Journal: Nature cancer

Article Title: Autophagy Inhibition by Targeting PIKfyve Potentiates Response to Immune Checkpoint Blockade in Prostate Cancer

doi: 10.1038/s43018-021-00237-1

Figure Lengend Snippet: (a) The percentage viabilities of DU145 cells treated with ESK981 or 167 other tyrosine kinase inhibitors (all at 300 nM) when compared to a DMSO vehicle control in a long term survival assay. The top five most inhibitory compounds, as well as cabozantinib and crizotinib (highlighted in orange), and their respective targets are indicated. ESK981 is highlighted in red. (b) Morphological differences of nuclear-restricted RFP-expressing DU145 cells treated with increasing concentrations of ESK981, crizotinib, or cabozantinib for 24 hours. Fluorescence and phase contrast images were taken by IncuCyte ZOOM from three independent experiments, with representative images shown. (c) A long-term survival assay was used to calculate the half-maximum inhibitory concentration (IC50) after two weeks of incubation with the serial dilutions of indicated drugs. IC50 of ESK981, crizotinib, and cabozantinib in a panel of prostate cancer cell lines are plotted as mean ± SEM from three independent experiments. (d) ESK981 was effective against enzalutamide (Enza)-resistant cell lines. LNCaP-AR and CWR-R1 enzalutamide-resistant cells were maintained in 5 μM and 20 μM enzalutamide medium, respectively, in vitro. Long-term survival (two weeks) was assayed by absorbance of crystal violet at OD.590. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated. (e) VCaP-RFP cells were cultured for three days in ultralow attachment plates to form 3D tumor spheroids prior to the indicated drug treatments. Increasing concentrations of ESK981 and cabozantinib were added over the indicated time period. Fluorescence intensity of 3D spheroids was measured by IncuCyte ZOOM.

Article Snippet: PC3 and DU145 cells were stably transfected with pMRX-IP-GFP-LC3-RFP-LC3ΔG (Addgene #84572), and single-cell clones were validated to avoid homologous recombination between the two LC3 fragments during retrovirus infection.

Techniques: Control, Clonogenic Cell Survival Assay, Expressing, Fluorescence, Concentration Assay, Incubation, In Vitro, Two Tailed Test, Cell Culture

Journal: Nature cancer

Article Title: Autophagy Inhibition by Targeting PIKfyve Potentiates Response to Immune Checkpoint Blockade in Prostate Cancer

doi: 10.1038/s43018-021-00237-1

Figure Lengend Snippet: (a) Morphology of DU145 and PC3 cells after siNC, siPIKFYVE, siPIP5K1C, or siPIK3CA transfection from three independent experiments. (b) mRNA levels of PIKFYVE, PIP5K1C, and PIK3CA were measured by qPCR after siRNA knockdown of indicated targets in DU145 and PC3 cells. Data were analyzed by two-tailed unpaired t test and presented as mean ± SEM from three independent experiments. P-value indicated. (c) Morphological changes of TRAMP-C2, ID8, and Ae17 cells after siNC or siPikfyve transfection from three independent experiments. (d) Autophagosome induction activity measured with CYTO-ID® assay in TRAMP-C2, ID8, and Ae17 cells after siRNA knockdown of Pikfyve. Data were analyzed by two-tailed unpaired t test and presented as mean ± SEM from four (TRAMP-C2 and ID8) and six (Ae17) independent experiments. P-value indicated.

Article Snippet: PC3 and DU145 cells were stably transfected with pMRX-IP-GFP-LC3-RFP-LC3ΔG (Addgene #84572), and single-cell clones were validated to avoid homologous recombination between the two LC3 fragments during retrovirus infection.

Techniques: Transfection, Knockdown, Two Tailed Test, Activity Assay

Journal: Nature cancer

Article Title: Autophagy Inhibition by Targeting PIKfyve Potentiates Response to Immune Checkpoint Blockade in Prostate Cancer

