Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: MTFR2-Mediated Fission Drives Fatty Acid and Mitochondrial Co-Transfer from Hepatic Stellate Cells to Tumor Cells Fueling Oncogenesis.

doi: 10.1002/advs.202416419

Figure Lengend Snippet: Figure 3. Activated HSCs enhance fatty acid (FA) synthesis and transfer to cancer cells, promoting proliferation. A) Western blot showing the ACC1 and CPT1A level of Huh7 cells and Huh7 cells cocultured with aHSCs (n = 3). B) Schematic of the experimental setup for FA chase pulse assay. HSCs were incubated in complete media (CM) with red fluorescent FA (Red C12) for 16 h (“pulse”). Following this, HSCs were cultured for an additional 72 h in CM or tumor-conditioned media (TCM) without the labeled FA (“chase”). Mitochondria and lipid drops (LDs) were stained before imaging. C,D) FA localization was assayed as described in (A) and chased in CM or TCM. C) LDs were labeled using BODIPY 493/503 (gray) and mitochondria were labeled using MitoTracker Far Red (green). Scale bar = 25 μm. D) Relative cellular localization of Red C12 was quantified by Pearson’s coefficient analysis

Article Snippet: For FA transfer assays, HSCs were preloaded with 1 × 10−3 m Red C12 (BODIPY 558/568 C12, MCE, Catalog No. HY-138226) in CM for 16 h, then rigorously washed three times with CM and equilibrated for 1 h to remove extracellular dye aggregates.

Techniques: Western Blot, Incubation, Cell Culture, Labeling, Staining, Imaging

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: MTFR2-Mediated Fission Drives Fatty Acid and Mitochondrial Co-Transfer from Hepatic Stellate Cells to Tumor Cells Fueling Oncogenesis.

doi: 10.1002/advs.202416419

Figure Lengend Snippet: Figure 5. Complementary roles of FAs transfer and mitochondrial transport in supporting tumor cell survival. A) Representative confocal images of Huh7 cells cocultured with aHSCs under L-778123 and NTL treatment. Maximum intensity projections (MIP) of confocal Z-stacks showing Huh7 cells (CellTrace green, green) cocultured with aHSCs pre-labeled with Red C12 fluorescent FA (red) (Upper panels). Corresponding 3D surface renderings (Imaris) of the boxed regions (Lower panels). Scale bars: 15 μm. B) Graph showing the fluorescence intensity of FAs in CellTrace green labeled Huh7 cells (Data are represented as mean ± SD, n = 6, at least 50 recipient cells analyzed per replicate, one-way ANOVA was performed). C) Flow cytometry analysis of fluorescence intensity of FAs in FITC-positive Huh7 cells. D) Confocal images showing the transportation of mitochondria from aHSCs to Huh7 cells. (Upper panels) MIP: Confocal Z-stacks of cocultured MitoTracker-labeled mitochondria in aHSCs (red) and CellTrace green-labeled Huh7 cells (green), with ActinTracker (cyan) highlighting tunneling nanotubes (TNTs). (Lower panels) 3D surface reconstruction (Imaris). Scale bars: 15 μm. E) Quantification of fluorescence intensity from Figure D, showing the uptake of HSC-derived mitochondria by Huh7 cells (Data are represented as mean ± SD, n = 6, at least 50 recipient cells analyzed per replicate, one-way ANOVA was performed). F) Fluorescence histograms of Huh7 cells (labeled

Article Snippet: For FA transfer assays, HSCs were preloaded with 1 × 10−3 m Red C12 (BODIPY 558/568 C12, MCE, Catalog No. HY-138226) in CM for 16 h, then rigorously washed three times with CM and equilibrated for 1 h to remove extracellular dye aggregates.

Techniques: Labeling, Flow Cytometry, Derivative Assay, Fluorescence

Journal: Cell & Bioscience

Article Title: The miR-34/449 clusters regulate the differentiation of ciliated cells in the oviduct via the Wnt/β-catenin signaling pathway

doi: 10.1186/s13578-025-01468-w

Figure Lengend Snippet: Wnt/β-catenin inhibitors rescue the ciliary differentiation defects in the miR-dKO oviductal epithelial organoids. A Schematic diagram of the oviductal epithelial organoids culture. B Representative images of the culture of oviductal epithelial organoids derived from the miR-dKO mice with or without Wnt signaling pathway inhibitors (XAV-939, NSC668036) and those derived from control (CON) mice. Scale bars: 200 μm. The number and size of the organoids were counted/measured on culture day 4 (B), day 8 ( D ), and day 16 ( E ). F The total number of organoids was counted on culture days 8 and 16. G Representative images of co-immunofluorescent staining of Ac-α-tubulin (indicating ciliated cells) and PAX8 (indicating secretory cells) on organoid sections of the control (CON) mice and the miR-dKO mice treated with the Wnt signaling pathway inhibitors or not for 2 months. Scale bars: 50 μm. The box in the lower column shows the higher magnification of the box area in the picture. H Quantification of Ac-α-tubulin + cells in the oviductal epithelial cells of the organoids. I Relative mRNA expression levels of β-catenin , Foxj1 , Pax8 , Dvl2 , Cdk1 , and Cyclin D1 . J The activity of the Wnt signaling pathway in the oviductal epithelial organoids before and after treatment with the Wnt/β-catenin signaling pathway inhibitor was detected by the TOPflash/FOPflash assay. Note that the blue columns in the figure represent the CON group, the purple columns represent the dKO group, the pink columns represent the dKO + XAV939 group and the green columns represent the dKO + NSC668036 group. All the data are presented as the mean ± SD, n = 3. *: p < 0.05; **: p < 0.01

