Journal: EMBO Molecular Medicine

Article Title: B cell lineage reconstitution underlies CAR-T cell therapeutic efficacy in patients with refractory myasthenia gravis

doi: 10.1038/s44321-024-00043-z

Figure Lengend Snippet: ( A ) A schematic overview of the time points at which patients receive different treatments before CAR-T infusion. # Patient MG-1 was treated with IVIG 2 g/Kg + intravenous pulse steroid 500 mg* 3days for myasthenia crisis. ( B ) A schematic overview of CAR-T treatment procedure. CAR T-cell kinetics are shown by the CAR copies per μg genomic DNA at serial time points post infusion detected by droplet digital PCR. ( C ) Representative plots showing FACS analysis stained for CAR-T cells with FITC-labeled human BCMA Fc tag protein and APC/Cy7 anti-human CD3 antibody in patient MG-1 at day 10 after CAR T-cell infusion. CAR T-cell percentage in circulating CD3 + T lymphocytes at serial time points after treatment. ( D ) Timelines of patients with cytopenia of grade 3 or higher at baseline and indicated time points after CAR T-cell infusion. BL baseline. Kinetic changes in numbers of circulating total white blood cells, neutrophils, monocytes and platelets. ( E ) Heatmap depicting protein levels of inflammatory mediators in blood following CAR T-cell infusion. Interleukin IL, TNF tumor necrosis factor, IFN interferon, CRP C-reactive protein, PCT procalcitonin. Average levels are normalized from the baseline. .

Article Snippet: Website links for the antibodies used in the flow cytometry are following: PerCP/Cyanine5.5 anti-human CD45 Antibody (Biolegend, 304028): https://www.biolegend.com/en-us/products/percp-cyanine5-5-anti-human-cd45-antibody-4240 ; FITC anti-human CD3 antibody (BD Biosciences, 561802): https://www.bdbiosciences.com/zh-cn/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/fitc-mouse-anti-human-cd3.561802 ; PE/Cyanine7 anti-Human CD4 (BD Biosciences, 560649): https://www.bdbiosciences.com/zh-cn/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/pe-cy-7-mouse-anti-human-cd4.560649 ; APC/Cyanine7 anti-Human CD8 (BD Biosciences, 557834): https://www.bdbiosciences.com/zh-cn/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/apc-cy-7-mouse-anti-human-cd8.557834 ; APC anti-human CD19 Antibody (Biolegend, 302212): https://www.biolegend.com/en-us/products/apc-anti-human-cd19-antibody-715 ; PE anti-human CD16 Antibody (Biolegend, 302056): https://www.biolegend.com/en-us/products/pe-anti-human-cd16-antibody-569 ; PE anti-human CD56 Antibody (Biolegend, 318306): https://www.biolegend.com/en-us/products/pe-anti-human-cd56-ncam-antibody-3796 ; FITC anti-Human CD38 (BD Biosciences, 567147): https://www.bdbiosciences.com/en-us/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/fitc-mouse-anti-human-cd38.567147 ; PerCP/Cyanine5.5 anti-Human CD27 (BD Biosciences, 560612): https://www.bdbiosciences.com/zh-cn/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/percp-cy-5-5-mouse-anti-human-cd27.560612 ; PerCP anti-Human CD45 (BD Biosciences, 347464): https://www.bdbiosciences.com/zh-cn/products/reagents/flow-cytometry-reagents/clinical-discovery-research/single-color-antibodies-ruo-gmp/percp-mouse-anti-human-cd45.347464 ; APC/Cyanine7 anti-human CD3 Antibody (Biolegend, 344818): https://www.biolegend.com/en-us/products/apc-cyanine7-anti-human-cd3-antibody-6940 ; FITC-labeled human BCMA Fc tag protein (Acrobiosystems, BCA-HF254): https://www.acrobiosystems.cn/P875-FITC-Labeled_Human_BCMA_%7C_TNFRSF17_Protein_Fc_Tag.html .

Techniques: Digital PCR, Staining, Labeling

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: Schematic of BCMA-Nb radiolabeling with (A) [ 18 F]FPy and (B) [ 131 I]I.