doi: 10.1038/s43018-021-00237-1

Figure Lengend Snippet: (a) Schematic illustration of the VCaP CRPC mouse xenograft experimental design. To generate castration-resistant VCaP, parental VCaP cells were injected subcutaneously into both flanks of intact male mice. When average VCaP tumors reached 200 mm3, mice were surgically castrated and VCaP tumors regressed due to loss of androgen. Castration-resistant VCaP tumors developed as VCaP tumors grew back to the size of pre-castration. Castration-resistant VCaP tumors were then randomized into three groups and treated with vehicle, 30 mg/kg, or 60 mg/kg ESK981 p.o., oral gavage. (b) Representative IHC images for proliferation marker Ki67 are shown after treatment with the indicated drugs for five days in VCaP tumors (left). Quantification of positive Ki67 percentage is shown on the right (right). Data were analyzed by two-tailed unpaired t test and presented as mean ± SEM. N=4 tumors per group. P-value indicated. (c) Representative individual tumors from vehicle and ESK981 groups in AR+ and ERG+ prostate PDX MDA-PCa-146-12 (left). Representative IHC showing Ki67 staining for vehicle and 30 mg/kg ESK981 groups of MDA-PCa-146-12 tumors (right) from three independent experiments. (d) Representative individual tumors from vehicle and ESK981 groups of DU145 tumors (left). Representative IHC showing Ki67 staining for the vehicle and 30 mg/kg ESK981 groups of DU145 tumors (right) from three independent experiments.

Article Snippet: PC3 and DU145 cells were stably transfected with pMRX-IP-GFP-LC3-RFP-LC3ΔG (Addgene #84572), and single-cell clones were validated to avoid homologous recombination between the two LC3 fragments during retrovirus infection.

Techniques: In Vivo, Injection, Marker, Two Tailed Test, Staining

Journal: Nature cancer

Article Title: Autophagy Inhibition by Targeting PIKfyve Potentiates Response to Immune Checkpoint Blockade in Prostate Cancer

doi: 10.1038/s43018-021-00237-1

Figure Lengend Snippet: (a) Castration-resistant VCaP tumors (VCaP CRPC) were established subcutaneously in castrated male SCID mice, and treated with vehicle, 30 mg/kg, or 60 mg/kg ESK981. Average tumor volumes were monitored twice per week, n=number of tumors (left). Individual VCaP CRPC tumor weights were measured at study endpoint, n=number of tumors (middle). Percent body weight changes and dosing schedule of VCaP CRPC model, n=number of mice (right). Data were analyzed by two-tailed unpaired t test and presented as mean ± SEM. N and P-value indicated. (b) Androgen receptor (AR)+ and ERG+ prostate patient-derived xenograft (PDX) MDA-PCa-146-12 were established subcutaneously in non-castrated SCID mice and treated with vehicle or 30 mg/kg ESK981. Average tumor volumes were monitored twice per week, n=number of tumors (left). Tumor weights from individual tumors at study endpoint, n=number of tumors (middle). Percent body weight changes of MDA-PCa-146-12 tumor-bearing mice, n=number of mice (right). Data were analyzed by two-tailed unpaired t test and presented as mean ± SEM. N and P-value indicated. (c) DU145 tumors were established subcutaneously in non-castrated SCID mice and treated with vehicle or 30 mg/kg ESK981. Average tumor volumes were monitored twice per week, n=number of tumors (left). Individual tumor weights were measured at study endpoint, n=number of tumors (middle). Percent body weight changes of DU145 tumor-bearing mice, n=number of mice (right). Data were analyzed by two-tailed unpaired t test and presented as mean ± SEM. N and P-value indicated. (d) Neuroendocrine (NEPC) prostate PDX MDA-PCa-146-10 were established in non-castrated SCID mice and treated with vehicle or 30 mg/kg ESK981. Tumor volumes were monitored twice per week, n=number of tumors (left). Individual tumor weights were measured at study endpoint, n=number of tumors (middle). Percent body weight changes of MDA-PCa-146-10 tumor-bearing mice, n=number of mice (right). Data were analyzed by two-tailed unpaired t test and presented as mean ± SEM. N and P-value indicated. (e) Representative H&E images from three independent experiments showing a dose-dependent induction of a vacuolization morphology from VCaP CRPC tumors treated with vehicle, 30 mg/kg, or 60 mg/kg ESK981 for five days.

Article Snippet: PC3 and DU145 cells were stably transfected with pMRX-IP-GFP-LC3-RFP-LC3ΔG (Addgene #84572), and single-cell clones were validated to avoid homologous recombination between the two LC3 fragments during retrovirus infection.

