Journal: Nature Communications

Article Title: Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal

doi: 10.1038/ncomms12326

Figure Lengend Snippet: ( a ) Representative western blot of stable HT29 line transduced with shSAE2 lentivirus showed lower SAE2, Oct-1 and ALDH1A1 protein levels than HT29 transduced with control non-silencing shRNA (shCtrl); GAPDH, loading control. Right panel: quantification of band intensity. ( b ) SAE2 knockdown decreased and SAE2 overexpression increased the occupancy of Oct-1 on the ALDH1A1 promoter as measured by ChIP assay. ( c ) SAE2 knockdown did not affect Oct-1 transcription as shown by real-time quantitative PCR measurement of mRNA of HT29 shCtrl and shSAE2 cells. ( d ) Representative western blot showing SAE2 knockdown reduced Oct-1 stability, as shown by treatment with 100 μg ml −1 CHX to block protein synthesis in HT29 shCtrl and shSAE2 cells; GAPDH, loading control. Quantification of band intensities (Oct-1 decay curve) in three independent experiments is shown below. ( e ) Representative western blot showing SAE2 knockdown-enhanced Oct-1 ubiquitination. HT29 shCtrl or shSAE2 cells were treated with or without proteasome inhibitor MG132 (10 μM) for 8 h, and IP was carried out using cell lysates, followed by western blot with an anti-Oct-1 or anti-ubiquitin antibody. NS, not significant; *** P <0.001.

Article Snippet: Myc-DDK-tagged human Oct-1 (POU2F1) plasmid was purchased from Origene.

Techniques: Western Blot, Transduction, shRNA, Over Expression, Real-time Polymerase Chain Reaction, Blocking Assay

Journal: Nature Communications

Article Title: Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal

doi: 10.1038/ncomms12326

Figure Lengend Snippet: ( a ) Mass spectrometry identified TRIM21 as an Oct-1-interacting protein. Representative silver stain of HCT116 cells transfected with Flag-tagged Oct-1 or empty vector (Ctrl) for 2 days, lysed and immunoprecipitated with anti-Flag antibody-conjugated M2 beads. Proteins identified in each group are indicated in the graphic. ( b ) Representative IP-western blot analysis of the interaction between endogenous TRIM21 and Oct-1 in HT29 cells. ( c ) Knockdown of TRIM21 delayed Oct-1 degradation. Representative western blot of Oct-1 level over time in HT29 cells transfected with control non-silencing siRNA (siCtrl) or siTRIM21 for 3 days, followed by 100 μg ml −1 CHX treatment; GAPDH, loading control. Oct-1 decay curve (right panel) was determined by quantifying three independent experiments. ( d ) TRIM21 knockdown resulted in increased Oct-1 protein level and decreased ubiquitination of Oct-1. Representative western blot of HT29 cells transfected with siCtrl and siTRIM21 treated with or without 10 μM MG132 for 8 h. IP was carried out with cell lysates, and Oct-1, TRIM21 and ubiquitin were detected; GAPDH, loading control. ( e ) Overexpression of TRIM21 decreased Oct-1 and increased Oct-1 ubiquitination in cells. Representative western blot of HT29 cells were transfected with empty vector (Ctrl) or Flag-TRIM21 for 2 days, then treated with or without 10 μM MG132 for 8 h. IP was carried out with cell lysates, and western blot was performed to detect Oct-1, TRIM21 and ubiquitin; GAPDH, loading control.

Article Snippet: Myc-DDK-tagged human Oct-1 (POU2F1) plasmid was purchased from Origene.

Techniques: Mass Spectrometry, Silver Staining, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Over Expression