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Radioactivity

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Kinetic binding assay of BCMA nanobody to human BCMA protein measured by biolayer interferometry using 100nM anti-BCMA-nanobody (B) Representative flow cytometry histograms of BCMA expression in MC38-BCMA and MC38-WT cells (C) Immunohistochemistry and Immunofluorescent staining of BCMA in excised tumors (D) Saturation binding assays of [ 18 F]FPy-BCMA Nb in MC38-BCMA cells (E) Representative PET/CT coronal images of MC38-BCMA and MC38-WT tumor model (150-250 mm 3 ), injected with 3.7 MBq [ 18 F]FPy-BCMA-Nb at 2 h post-injection. PET scans are normalized to the same scale. Data are presented as mean ± SD (n = 6 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Binding Assay, Flow Cytometry, Expressing, Immunohistochemistry, Staining, Positron Emission Tomography-Computed Tomography, Injection

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Flow cytometry analysis of BCMA expression in H929 and RPMI8226 cell lines. (B) Immunofluorescent staining of H929 cells using anti-BCMA antibody (green) and DAPI (blue). Saturation binding assay to assess the specific binding and dissociation constant of [ 18 F]FPy-BCMA-Nb in (C) H929 and (D) RPMI8226 cells. (E) Internalization of [ 18 F]FPy-BCMA-Nb in H929 cells over 180 minutes. (F) 3 h post-injection uptake of [ 18 F]FPy-BCMA-Nb in the femur/knee region and (G) spine of the human H929 and RPMI8226 MM model compared to healthy mice and blocking. White arrows indicate tumor cells in the bone marrow. Relative quantitative uptake of the tracer in the H929 human MM model with higher BCMA expression in spine, femur, arm, skull bone marrow, and kidney under (H) pre-blocking and (I) post-blocking conditions . Data are presented as mean ± SD (n = 6 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Flow Cytometry, Expressing, Staining, Saturation Assay, Binding Assay, Injection, Blocking Assay

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Schematic illustration of the timeline, treatment regimen and efficacy study for the radiotherapy study. (B) Representative BLI images of mice pre- and post-treatment regimen and (C) overall survival percentage across all groups, including Control (PBS), 7.4 MBq and 18.5 MBq of [ 131 I]I-BCMA-Nb, compared to 18.5 MBq of [¹³¹I]I-non-specific-Nb treated cohorts, as determined by Kaplan-Meier analysis. Data are presented as mean ± SD (n = 8 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Control

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Representative Sagittal [ 18 F]FDG PET images of healthy mice compared to treatment groups (PBS, 7.4 MBq and 18.5 MBq of [ 131 I]I-BCMA-Nb,) acquired pre- and post-treatment regimen. Quantitative analysis of [ 18 F]FDG uptake in bone marrow of the (B) spine, (C)femur, (D) arm, and (E) skull across all groups pre-, during and post-treatment. Gray areas are defined as baseline based on [ 18 F]FDG of healthy mice. Data are presented as mean ± SD (n = 8 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques:

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: Changes in (A) body weight during the treatment regimen. (B) Complete cell blood counts for mice pre- and post-treatment in all groups including PBS, injected with 7.4 MBq and 18.5 MBq of [ 131 I]I-BCMA-Nb and 18.5 MBq of [ 131 I]I-non-specific-Nb. Orange-shaded cells indicate values significantly different from standard NSG mouse ranges (p<0.05). Data are presented as mean ± SD (n = 8 mice per group).

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Injection

Journal: Theranostics

Article Title: Precision radiolabeled B-cell maturation nanobody for targeted PET imaging and radioligand therapy of disseminated multiple myeloma

doi: 10.7150/thno.126920

Figure Lengend Snippet: (A) Immunohistochemistry staining and (B) quantitative analysis of BCMA, (C) immunofluorescence staining for and (D) quantitative analysis of Ki67 proliferation marker in femur and spine bone marrow sections of H929 human MM-bearing mice, before and after treatment, compared with healthy mice.

Article Snippet: Biolayer interferometry studies were conducted similarly to the published procedure using 1ug/mL of biotinylated human BCMA protein (ACROBiosystems, BCA-H82E4) and 100nM concentration of BCMA-Nb in PBS as an eluent .