Techniques: Two Tailed Test, Derivative Assay

Journal: Nature cancer

Article Title: Autophagy Inhibition by Targeting PIKfyve Potentiates Response to Immune Checkpoint Blockade in Prostate Cancer

doi: 10.1038/s43018-021-00237-1

Figure Lengend Snippet: (a) Representative morphologies of DU145-RFP cells from three independent experiments treated with either ESK981, autophagy inhibitors (3-methyladenine [3-MA], chloroquine [CQ], bafilomycin A1 [BF]), or a combination of ESK981 and one additional autophagy inhibitor for six hours. Red indicates nuclei. (b) VCaP and LNCaP cells were treated with increasing concentrations of ESK981 for 24 hours. Autophagosome induction activity was measured with CYTO-ID®, and the quantification of autophagosomes are shown on the right. Rapamycin served as a positive control for autophagy induction. Data were analyzed by two-tailed unpaired t test from three (LNCaP) and four (VCaP) independent experiments and presented as mean ± SEM. **p<0.01; ***p<0.001. (c) Autophagosome induction activity of ESK981 (in red), measured with CYTO-ID®, when compared to an autophagy-related compound library consisting of 154 compounds (top) or a tyrosine kinase inhibitor library consisting of 167 compounds (bottom). DU145 cells were treated with ESK981 or the other compounds (all at 300 nM) for 24 hours. The top five compounds and their respective targets are indicated. VX-680 and rapamycin are highlighted in orange in autophagy-related compound library. Crizotinib and cabozantinib are highlighted in orange in tyrosine kinase inhibitor library. Targets of highlighted compounds are indicated in parentheses. (d) The indicated prostate cancer cell lines were treated with increasing concentrations of ESK981 for 24 hours. LC3 levels were assessed by western blot, with GAPDH serving as a loading control. (e) Representative images of GFP-LC3 puncta in DU145 cells with 300 nM ESK981 treatment for various times. Quantifications of GFP-LC3 puncta from Ctrl (n=20 analyzed cells), 1h (n=20 analyzed cells), 4h (n=20 analyzed cells), and 24h (n=16 analyzed cells) are shown on the right. Data were analyzed by two-tailed unpaired t test and presented as mean ± SEM. P-value indicated.

Article Snippet: PC3 and DU145 cells were stably transfected with pMRX-IP-GFP-LC3-RFP-LC3ΔG (Addgene #84572), and single-cell clones were validated to avoid homologous recombination between the two LC3 fragments during retrovirus infection.

Techniques: Activity Assay, Positive Control, Two Tailed Test, Drug discovery, Western Blot, Control

Journal: Nature cancer

Article Title: Autophagy Inhibition by Targeting PIKfyve Potentiates Response to Immune Checkpoint Blockade in Prostate Cancer

doi: 10.1038/s43018-021-00237-1

Figure Lengend Snippet: (a) DU145 cells with the indicated drug treatment for 24 hours. Autophagosome induction activity was visualized by CYTO-ID® assay from three independent experiments. Rapa, rapamycin. (b) VCaP cells were treated with 300 nM ESK981 for the indicated time points, and LC3 protein levels were assessed by western blot from three independent experiments. (c) VCaP cells were treated with ESK981 (ESK), crizotinib (Crizo), and cabozantinib (Cabo) at the indicated concentrations. Protein levels of LC3 were examined after 24 hours of treatment from three independent experiments. (d) Protein levels of Atg8 in yeast prd5Δ cells after ESK981 (ESK) or cabozantinib (Cabo) treatment under nitrogen deprivation conditions. NT, no treatment. Data were analyzed by two-tailed unpaired t test from four independent experiments and presented as mean ± SEM. P value indicated. (e) Protein levels of indicated protein post various siRNA knockdown in VCaP and LNCaP cells with or without 300 nM ESK981 or 1 μM sunitinib treatment for 24 hours from three independent experiments.

Article Snippet: PC3 and DU145 cells were stably transfected with pMRX-IP-GFP-LC3-RFP-LC3ΔG (Addgene #84572), and single-cell clones were validated to avoid homologous recombination between the two LC3 fragments during retrovirus infection.