Journal: Nature Communications

Article Title: Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal

doi: 10.1038/ncomms12326

Figure Lengend Snippet: ( a ) Representative IHC staining indicates higher TRIM21 level corresponding with lower Oct-1 in shSAE2 group tumour tissue compared with shCtrl group (scale bar, 100 μm). Tumour tissues were from LDA assay described in . ( b ) Western blot (left) and quantification of SAE2, Oct-1, TRIM21 and ALDH levels in the same tumour tissues as a ; GAPDH, loading control. ‘1, 2 and 3' indicate tumour tissues from different mouse. ( c ) TRIM21 promoter activity was inhibited by overexpression of SAE2 and Ubc9 but increased by SENP1 overexpression as determined by luciferase reporter assay. HT29 cells were transfected with empty vector (Ctrl) or SAE2, UBC9 or SENP1 expression plasmid together with TRIM21 promoter luciferase reporter and Renilla plasmids. Dual-luciferase activity was measured after 48 h and normalized results were analysed with two-tailed Student's t -test. ( d ) TRIM21 mRNA level was suppressed by SAE2 or Ubc9 overexpression and enhanced with SENP1 overexpression in HT29 cells as determined by quantitative PCR (qPCR). ( e ) IRF1 SUMOylation site mutant K78R induced higher TRIM21 mRNA level than wild-type (WT) IRF1 as determined by qPCR. ( f ) TRIM21 protein level was suppressed on SAE2 or Ubc9 overexpression but enhanced with SENP1 overexpression as indicated by western blot; GAPDH, loading control. ( g ) Western blot showed overexpression of K78R mutant-induced higher TRIM21 protein level than WT IRF1 in shCtrl HT29 cells; GAPDH, loading control. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Myc-DDK-tagged human Oct-1 (POU2F1) plasmid was purchased from Origene.

Techniques: Immunohistochemistry, Western Blot, Activity Assay, Over Expression, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Expressing, Two Tailed Test, Real-time Polymerase Chain Reaction, Mutagenesis

Journal: Nature Communications

Article Title: Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal

doi: 10.1038/ncomms12326

Figure Lengend Snippet: ( a ) Overexpression of Oct-1 partially compensated for the reduction of CSC frequency by SAE2 knockdown in HT29 cells as determined by LDA. Stable cell lines were generated with lentivirus expressing pLenti CMV-hygro empty vector (EV) or pLenti CMV-Flag-Oct-1 in HT29 shCtrl and shSAE2 cells as shCtrl+EV, shSAE2+EV and shSAE2+Oct-1. ( b ) Representative western blot of the stable lines to confirm expression with Oct-1 and Flag-tag antibodies; GAPDH, loading control. ( c ) Overexpression of Oct-1 in SAE2 or Ubc9 knockdown cells restored ALDH + cells population in HT29 cells as measured by FACS analysis using the AldeFluor kit. HT29 cells were transfected with control non-targeting siRNA (SiCtrl), SAE2-targeting siRNA (SiSAE2) or Ubc9-targeting siRNA (SiUbc9) followed by Flag-Oct-1 plasmid or control empty vector transfection. After 3 days, cells were collected for FACS analysis using the AldeFluor kit. ( d ) Representative western blot of the samples from c ; quantification of the ALDH1A1 band intensity is shown on the bottom. ( e ) Knockdown of Ubc9 reduced CSC frequency, as shown by LDA using spheroid formation using HT29 stable cell lines expressing two different UBC9-targeting shRNA (shUBC9#1 and shUBC9#2). ( f ) Schematic diagram showing the mechanism of how SUMOylation regulates CSCs through Oct-1, TRIM21 and IRF1. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Myc-DDK-tagged human Oct-1 (POU2F1) plasmid was purchased from Origene.

Techniques: Over Expression, Stable Transfection, Generated, Expressing, Plasmid Preparation, Western Blot, FLAG-tag, Transfection, shRNA