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Marker

Journal: STAR Protocols

Article Title: Multi-CUT&Tag to simultaneously profile multiple chromatin factors

doi: 10.1016/j.xpro.2021.101100

Figure Lengend Snippet:

Article Snippet: Dimethylformamide (DMF) , Cell Signaling Technology , Cat#12767S.

Techniques: Virus, Bacteria, Recombinant, Produced, Saline, Protease Inhibitor, Isolation, Magnetic Beads, Control, Library Quantification, Sequencing, Software

Journal: Nature biomedical engineering

Article Title: Enhanced intratumoural activity of CAR T cells engineered to produce immunomodulators under photothermal control

doi: 10.1038/s41551-021-00781-2

Figure Lengend Snippet: a , Schematic representation of transduced primary human T cells and thermal delivery profiles. b , Gluc activity of the 7H-YB thermal switch in primary human T cells after continuous (light grey) and pulsed (dark grey) heat treatments with temperatures, total durations and heating profiles as indicated. * P = 0.033; ** P = 0.0021; two-tailed t -test; mean ± s.e.m. is depicted; n = 3 biologically independent wells. c , PI and AnV flow staining of CD3 + T cells. Bars represent viable populations (PI − AnV − ) normalized to unheated samples. * P = 0.0455; ** P = 0.0034; **** P < 0.0001; two-way ANOVA and Šidák post-test and correction; mean ± s.e.m. is depicted; n = 3 biologically independent wells; FSC-A, forward scatter area; SSC-A, side scatter area. Two independent experiments were performed with similar results. d , CellTrace Violet (CTV) flow histograms of T cells after heat treatments and incubation with CD3/28 beads at a 3:1 bead to T cell ratio. Values in histogram boxes represent percentage in indicated gate. Two independent experiments were performed with similar results. e , Number of cells in lower well of a transwell plate containing CXCL12. T cells were heated and loaded into the top well before sampling at indicated timepoints. Two-way ANOVA and Tukey post-test and correction; mean ± s.e.m. is depicted; n = 3 biologically independent wells. Two independent experiments were performed with similar results. f , Percent cytotoxicity observed in CD19− or CD19+ luciferized K562 cells after incubation with T cells constitutively expressing CARs after heating, with effector to target ratios as indicated. * P = 0.0166 at a 1:1 CAR T cell to target cell ratio; * P = 0.0251 at a 5:1 CAR T cell to target cell ratio; two-way ANOVA and Šidák post-test and correction; mean ± s.e.m. is depicted; n = 3 biologically independent wells. Two independent experiments were performed with similar results.

Article Snippet: To detect CD19 CAR expression, biotinylated CD19 (10 μg ml −1 ; Acro Biosystems CD9-H8259) and streptavidin-APC (ThermoFisher S868) were used according to the manufacturer’s instructions.

Techniques: Activity Assay, Two Tailed Test, Staining, Incubation, Sampling, Expressing

Journal: Nature biomedical engineering

Article Title: Enhanced intratumoural activity of CAR T cells engineered to produce immunomodulators under photothermal control

doi: 10.1038/s41551-021-00781-2

Figure Lengend Snippet: a , Thermal and luminescence images of wells containing TS-Fluc T cells after irradiation with NIR laser light. Thermal images (left) were acquired using a FLIR thermal camera while luminescence images (right) were acquired using an IVIS Spectrum CT system 6 h after heating. b , Schematic representation of TS-Fluc αCD19 CAR construct transduced into primary human T cells before transfer into NSG mice with two flank (K562 or Raji) tumours followed by photothermal heating of single tumour. c , Thermal images of mouse during laser irradiation of tumour site at 0 and 3 min. d , Kinetic traces (coloured lines) showing average skin temperature of a 3 × 3 pixel region of interest (ROI) centred on laser site. Shaded regions show s.d. of three heating runs. e , Left: luminescence images of heated mice bearing either K562 (CD19−) or Raji (CD19+) tumours. Signal indicates luciferase activity by transferred TS-Fluc T cells. Dotted circles represent ROIs. Right: luminescence of each tumour site relative to the luminescence from the unheated tumour in the same animal. A separate experiment with repeated heating of Raji tumours was conducted to confirm reproducibility of experimental results ( Supplementary Fig. 8 ). f , Mice bearing two Raji (CD19+) tumours with one site heated. Left: luminescence images of excised tumours (heated and unheated) and spleen. Right: quantification of luminescence following heat treatments (0, 6, 12, 18, 24 h). **** P < 0.0001; two-way ANOVA and Tukey post-test and correction; mean ± s.e.m. is depicted; n = 4 to 5 biologically independent mice.