Techniques: Activity Assay, Western Blot, Two Tailed Test, Knockdown

Journal: Nature cancer

Article Title: Autophagy Inhibition by Targeting PIKfyve Potentiates Response to Immune Checkpoint Blockade in Prostate Cancer

doi: 10.1038/s43018-021-00237-1

Figure Lengend Snippet: (a) Representative TEM micrographs of DU145 cells after 300 nM ESK981 treatment for 24 hours from three independent experiments. Micrograph of ESK981-treated cell shows mostly clear vacuoles adjacent to an autophagic vacuole, which is magnified in the red dashed box. Red arrow indicates a mostly clear vacuole. N, nucleus. (b) Representative micrographs of MDA-PCa-146-12 PDX tumors taken by TEM after five days of treatment from three independent experiments. Red arrows indicate vacuoles in ESK981 group, and yellow arrows indicate cellular materials inside the vacuole. N, nucleus. (c) Representative immunofluorescence staining of LAMP1 in DU145 cells treated with control or 300 nM ESK981 for 24 hours from three independent experiments. (d) Lysosomal activity was quantified by FACS after staining with LysoTracker Green. VCaP, LNCaP, PC3, and DU145 cells were treated with increasing concentrations of ESK981 for 24 hours (left). VCaP, LNCaP, PC3, and DU145 cells were treated with DMSO, ESK981 (300 nM), bafilomycin A1 (100 nM), or ESK981-bafilomycin A1 combination for 24 hours (right). (e) Ratio of GFP/RFP signal in PC3 and DU145 GFP-LC3-RFP-LC3ΔG stable expressing cells with the indicated treatment for 24 hours. Data were analyzed by two-tailed unpaired t test from four independent experiments and presented as mean ± SEM. *p<0.05; **p<0.01. (f) Paired MEF cells with either Atg5 wild type (Atg5+/+) or Atg5 knockout (Atg5−/−) were treated with 300 nM ESK981 for 24 hours. Representative morphologies are shown in phase contrast microscopy (left) from three independent experiments. ATG5 and LC3 protein levels were examined by western blot (right).

Article Snippet: PC3 and DU145 cells were stably transfected with pMRX-IP-GFP-LC3-RFP-LC3ΔG (Addgene #84572), and single-cell clones were validated to avoid homologous recombination between the two LC3 fragments during retrovirus infection.

Techniques: Immunofluorescence, Staining, Control, Activity Assay, Expressing, Two Tailed Test, Knock-Out, Microscopy, Western Blot

Journal: Nature cancer

Article Title: Autophagy Inhibition by Targeting PIKfyve Potentiates Response to Immune Checkpoint Blockade in Prostate Cancer

doi: 10.1038/s43018-021-00237-1

Figure Lengend Snippet: (a) CXCL10 protein levels measured by ELISA in conditioned media from VCaP cells treated with ESK981 or various autophagy inducers for 24 hours. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated. (b) CXCL10 mRNA levels measured by quantitative PCR (qPCR) in VCaP, PC3, and DU145 cells with the indicated treatment for 24 hours. IFNγ, interferon gamma. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated. (c) IC50 of ESK981, crizotinib, and cabozantinib determined in Myc-CaP cells. (d) Protein levels of LC3 after 50 nM, 100 nM, and 300 nM ESK981 treatment for 24 hours in Myc-CaP cells from three independent experiments. (e) Ratio of GFP/RFP signal in Myc-CaP GFP-LC3-RFP-LC3ΔG stable expressing cells with the indicated treatment for 24 hours. Data were analyzed by two-tailed unpaired t test from four independent experiments and presented as mean ± SEM. P-value indicated.

Article Snippet: PC3 and DU145 cells were stably transfected with pMRX-IP-GFP-LC3-RFP-LC3ΔG (Addgene #84572), and single-cell clones were validated to avoid homologous recombination between the two LC3 fragments during retrovirus infection.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: Nature cancer