Journal: bioRxiv

Article Title: Microfluidic Mechanical Reactivation of Aged Stem Cells

doi: 10.64898/2026.02.25.707893

Figure Lengend Snippet: (A) Representative fluorescence images and comparative analysis of intracellular ROS levels among P6, P11, P18, and P18 μ-CPR cells. Scale bar = 500 µm. (B) Representative fluorescence images showing intracellular ROS levels in P18 μ-CPR under three flow conditions ( Re = 89, 267, and 446). Scale bar = 500 µm. (C) RT-qPCR analysis of stemness-related genes ( OCT4 , SOX2 , and KLF4 ) after 3 days. Expression levels are normalized to P7 WJ-MSCs and presented as relative ratios for the P18 μ-CPR groups ( Re = 89, 267, and 446). (D) Representative immunofluorescence images showing the expression of stemness markers (OCT4, SOX2, and SSEA4) in P18 and P18 μ-CPR cells. Nuclei were stained with DAPI. Scale bar = 20 µm. (E–G) Quantification of fluorescence intensity (fold change) for OCT4 (E), SOX2 (F) , and SSEA4 (G) in P18 and P18 μ-CPR cells. (H) Quantification of Western blot band intensities (fold change) for stemness markers (OCT4, SOX2, and SSEA4) and the proliferation marker PCNA in P18 μ-CPR cells relative to the P18 control. (I) Immunoblotting of OCT4, SOX2, SSEA4, PCNA, and β-actin in μ-CPR and control MSCs. (J) Relative quantification of senescent cells using SA-β-Gal staining in P6, P11, P18, and P18 μ-CPR cells. The graph presents the percentages of SA-β-Gal-positive cells. Scale bar = 500 µm. (K, L) Evaluation of intracellular γH2AX expression and its quantification in P6, P11, P18, and P18 μ-CPR cells. Scale bar = 20 µm. Data are presented as the mean ± SEM of three independent experiments. Statistical analysis was performed using one-way ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Membranes were blocked using 5% skimmed milk in Tris-buffered saline for 1 h, then incubated overnight at 4 °C with the appropriate primary antibodies (Abs): OCT4 (Santa Cruz Biotec, sc-5279), SOX2 (CST, 23064), SSEA4 (Santa Cruz Biotec, sc-21704), PCNA (CST, 2586), and anti-β-actin (CST, 4970S).

Techniques: Fluorescence, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Western Blot, Marker, Control, Quantitative Proteomics

Journal: Cell Stem Cell

Article Title: Distinct Molecular Trajectories Converge to Induce Naive Pluripotency

doi: 10.1016/j.stem.2019.07.009

Figure Lengend Snippet:

Article Snippet: Monoclonal rabbit anti-Oct4 , Cell Signaling Technology , Cat#83932; RRID: AB_2721046.

Techniques: Recombinant, cDNA Synthesis, SYBR Green Assay, Negative Control, Software

Journal: British Journal of Cancer

Article Title: CSN6–TRIM21 axis instigates cancer stemness during tumorigenesis

doi: 10.1038/s41416-020-0779-9

Figure Lengend Snippet: a DLD-1 cells were transfected with Flag-CSN6 plasmids. The protein level of TRIM21 was immunoblotted with anti-TRIM21 antibodies. b DLD-1 cells were infected with scrambled or CSN6-specific shRNA lentivirus. The protein level of TRIM21 was immunoblotted with anti-TRIM21 antibodies. c Quantitative RT-PCR analysis was performed to measure the mRNA levels of Trim21 in cells transfected with empty vector or Flag-CSN6. d Flag-TRIM21 was expressed in HCT116 cells. Flag-TRIM21 was immunoprecipitated with anti-Flag, and the associated CSN6 was detected by western blotting. e HEK293T cell lysates were immunoprecipitated with an anti-TRIM21 antibody and immunoblotted with anti-TRIM21 and anti-CSN6 antibodies. f HEK293T cells were co-transfected with Flag-TRIM21 and the HA-CSN6-WT, CSN6-N terminal or CSN6-C terminal construct. Cell lysates were immunoprecipitated with anti-Flag and subsequently immunoblotted with anti-Flag and anti-HA antibodies. Heavy heavy immunoglobulin chain, Light light immunoglobulin chain. g HEK293T cells were co-transfected with HA-CSN6 and the Flag-TRIM21-WT, TRIM21-N terminal or TRIM21-C terminal construct. Cell lysates were immunoprecipitated with anti-Flag and subsequently immunoblotted with anti-Flag and anti-HA antibodies. h Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in HCT116 cells carrying scrambled or TRIM21-specific siRNA. Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in HCT116 cells carrying empty vector or Flag-TRIM21 plasmids. i Sphere-formation assay of DLD-1 cells transduced with control or Flag-TRIM21 lentivirus. j HCT116 cells were transfected with Flag-CSN6 plasmids. The protein level of TRIM21, OCT1 and ALDH1A1 was immunoblotted with anti-TRIM21, anti-OCT1 and anti-ALDH1A1 antibodies. k Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in HCT116 cells with TRIM21 knockout. l Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in HCT116 cells stably expressing empty vector or Flag-CSN6 and rescued with Flag-TRIM21 plasmids. m Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in HCT116 cells stably expressing scrambled or CSN6-specific shRNA and rescued with TRIM21-specific siRNA. n The knockout efficiency of TRIM21 in HCT116 cells by TRIM21 sgRNAs was evaluated by western blotting. Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in control or HCT116 cells with TRIM21 knockout and then transfected with empty vector or Flag-CSN6. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: We used antibodies against Flag-Tag (Cell Signaling, #8146), HA-Tag (Cell Signaling, #3724), Myc-Tag (Cell Signaling, #2278), CSN6 (Enzo, BML-PW8295), TRIM21 (abcam, ab119859), OCT1 (Cell Signaling, #8157) and ALDH1A1 (Cell Signaling, #54135).