Article Snippet: To detect CD19 CAR expression, biotinylated CD19 (10 μg ml −1 ; Acro Biosystems CD9-H8259) and streptavidin-APC (ThermoFisher S868) were used according to the manufacturer’s instructions.

Techniques: Activation Assay, In Vivo, Irradiation, Construct, Luciferase, Activity Assay

Journal: Nature biomedical engineering

Article Title: Enhanced intratumoural activity of CAR T cells engineered to produce immunomodulators under photothermal control

doi: 10.1038/s41551-021-00781-2

Figure Lengend Snippet: a , Schematic of co-culture assay of heated TS-IL-15 αCD19 CAR T cells and CFSE-labelled wild-type cells. CD3/28 beads were added at 1:10 bead to T cell ratio. b , Representative flow histograms (left), quantified proliferation (middle) and division indices (right) of the CFSE-labelled wild-type T cell population, as calculated by FlowJo proliferation tool, after a 30 min thermal treatment at indicated temperatures. *** P = 0.0009; * P = 0.0352; two-tailed t -test; mean ± s.e.m. is depicted; n = 3 biologically independent wells. c , IL-15 superagonist concentrations in supernatant of TS-IL-15 αCD19 CAR T cells following heat treatments. Temperature and duration of treatments are as indicated. ** P = 0.0032, **** P < 0.0001; two-way ANOVA and Tukey post-test and correction; mean ± s.e.m. is depicted; n = 3 biologically independent wells, comparisons displayed between sample and unheated control. Two independent experiments were performed with similar results. d , Schematic of TS-IL-15 αCD19 CAR vector used to transduce primary human T cells before transfer into tumour-bearing NSG mice. e , Tumour growth curves of CD19+ K562s following transfer of TS-IL-15 αCD19 CAR T cells on day 0 and heat treatments on days 2, 6, 9, 13 and 16. *** P = 0.0004 between cells only and untreated cohort on day 23; * P = 0.0157 between cohorts receiving cells and heat versus cells only on day 20; ** P = 0.0047 between cohorts receiving cells and heat versus cells only on day 23; ** P = 0.0012 between cohorts receiving cells and heat versus cells only on day 27; two-way ANOVA and Tukey post-test and correction; mean ± s.e.m. is depicted; n = 7 biologically independent mice. Three independent experiments were performed with similar results. f , Survival curves of tumour-bearing mice in d and e following transfer of TS-IL-15 αCD19 CAR T cells and heat treatments. ** P = 0.000022, log-rank (Mantel–Cox) test with correction for six multiple comparisons, n = 7 biologically independent mice. g , Schematic of TS-IL-15 vector transduced into primary murine Pmel-1 T cells transferred into tumour-bearing C57BL/6 J mice. h , Tumour growth curves following inoculation of B16-F10 after transfer of TS-IL-15 Pmel-1 T cells on day 0 and heat treatments on days 1 and 3. * P = 0.0438 between untreated and heat-only cohorts on day 10; * P = 0.0166 between cohorts receiving cells and heat versus cells only on day 12; ** P = 0.0058 between cohorts receiving cells and heat versus cells only on day 15; two-way ANOVA and Tukey post-test and correction; mean ± s.e.m. is depicted; n = 6 to 7 biologically independent mice. i , Survival curves of tumour-bearing mice in g and h following transfer of TS-IL-15 Pmel-1 T cells and heat treatments. ** P = 0.000043, log-rank (Mantel–Cox) test with correction for six multiple comparisons, n = 6 to 7 biologically independent mice.

Article Snippet: To detect CD19 CAR expression, biotinylated CD19 (10 μg ml −1 ; Acro Biosystems CD9-H8259) and streptavidin-APC (ThermoFisher S868) were used according to the manufacturer’s instructions.