Article Title: Autophagy Inhibition by Targeting PIKfyve Potentiates Response to Immune Checkpoint Blockade in Prostate Cancer

doi: 10.1038/s43018-021-00237-1

Figure Lengend Snippet: (a) Gene ontology analysis for top elevated genes after 300 nM ESK981 in VCaP cells for 6 and 24 hour treatments. The top three processes are listed. (b) Heatmap representation of untargeted lipidomics analysis after 300 nM ESK981 treatment for 6 and 24 hours in VCaP cells. N=3 technical replicates per group. The PE class is highlighted in red. (c) Percent inhibition of 1 μM ESK981 against a panel of 22 lipid kinases. Data are presented as mean with individual data points. (d) Representative dissociation constant (Kd) curve of ESK981 against lipid kinases PIKfyve, PIP5K1C, PIP5K1A, and PIK3CA. (e) Representative morphology of DU145-RFP cells with control siRNA or PIKFYVE siRNA with three independent experiments. (f) Fluorescent images showing autophagosome levels measured with CYTO-ID® assay in DU145, PC3, LNCaP, and VCaP after siRNA knockdown of PIKFYVE (top). PIKFYVE mRNA levels were quantified by qPCR in indicated cells after siNC or siPIKFYVE knockdown (bottom). Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated. (g) Quantification of autophagosome induction activity measured with CYTO-ID® assay in DU145, PC3, LNCaP, and VCaP after siRNA knockdown of PIKFYVE or treatment with ESK981. Data were analyzed by two-tailed unpaired t test from three independent experiments for VCaP siRNA and four independent experiments for the remaining conditions. Data are presented as mean ± SEM. P-values indicated. (h) Cellular thermal shift assay (CESTA) of VCaP cells treated with control, 1 μM ESK981, or 1 μM apilimod for 2 hours with three independent experiments. (i) CXCL10 mRNA levels in VCaP or PC3 cells after siRNA knockdown of a non-targeting control (siNC) or PIKFYVE (siPIKFYVE) with the indicated treatment for 24 hours. Data were analyzed by two-tailed unpaired t test from three independent experiments and presented as mean ± SEM. P-value indicated.

Article Snippet: PC3 and DU145 cells were stably transfected with pMRX-IP-GFP-LC3-RFP-LC3ΔG (Addgene #84572), and single-cell clones were validated to avoid homologous recombination between the two LC3 fragments during retrovirus infection.

Techniques: Inhibition, Control, Knockdown, Two Tailed Test, Activity Assay, Thermal Shift Assay

Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet: PLAT-E cells were transfected with retroviral pMX-Flag-NEMO- WT -RFP (NM-WT) and pMX-flag-NEMO- K270 -RFP (NM-KA) expression vector. ( A ) Fluorescence images showing distribution of NM-WT-RFP in cytoplasm compared to puncta (yellow arrows) (juxtaposed to nuclei- DAPI stained) formation in case of NM-KA-RFP in PLAT-E cells. ( B ) Western blot for LC3 using WES (protein simple). BMMs were cultured for 2 days with RANKL (preOC) followed by 6 hr of serum starvation and western blotting. Fold change of LC3 relative to actin is indicated on top. ( C ) Quantification of LC3+ cells per high magnification field. ( D ) For flow cytometry, BMMs were transduced with pMX-GFP-LC3-RFP retrovirus generated in PLAT-E packing cells, and flow analysis was done to detect GFP signal or LC3 flux. Contour plots showing LC3-GFP+ expressing cells in NM-WT and NM-KA preOC (Blue: NM-WT without serum starvation, Red: NM-KA without serum starvation, and Black: after 6 hr of serum starvation), ( E ) Histograms representing shift in LC3-GFP+ cells following induction of autophagy (Red histogram: background signal in uninfected cells, Blue histogram: No serum starvation or 10% FBS control, yellow: 6 hr serum starvation, and pink: chloroquine), ( F ) Change in Mean fluorescent intensity (MFI) showing LC3-GFP signal in NM-WT and NM-KA preOC cells post autophagy induction. LysM-cre-NEMO-WT-f/f (NM-WT), LysM-cre-NEMO-K270A-f/f (NM-KA) mice. (*p<0.05). (*p<0.05, **p<0.01 and ***p<0.001). Figure 4—source data 1. Western blot for LC3 using WES (protein simple). Figure 4—source data 2. Quantification of LC3+ cells. Figure 4—source data 3. LC3-GFP FACS analysis.

Article Snippet: Recombinant DNA reagent , PMRX-GFP-LC3-RFP retrovirus , AddGene , Cat# 84573 , LC3 wth GFP and RFP on PMRX backbone.