Techniques: Transfection, Infection, shRNA, Quantitative RT-PCR, Plasmid Preparation, Immunoprecipitation, Western Blot, Construct, Tube Formation Assay, Transduction, Control, Knock-Out, Stable Transfection, Expressing

Journal: British Journal of Cancer

Article Title: CSN6–TRIM21 axis instigates cancer stemness during tumorigenesis

doi: 10.1038/s41416-020-0779-9

Figure Lengend Snippet: a HEK293T cells were transfected with His-Ubi together with Flag-TRIM21 and HA-CSN6. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with Ni-NTA beads and immunoblotted with anti-Flag and anti-HA antibodies. b HEK293T cells were transfected with HA-Ubi and Flag-TRIM21 and subsequently treated with shCSN6. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with the anti-Flag and anti-HA antibodies. c In DLD-1 cells expressing scrambled or CSN6-specific shRNA, CSN6 knockdown reduced the turnover rate of the TRIM21 protein and increased the turnover rate of OCT1. CHX cycloheximide. d DLD-1 cells were seeded into six-well plates. Forty-eight hours later, the cells were treated with MG132 at the indicated time points. e HEK293T cells were co-transfected with HA-Ubi and Flag-TRIM21. Forty-eight hours later, MG132 or NH 4 Cl was added to the cells at the indicated time points. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-Flag and anti-HA antibodies. f HCT116 cells were co-transfected with HA-CSN6 and Flag-TRIM21-WT or Flag-TRIM21-LD (C16A, C31A and H33W) mutant. Cell lysates were immunoblotted with anti-Flag and anti-HA antibodies. g The knockout efficiency of TRIM21 in HEK293T cells by TRIM21 sgRNAs was evaluated by western blotting. h HEK293T cells were co-transfected with the HA-Ubi-WT, Ubi-K48 only or Ubi-K63 only construct and the Flag-TRIM21-WT or Flag-TRIM21-LD construct. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-Flag and anti-HA antibodies. i HEK293T KO cells were co-transfected with the indicated HA-Ubi constructs and Flag-TRIM21 or the Flag-TRIM21 ΔRING mutant. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-HA antibodies. j Prediction of TRIM21 ubiquitination sites by UbiSite ( http://140.138.144.145/~ubinet/index.php ) and UbPred ( http://www.ubpred.org/index.html ). k HEK293T cells were co-transfected with HA-Ubi and Flag-TRIM21-WT or its mutants. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-Flag and anti-HA antibodies. l HCT116 cells were co-transfected with HA-CSN6 and Flag-TRIM21-WT or the Flag-TRIM21 K214R/K217R mutant. Cell lysates were immunoblotted with anti-Flag and anti-HA antibodies. m HCT116 cells were co-transfected with HA-CSN6 and Flag-TRIM21 or the Flag-TRIM21 K214R/K217R mutant and were then treated with cycloheximide. Cell lysates were immunoblotted with anti-Flag and anti-HA antibodies. n HEK293T KO cells were co-transfected with the indicated HA-Ubi constructs and Flag-TRIM21-WT or the Flag-TRIM21 K214R/K217R mutant. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-HA antibodies.

Article Snippet: We used antibodies against Flag-Tag (Cell Signaling, #8146), HA-Tag (Cell Signaling, #3724), Myc-Tag (Cell Signaling, #2278), CSN6 (Enzo, BML-PW8295), TRIM21 (abcam, ab119859), OCT1 (Cell Signaling, #8157) and ALDH1A1 (Cell Signaling, #54135).