Article Snippet: For organoids treated with Wnt signaling pathway inhibitor XAV-939(MedChemExpress, catalog no. HY-15147, USA) or NSC668036 (MedChemExpress, catalog no. HY-117666, USA), XAV-939 or NSC668036 was added at a concentration of 3 μM or 10 μM into BET organoid medium and organoids were continuously exposed to the inhibitors over the culture period.

Techniques: Derivative Assay, Control, Staining, Expressing, Activity Assay

Journal: Science Advances

Article Title: A deubiquitination module essential for T reg fitness in the tumor microenvironment

doi: 10.1126/sciadv.abo4116

Figure Lengend Snippet: ( A ) USP mRNA level in iT reg cells in control T cell medium compared to addition of tumor cell–treated medium at 50:50 with T cell medium for 24 hours. Usp22 : control: n = 14, B16: n = 10, LLC1: n = 5, and EG7: n = 4. Usp21 : control: n = 12, B16: n = 8, LLC1: n = 3, and EG7: n = 3. Usp7 : control: n = 10, B16: n = 7, LLC1: n = 4, and EG7: n = 5. ( B ) USP protein level in iT reg cells in control T cell medium compared to addition of tumor cell–treated medium at 50:50 with T cell medium for 24 hours. The relative protein expression levels were quantified using Bio-Rad Image software and normalized with the loading controls. ( C ) USP mRNA level in iT reg cells with the addition of a TGF-β inhibitor in tumor cell medium. Usp22 : control: n = 22, B16: n = 15, B16 + inhibitor (inh): n = 5, LLC1: n = 10, LLC1 + inh: n = 5, EG7: n = 7, and EG7 + inh: n = 5. Usp21 : control: n = 20, B16: n = 13, B16 + inh: n = 5, LLC1: n = 8, LLC1 + inh: n = 4, EG7: n = 7, and EG7 + inh: n = 5. Usp 7: control: n = 14, B16: n = 10, B16 + inh: n = 5, LLC1: n = 8, LLC1 + inh: n = 3, EG7: n = 8, and EG7 + inh: n = 6. ( D ) SMAD2, SMAD3, and SMAD4 binding capacity along the Usp 22 promoter under TGF-β inhibition. SMAD2: n = 4 to 5, SMAD3: n = 3, and SMAD4: n = 3. (A to C) All mRNA values were calculated relative to untreated WT iT reg cells. (A to D) Ordinary one-way ANOVA with multiple comparisons was performed to determine significance. All data are presented as means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The TGF-β inhibitor LY 3200882 (MedChem Express, catalog no. HY-103021) was added at 25 μg/ml where indicated.

Techniques: Expressing, Software, Binding Assay, Inhibition

Journal: Science Advances

Article Title: A deubiquitination module essential for T reg fitness in the tumor microenvironment

doi: 10.1126/sciadv.abo4116

Figure Lengend Snippet: ( A ) Structure of compound CS30 (Usp22i-S02). ( B ) Foxp3 MFI in WT and 22KO of T reg cells after treatment with Usp22i-S02 (20 μg/kg) in vivo ( n = 3). ( C ) Representative flow cytometry plot of Foxp3 + CD25 + MFI of CD4 + peripheral cells of mice treated with Usp22i-S02 (20 μg/kg) relative to control ( n = 5). ( D ) Graphical representation of Foxp3 MFI upon Usp22i-S02 administration ( n = 5). ( E ) Tumor growth curve of LLC1 cells subcutaneously injected in the flank of WT mice with or without the addition of the Usp22 inhibitor (20 mg/kg per time) starting at day 15 in 100 μl of oil ( n = 4). ( F and G ) Representative flow cytometry plot and graphical representation of % infiltration of CD4 + and CD8 + T cells within the tumor ( n = 4). ( H ) Representative histogram plot and graphical representation of itT reg Foxp3 MFI ( n = 4). ( I ) MFI of itT reg -suppressive markers ( n = 3 to 4). ( J ) Percent Foxp3 + IFN-γ + itT reg cells in control and Usp22-S02–treated mice ( n = 3 to 4). ( K ) Proposed model: TME-specific factors can drive increased levels of Usp22 and Usp21 potentially through modulation of TGF-β signaling, HIF1-α, AMPK, and mTOR activity to render T reg cells more stable in the tumor microenvironment. (D, E, and G to I) Two-way ANOVA with multiple comparisons between rows was performed to determine statistical significance. (J) Unpaired two-tailed t test was performed to determine significance. All data are presented as means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The TGF-β inhibitor LY 3200882 (MedChem Express, catalog no. HY-103021) was added at 25 μg/ml where indicated.