Techniques: Transfection, Retroviral, Expressing, Plasmid Preparation, Fluorescence, Staining, Western Blot, Cell Culture, Flow Cytometry, Transduction, Generated, Control

Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet: ( A ) Pre-OC (RANKL-treated BMMs) from NM-WT and NM-KA mice were pelleted and processed for electron microscopic and Immunofluorescence (IF) analysis after 6 hr of serum starvation. Representative Electron microscopic images (x7500) showing nucleus, Cell membrane (CM) lysosome ( L ) in NM-WT preOC and cytoplasmic aggregates (yellow arrow) in NM-KA preOC. ( B ) Representative IF images for NEMO (red), LC3 (green) and NEMO-LC3 colocalization (yellow). Arrows indicate accumulation of NEMO in LC3 positive vacuole-like structures. ( C ) Representative western blot showing expression of LC3 from BMMs starved and stimulated with RANKL as shown. ( D ) Representative Western blot for mTOR expression in BMMs from NM-WT and NM-KA mice treated as shown. ( E ) Pre-osteoclasts were treated with chloroquine as indicated and number of TRAP+ multi nucleated osteoclasts (MNC) per well were counted in triplicate wells from three independent experiments (*p<0.05).

Article Snippet: Recombinant DNA reagent , PMRX-GFP-LC3-RFP retrovirus , AddGene , Cat# 84573 , LC3 wth GFP and RFP on PMRX backbone.

Techniques: Immunofluorescence, Membrane, Western Blot, Expressing

Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet: BMMs from NM-WT and NM-KA mice were transduced with viral particles (generated by transfecting pMX- retroviral vectors in PLAT-E cells) expressing ISG15 and cultured in the presence of MCSF (10 ng/ml) and RANKL (50 ng/ml) for 4 days. ( A ) Representative TRAP staining for osteoclast (n = 6) and ( B ) quantification of TRAP positive OCs. ( C ) BMMs from NM-WT and NM-KA mice were transduced with ISG15 and pMRX-GFP-LC3-RFP retrovirus generated in PLAT-E packing cells. The cells were cultured for 2 days (preOC) followed by 6 hr of serum starvation and flow analysis to detect GFP signal or LC3 flux. ( C ) Histograms representing shift in LC3-GFP+ cells following induction of autophagy. Blue histogram: serum starvation, yellow histogram: serum starvation + ISG15 expression ( D ) Change in Mean Fluorescent Intensity (MFI) showing LC3-GFP signal. ( E ) Wild type BMMs transduced with viral particles (generated by transfecting pMX- retroviral vectors in PLAT-E cells) expressing NEMO+/-ISG15, NEMO-K270A+/-ISG15 NEMO-WT::ISG15 (fused) and NEMO-K270A::ISG15 (fused) protein and cultured in the presence of MCSF (10 ng/ml) and RANKL (50 ng/ml) for 4 days.( E ) Representative TRAP staining for osteoclast (n = 3) and ( F ) quantification of TRAP positive OCs. ( G ) NEMO puncta regulation by ISG15: Live images of preOC expressing RFP-NEMO WT +/-ISG15, RFP-NEMO K270A +/-ISG15, GFP-NEMO WT ::ISG15 and GFP-NEMO K270A ::ISG15 fusion protein. Yellow arrows indicate NEMO K270A puncta. ISG15 panel which is not tagged serves as background control. ( H ) Quantification of LC3 puncta+ preOC cells shown in . ( I ) WB for LC3 in preOC expressing NEMO WT +/-ISG15, NEMO K270A +/-ISG15, NEMO WT ::ISG15 and NEMO K270A ::ISG15 fusion protein. (*p<0.05, **p<0.01 and ***p<0.001). (::) denotes fusion. ( J ) Representative IF images (NEMO (Red); LAMP1(green)). NEMO localization in preOC expressing NEMO WT +/-ISG15, NEMO K270A +/-ISG15, NEMO WT ::ISG15 and NEMO K270A ::ISG15 fusion protein. Green arrow- Lysosome and Yellow arrow-localization of NEMO in Lysosome. Figure 7—source data 1. quantification of TRAP positive OCs. Figure 7—source data 2. LC3-GFP FACS analysis. Figure 7—source data 3. Quantification of TRAP positive OCs. Figure 7—source data 4. Quantification of LC3 positive puncta in pre-OC cells shown in . Figure 7—source data 5. Western blot for LC3 expression in preOC expressing different NEMO and ISG15 constructs.

Article Snippet: Recombinant DNA reagent , PMRX-GFP-LC3-RFP retrovirus , AddGene , Cat# 84573 , LC3 wth GFP and RFP on PMRX backbone.

