infected (control). Phagosomes, lysosomes, and phagolysosomes are indicated by open arrowheads, closed arrowheads, and arrows, respectively. Bar = 2.0 μm. Experiments were performed in duplicate with four mice in each group. Significant differences between Nrf2 −/− mice and wild-type mice after MAC infection are shown (#, P < 0.05). Data are means and SEM.

Article Snippet: Mouse Nramp1 cDNA open reading frame (ORF) clones (MR227222) were purchased from OriGene.

Techniques: Bacteria, Infection, Staining, Labeling, Transfection, Control

Journal: mBio

Article Title: Nrf2 Regulates Granuloma Formation and Macrophage Activation during Mycobacterium avium Infection via Mediating Nramp1 and HO-1 Expressions

doi: 10.1128/mBio.01947-20

Figure Lengend Snippet: Treatment with sulforaphane (SFN) decreases Mycobacterium growth by upregulating the expression of Nramp1 and HO-1. (A) Mycobacterial outgrowths in the lung, liver, and spleen of wild-type mice 1 month after intranasal inoculation of 1 × 10 7 CFU of MAC with or without treatment with sulforaphane. The results are expressed as CFU per organ. (B) Expression of Nramp1 and HO-1 in the lungs of wild-type mice 1 month after intranasal inoculation of 1 × 10 7 CFU of MAC (solid bars). Control mice were administered saline intranasally and the same amount of dimethyl sulfoxide (DMSO) with PBS intraperitoneally (open bars). Experiments were performed in duplicate with four mice in each group. Differences between SFN-treated Nrf2 −/− mice and control Nrf2 −/− mice after MAC infection were significant (*, P < 0.05). Data are means and SEM.

Article Snippet: Mouse Nramp1 cDNA open reading frame (ORF) clones (MR227222) were purchased from OriGene.

Techniques: Expressing, Control, Saline, Infection

Journal: mBio

Article Title: Nrf2 Regulates Granuloma Formation and Macrophage Activation during Mycobacterium avium Infection via Mediating Nramp1 and HO-1 Expressions

doi: 10.1128/mBio.01947-20

Figure Lengend Snippet: Schematic presentation of the role of Nrf2 in MAC infection. In Nrf2-competent animals, infection with MAC bacteria activates Nrf2 to induce the expression of Nramp1 and HO-1 genes in the lung tissue. Nramp1 is especially induced by Nrf2 in infected alveolar macrophages. HO-1 is associated with organized granuloma formation, whereas Nramp1 is involved in promoting phagosome-lysosome fusion in alveolar macrophages and eventually in organized granuloma formation as well. If Nrf2 is not activated in alveolar macrophages or lungs in response to MAC infection, the phagosome-lysosome fusion and granuloma formation are inhibited. Consequently, MAC bacteria can survive in alveolar macrophages and granulomatous lesions. Thus, the expression of Nramp1 and HO-1 regulated by Nrf2 is among the important host factors in MAC infection.

Article Snippet: Mouse Nramp1 cDNA open reading frame (ORF) clones (MR227222) were purchased from OriGene.

Techniques: Infection, Bacteria, Expressing