Techniques: Control, Co-culture Assay, Two Tailed Test, Plasmid Preparation, Transduction

Journal: Nature biomedical engineering

Article Title: Enhanced intratumoural activity of CAR T cells engineered to produce immunomodulators under photothermal control

doi: 10.1038/s41551-021-00781-2

Figure Lengend Snippet: a , Schematic of TS-BiTE and TS-Fluc thermal switches containing heat-triggered BiTE or Fluc reporters. Both constructs contained constitutive αCD19 CARs. b , Histograms for HisTag flow staining in TS-BiTE and TS-Fluc primary T cells following heating. c , NKG2DL flow staining on primary human T cells and K562s using an NKG2D-Fc chimera and an αFc-A488 secondary antibody. Stain, full staining; 2° Ctrl, secondary antibody only. d , Schematic depicting BiTE-mediated targeting of K562 target cells lacking the CAR target antigen via BiTE binding to NKG2DL and CD3. e , Flow-gating strategy for defining bystander cells on the basis of CD19 CAR expression using phycoerythrin (PE) in Jurkat co-culture assays with K562s. UTD controls were gated on the lower (CAR−) population for graphing in g . f , g Flow staining of CD69 on Jurkat T cells following heating and incubation with K562s. TS-BiTE CAR+ histograms ( f ) and summary data of indicated populations ( g ) are graphed. Statistics show comparison to UTD; ** P = 0.0025 between bystander and UTD populations after 30 min heating; ** P = 0.0044 between bystander and UTD populations after 45 min heating; **** P < 0.0001; two-way ANOVA and Tukey post-test and correction; mean + s.e.m. is depicted; n = 3 biologically independent wells. Two independent experiments were performed with similar results. MFI, median fluorescent intensity. h , Cytokine concentrations in supernatant of primary human T cells after heat treatments and incubation with K562 cells. T cells were either UTD or transduced with the indicated thermal switches. **** P < 0.0001; two-way ANOVA and Tukey post-test and correction; mean + s.e.m. is depicted; n = 3 biologically independent wells. i , Cytotoxicity against K562s as quantified by luciferase assay after incubation with primary human T cells. T cells were either UTD or transduced with the indicated thermal switches. ** P = 0.0043 between UTD and TS-BiTE cells; **** P < 0.0001; two-way ANOVA and Tukey post-test and correction; comparisons are to UTD control; mean + s.e.m. is depicted; n = 3 biologically independent wells.

Article Snippet: To detect CD19 CAR expression, biotinylated CD19 (10 μg ml −1 ; Acro Biosystems CD9-H8259) and streptavidin-APC (ThermoFisher S868) were used according to the manufacturer’s instructions.

Techniques: Construct, Staining, Binding Assay, Expressing, Co-Culture Assay, Incubation, Comparison, Transduction, Luciferase, Control

Journal: Cell Death & Disease

Article Title: Dimethyl fumarate promotes the degradation of HNF1B and suppresses the progression of clear cell renal cell carcinoma

doi: 10.1038/s41419-025-07412-7

Figure Lengend Snippet: A CCK-8 assays of the proliferation of 786-O and RCC4 cells treated with a serial dose of DMF as indicated. The data represent the mean ± SD of n = 3 independent experiments. Statistical differences were determined by one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001. B Colony formation assays and statistical analysis of the proliferation of 786-O and RCC4 cells treated with a serial dose of DMF as indicated. The data represent the mean ± SD of n = 3 independent experiments. Statistical differences were determined by one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, *** P < 0.0001. C Western blot analysis of 786-O and RCC4 cells stably overexpressing Flag-HNF1B. D Western blot analysis of exogenous Flag-HNF1B in 786-O and RCC4 cells treated with or without DMF (50 μM, 12 h). E Western blot analysis of endogenous HNF1B in 786-O and RCC4 cells treated with or without DMF (50 μM, 12 h). F Western blot analysis of HNF1B treated with 50 μM DMF for different times as indicated. G Western blot analysis of HNF1B treated with increasing concentrations of DMF (0 to 100 μM) for 12 h. qPCR analysis of CRB3 , KIF12 and PKHD1 levels in 786-O ( H ) and RCC4 ( I ) cells treated with or without 50 μM DMF for 12 h.

Article Snippet: DMF (624-49-7), CCK-8 (C0005), sunitinib (557795-19-4), MLN4924 (905579-51-3) were purchased from TargetMol (Shanghai, China).