Techniques: Transduction, Generated, Retroviral, Expressing, Cell Culture, Staining, Control, Western Blot, Construct

Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet: ( A ) Representative IF images for LC3 puncta+ cells (arrow) in preOC expressing NEMO WT +/-ISG15, NEMO K270A +/-ISG15, NEMO WT ::ISG15 and NEMO K270A ::ISG15 fusion protein (quantified in ).

Article Snippet: Recombinant DNA reagent , PMRX-GFP-LC3-RFP retrovirus , AddGene , Cat# 84573 , LC3 wth GFP and RFP on PMRX backbone.

Techniques: Expressing

Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , PMRX-GFP-LC3-RFP retrovirus , AddGene , Cat# 84573 , LC3 wth GFP and RFP on PMRX backbone.

Techniques: Luciferase, Recombinant, Retroviral, Plasmid Preparation, FLAG-tag, Mutagenesis, Construct, Transfection, Activity Assay, BIA-KA, Quantitation Assay, Lysis, Western Blot, Electron Microscopy, Multiplex Assay, Marker, Isolation, SYBR Green Assay, Real-time Polymerase Chain Reaction, Sequencing, Software

Journal: Autophagy

Article Title: USP4 depletion-driven RAB7A ubiquitylation impairs autophagosome-lysosome fusion and aggravates periodontitis

doi: 10.1080/15548627.2024.2429371

Figure Lengend Snippet: Impaired autophagy and decreased RAB7A expression are found in periodontitis patients. (A) Western blot analysis of autophagy regulator RAB7A and autophagy markers (LC3, SQSTM1) in healthy and periodontitis gingiva ( n = 3 for each group). Anti-RAB7A (cell signaling technology 95,746), anti-LC3 (cell signaling technology 12,741), anti-SQSTM1 (cell signaling technology 88,588). (B) immunofluorescence analysis of autophagy regulator RAB7A, LC3, SQSTM1, LAMP1 in lamina propria of healthy and periodontitis gingiva ( n = 3 for each group). Scale bars: 50 μm; zoom: 20 μm. Anti-RAB7A (cell signaling technology 95,746). anti-LC3 (cell signaling Technology,12741), anti-SQSTM1 (cell signaling Technology,88588). Data were presented as mean ± SEM. p -values were calculated through two-tailed Student’s t -tests.

Article Snippet: Scale bars: 10 μm. (E-F) the number of GFP + RFP + LC3 (autophagosome) and GFP − RFP + LC3 (autolysosome) was calculated from multicell images.

Techniques: Expressing, Western Blot, Immunofluorescence, Two Tailed Test

Journal: Autophagy

Article Title: USP4 depletion-driven RAB7A ubiquitylation impairs autophagosome-lysosome fusion and aggravates periodontitis

doi: 10.1080/15548627.2024.2429371

Figure Lengend Snippet: RAB7A agonist ML098 promotes autophagy and inhibits alveolar bone loss in periodontitis mice. (A) mouse experimental periodontitis scheme. The 6-8-week-old mice were randomly divided into three groups. The control group was not treated. The periodontitis group was subjected to ligature and P. g . on the maxillary second molars with or without RAB7A agonist ML098 (1 mg/kg) intraperitoneal injection (every 2 days). Mice were harvested 10 days later for periodontal tissue analysis. (B) Representative micro-ct analysis of alveolar bone in normal control mice, periodontitis mice, and periodontitis mice with ML098 group. Scale bars: 500 μm. (C) micro-ct analysis of the CEJ-ABC distance at the palatal side of the second molar (indicated by red lines) in three groups of mice ( n = 5 for each group). CEJ-ABC, the cementoenamel junction to the alveolar bone crest. (D) Representative Immunofluorescence staining of autophagy regulator RAB7A and autophagy maker LC3 and SQSTM1 in three groups of mice. Scale bars: 50 μm; zoom: 10 μm. Anti-RAB7A (cell signaling Technology,95746), anti-LC3 (cell signaling Technology,12741), anti-SQSTM1 (CST 88,588). (E, F, G) expression of RAB7A, LC3, and SQSTM1 in the gingiva of three groups of mice ( n = 5 for each group). Data were presented as mean ± SEM. p -values were calculated through one-way ANOVA test.