Techniques: Transfection, Expressing, shRNA, Knockdown, Mutagenesis, Knock-Out, Western Blot, Construct, Ubiquitin Proteomics

Journal: British Journal of Cancer

Article Title: CSN6–TRIM21 axis instigates cancer stemness during tumorigenesis

doi: 10.1038/s41416-020-0779-9

Figure Lengend Snippet: a HCT116 cells were transfected with the indicated plasmids. Cell lysates were immunoblotted with anti-Flag and anti-OCT1 antibodies. b TRIM21 knockout cells were transfected with the indicated plasmid, and cell lysates were immunoblotted with anti-Flag and anti-OCT1 antibodies. c HEK293T cells were transfected with HA-Ubi and the indicated plasmids. Cell lysates were subjected to IP with anti-Flag and immunoblotted with anti-HA antibodies. d HEK293T cells were transfected with His-Ubi and the indicated plasmids. Cell lysates were subjected to PD with Ni-NTA beads and immunoblotted with anti-Flag and anti-CSN6 antibodies. e HEK293T cells were transfected with the indicated plasmids. Cell lysates were subjected to PD with Ni-NTA beads and immunoblotted with anti-Flag antibodies. f HEK293T cells were transfected with HA-Ubi and Flag-OCT1 and subsequently treated with shCSN6 and siTRIM21. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-Flag and anti-HA antibodies. g DLD-1 cells were engineered to stably express scrambled or CSN6-specific shRNA and were then rescued with Flag-OCT1 plasmids. Sphere formation was measured. Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 . * P < 0.05 and ** P < 0.01.

Article Snippet: We used antibodies against Flag-Tag (Cell Signaling, #8146), HA-Tag (Cell Signaling, #3724), Myc-Tag (Cell Signaling, #2278), CSN6 (Enzo, BML-PW8295), TRIM21 (abcam, ab119859), OCT1 (Cell Signaling, #8157) and ALDH1A1 (Cell Signaling, #54135).

Techniques: Transfection, Knock-Out, Plasmid Preparation, Stable Transfection, shRNA, Quantitative RT-PCR

Journal: British Journal of Cancer

Article Title: CSN6–TRIM21 axis instigates cancer stemness during tumorigenesis

doi: 10.1038/s41416-020-0779-9

Figure Lengend Snippet: a Frequency of CSCs in DLD-1 cells transduced with control non-silencing shRNA (shCtrl) or shCSN6 lentivirus, as measured by an LDA in vivo. Tumour morphology in each group. b Quantification of tumour initiation. c Tumour growth curve for mice subcutaneously injected with control or shCSN6 DLD-1 cells (1 × 10 6 cells per mouse, six mice per group). d RNA was extracted from tumour tissues. Quantitative RT-PCR analysis was performed to measure the mRNA level of Aldh1a1 . The data are presented as the means ± SDs. e Protein was extracted from the tumour tissues described in c and immunoblotted with the indicated antibodies. f Representative IHC staining for CSN6, TRIM21, OCT1 and ALDH1A1 in tumour tissues from mice. The scale bars represent 100 μm. g Representative IHC staining for CSN6, TRIM21 and ALDH1A1 in human CRC TMAs. Case 1 is representative of a patient with CSN6-high colon cancer. Case 2 is representative of a patient with non-CSN6-high colon cancer. The scale bars represent 100 μm. h Quantification of staining intensities from sections in g . CSN6 and TRIM21 show a negative correlation, while CSN6 and ALDH1A1 show a positive correlation. i Kaplan–Meier survival curves of overall survival time based on CSN6 and TRIM21 expression from TMA analysis. * P < 0.05 and *** P < 0.001.

Article Snippet: We used antibodies against Flag-Tag (Cell Signaling, #8146), HA-Tag (Cell Signaling, #3724), Myc-Tag (Cell Signaling, #2278), CSN6 (Enzo, BML-PW8295), TRIM21 (abcam, ab119859), OCT1 (Cell Signaling, #8157) and ALDH1A1 (Cell Signaling, #54135).

Techniques: Transduction, Control, shRNA, In Vivo, Injection, Quantitative RT-PCR, Immunohistochemistry, Staining, Expressing