Techniques: CCK-8 Assay, Western Blot, Stable Transfection

Journal: Cell Death & Disease

Article Title: Dimethyl fumarate promotes the degradation of HNF1B and suppresses the progression of clear cell renal cell carcinoma

doi: 10.1038/s41419-025-07412-7

Figure Lengend Snippet: A qPCR analysis of HNF1B level in 786-O and RCC4 cells treated with or without 50 μM DMF for 12 h. B HNF1B mRNA stability under DMF treatment (50 μM) was determined in 786-O and RCC4 cells treated with 5 μg/mL actinomycin D for different time points as indicated. n = 3 biologically independent experiments per group. C The half-life of HNF1B protein under DMF treatment was determined in the presence of 100 μg/mL cycloheximide (CHX) in 786-O and RCC4 cells. Western blot analysis of HNF1B level in 786-O ( D ) and RCC4 ( E ) cells treated with DMF in the absence or presence of lysosome inhibitor NH 4 Cl (20 mM, 12 h), or proteasome inhibitor MG132 (10 μM, 12 h). The quantifications are shown below. The data represent the mean ± SD of n = 3 independent experiments. Statistical differences were determined by one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001. F Flag-HNF1B and HA-Ub were co-transfected into HEK-293T cells with or without DMF treatment. HNF1B ubiquitination level was determined by western blot analysis. G Western blot analysis of endogenous HNF1B ubiquitination level in 786-O and RCC4 cells treated with DMF treatment. H Western blot analysis of HNF1B level in 786-O and RCC4 cells treated with DMF in the absence or presence of 0.5 μM MLN4924. I Identification of potential HNF1B-interacting deubiquitinases using BioGRID. J HEK-293T cells were transfected with indicated constructs for 48 h. Cells were collected for coimmunoprecipitation analysis. K Western blot analysis of Flag-HNF1B in HEK-293T cells stably expressing Flag-hnf1b transfected with HA-BAP1, HA-USP54, or HA-OTUD3. L Co-IP analysis of interactions between Flag-HNF1B and HA-OTUD3 in HEK-293T cells with or without DMF treatment.

Article Snippet: DMF (624-49-7), CCK-8 (C0005), sunitinib (557795-19-4), MLN4924 (905579-51-3) were purchased from TargetMol (Shanghai, China).

Techniques: Western Blot, Transfection, Ubiquitin Proteomics, Construct, Stable Transfection, Expressing, Co-Immunoprecipitation Assay

Journal: Cell Death & Disease

Article Title: Dimethyl fumarate promotes the degradation of HNF1B and suppresses the progression of clear cell renal cell carcinoma

doi: 10.1038/s41419-025-07412-7

Figure Lengend Snippet: A 786-O and RCC4 cells were individually divided into four groups, treated with vehicle, DMF, sunitinib, DMF plus sunitinib, respectively, then subjected to CCK-8 analysis to determine cell proliferation. B Colony formation assays and statistical analysis of the proliferation of 786-O and RCC4 cells treated with vehicle, DMF, sunitinib, DMF plus sunitinib, respectively. The data represent the mean ± SD of n = 3 independent experiments. Statistical differences were determined using ordinary one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant. C Schematic representation of the animal experiment. D 786-O cells were employed to mouse xenograft model and the mouse was administrated with DMF and/or sunitinib as indicated. Tumors were dissected from each group. E Tumor growth of each group was monitored. Statistical differences were determined using ordinary one-way ANOVA. **** P < 0.0001, ns, not significant. F The weight of tumors from each group was analyzed. Statistical differences were determined using ordinary one-way ANOVA. *** P < 0.001, **** P < 0.0001, ns, not significant. G IHC staining of HNF1B and Ki67 in xenograft tumors. H Quantifications of IHC images. Statistical differences were determined using ordinary one-way ANOVA. **** P < 0.0001, ns, not significant. I Working model depicting the mechanism of DMF-mediated HNF1B degradation and ccRCC inhibition.

Article Snippet: DMF (624-49-7), CCK-8 (C0005), sunitinib (557795-19-4), MLN4924 (905579-51-3) were purchased from TargetMol (Shanghai, China).

Techniques: CCK-8 Assay, Immunohistochemistry, Inhibition