Techniques: In Vivo, Flow Cytometry, Injection, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: MALT1 protease inhibition restrains glioblastoma progression by reversing tumor-associated macrophage-dependent immunosuppression

doi: 10.1101/2024.09.26.614808

Figure Lengend Snippet: (a) Induction of MALT1 protease activity or (b) NF-κB activity in RAW264.7 macrophages co-cultured with GL261-GBM cells. Macrophages were pretreated for 2 hours with or without the MALT1-protease inhibitor MLT-748 (MALT1i, 5 µM) or the CARD9 inhibitor BRD5529 (CARD9i, 50 µM) prior to co-culture. MALT1-GloSensor luciferase reporter macrophages and RAW-Blue macrophages which harbor an NF-κB-inducible secreted embryonic alkaline phosphatase reporter gene were used to measure MALT1 protease or NF-κB activity, respectively, after 48 hours of co-culture. Results are shown as fold change relative to control macrophages cultured alone. (c) Cytokines and chemokines in the cell culture supernatant were measured by cytometric bead array 48 h after co-culturing primary mouse macrophages with GL261-GBM cells in the absence or presence of MALT1i. Cytokine levels are presented as a heat map indicating fold change relative to the control macrophages alone. *Significant difference compared to macrophage control alone. # Significant difference compared to macrophage + GBM co-culture. (d,e) Changes in macrophage M1/M2-like polarization markers after co-culture with GBM cells. (d) RAW264.7 mouse macrophages were co-cultured with GL261 mouse GBM cells in the absence or presence of MLT-748 (MALT1i, 5 µM). (e) RAW264.7 macrophages were cocultured with GL261 cells in the absence or presence of BRD5529 (CARD9i, 50 µM). Macrophages within the co-cultures were identified by gating for CD11b positive cells. (f-h) The Incucyte® live-cell analysis system was used to measure tumor cell killing in real-time. A dual color monitoring system tracked tumor cell killing by using fluorescently labeled tumor cells (red-Cell Tracker) and caspase-3/7 reagent (green) to track apoptosis. (f) Fluorescent images of RAW264.7 macrophages co-cultured with GBM-GL261 tumor cells -/+ MALT1i. Apoptotic tumor cells are indicated by yellow. (g) Co-cultures of mouse RAW264.7 macrophages with GL261-GBM cells were performed to compare macrophage-dependent tumor cell apoptosis in the absence/presence of MALT1i. (h) Controls demonstrate that MALT1i has no effect on apoptosis of GL261-GBM tumor cells or RAW264.7 macrophages when either are cultured alone. All values are represented as mean ± SD. Data were analyzed by 1-way ANOVA, followed by Tukey’s multiple-comparisons. P values are indicated in the figure.

Article Snippet: In experiments with the MALT1 inhibitor, macrophages were exposed to MLT-748 (5 µM, catalog no.: HY-115466, Med Chem Express) or BRD5529 (50 µM, catalog no.: HY-115497) for 2 hours prior to addition of tumor cells and during the entire co-culture time.

Techniques: Activity Assay, Cell Culture, Protease Inhibitor, Co-Culture Assay, Luciferase, Control, Cell Analysis, Labeling

Journal: bioRxiv

Article Title: MALT1 protease inhibition restrains glioblastoma progression by reversing tumor-associated macrophage-dependent immunosuppression

doi: 10.1101/2024.09.26.614808

Figure Lengend Snippet: ( a ) Schematic of macrophage cell migration assay. RAW264.7 macrophage cell migration was monitored in a real-time scratch assay, using the IncuCyte system. Blue colored cells are meant to represent macrophages that have migrated into the scratch wound. (b) Conditioned media (CM) from GL261 GBM cells enhances migration of macrophages in comparison to DMEM control. ( c-e ) Macrophages were exposed to GL261-GBM CM and treated with increasing concentrations of MLT-748 (c), mepazine (d) or BRD5529 (e). Control cells were exposed to GL261-CM with equivalent volumes of DMSO. Wound density (closure) is plotted as a continuous function of time. ( f) Schematic of transwell migration assay. (g) GL261 GBM tumor cells induce the migration of primary macrophages isolated from WT mice. (h) MALT1 protease inhibitors (MLT-748 or mepazine) and CARD9 inhibitor (BRD5529) each prevent GBM cell-induced macrophage transwell migration. (i) Primary macrophages from WT, MALT1-PD or MALT1-KO were tested for chemotactic migration towards GL261-GBM cells in the bottom chamber. Data shown is from three independent experiments. The P values were calculated by One-way ANOVA.

Article Snippet: In experiments with the MALT1 inhibitor, macrophages were exposed to MLT-748 (5 µM, catalog no.: HY-115466, Med Chem Express) or BRD5529 (50 µM, catalog no.: HY-115497) for 2 hours prior to addition of tumor cells and during the entire co-culture time.

Techniques: Cell Migration Assay, Migration, Wound Healing Assay, Comparison, Control, Transwell Migration Assay, Isolation