Article Snippet: Scale bars: 10 μm. (E-F) the number of GFP + RFP + LC3 (autophagosome) and GFP − RFP + LC3 (autolysosome) was calculated from multicell images.

Techniques: Control, Injection, Micro-CT, Immunofluorescence, Staining, Expressing

Journal: Autophagy

Article Title: USP4 depletion-driven RAB7A ubiquitylation impairs autophagosome-lysosome fusion and aggravates periodontitis

doi: 10.1080/15548627.2024.2429371

Figure Lengend Snippet: Autophagy impairment is mediated by blocking autophagosome-lysosome fusion in macrophages. (A-B) time-dependent (left panel) and dose-dependent (right panel) assay of P. g .stimulation were performed on THP-1-derived macrophages. Western blot analysis was performed for the expression RAB7A and active RAB7A, autophagy markers (SQSTM1 and LC3), autophagy initiation-related proteins (BECN1, MTOR, and p-mtor). One representative blot is shown in two independent experiments. (C) GFP-RFP-LC3 THP-1 cells were stimulated with P. g . (MOI = 100), and representative time-lapse confocal images were captured to detect autophagic flux. Red dots represent autolysosomes and yellow dots autophagosomes. Scale bars: 10 μm. Zoom, 5 μm. (D) GFP-RFP-LC3 THP-1 cells were treated with P. g . (MOI = 100), Rapa (10 nM), and Baf A1 (50 nM) for 8 h to detect autophagy flux. Representative images of fluorescent LC3 puncta were shown. Red dots represent autolysosomes and yellow dots autophagosomes. Scale bars: 10 μm. (E-F) the number of GFP + RFP + LC3 (autophagosome) and GFP − RFP + LC3 (autolysosome) was calculated from multicell images. Data were presented as mean ± SEM. p-values were calculated through one-way ANOVA test. Rapa, rapamycin. Baf A1, bafilomycin A 1 .

Article Snippet: Scale bars: 10 μm. (E-F) the number of GFP + RFP + LC3 (autophagosome) and GFP − RFP + LC3 (autolysosome) was calculated from multicell images.

Techniques: Blocking Assay, Derivative Assay, Western Blot, Expressing

Journal: Autophagy

Article Title: USP4 depletion-driven RAB7A ubiquitylation impairs autophagosome-lysosome fusion and aggravates periodontitis

doi: 10.1080/15548627.2024.2429371

Figure Lengend Snippet: Autophagosome-lysosome fusion is blocked by decreased GTP-RAB7A. (A) Western blot analysis of total RAB7A and GTP-RAB7A (active form, immunoprecipitated by anti-GTP-RAB7A antibody) in control vs. P. g -stimulated THP-1 cells (MOI = 100, 8 h). One representative blot is shown in three independent experiments. Anti-RAB7A (cell signaling Technology,95746), anti-GTP-RAB7A (NewEast 26,923). (B) quantity analysis of the ratio of GTP-RAB7A expression to total RAB7A. (C) Western blot analysis of co-ip from THP-1 cells transfected and treated as indicated. RAB7A pulled down by GST-RILP represents GTP-RAB7A. (D) confocal microscopy analysis for colocalizations of RAB7A with RILP in control vs. P. g -stimulated THP-1 cells (MOI = 100, 8 h). RAB7A (red) and RILP (green) were stained using anti-RAB7A and anti-rilp antibodies. Scale bars: 10 μm. (E) colocalization plots report Mander’s overlap quantified from multicell images (black dots). (F) Western blot analysis of SQSTM1, LC3, total RAB7A and GTP-RAB7A (immunoprecipitated by anti-GTP-RAB7A antibody) in four groups as indicated. CID-1067700 (100 μM), ML098 (100 nM), baf A1 (50 nM). (G) GFP-RFP-LC3 THP-1 cells were treated with P. g . (MOI = 100), ML098 (100 nm), and CID-1067700 (100 μM) for 8 h and the colocalization of GFP-LC3 and RFP-LC3 was captured to detect the autophagy. Red dots represent autolysosomes and yellow dots autophagosomes. Scale bars: 10 μm. (H-I) the number of GFP + RFP + LC3 (autophagosome) and to GFP − RFP + LC3 (autolysosome) was calculated from multicell images. One representative blot is shown in two independent experiments. Data were presented as mean ± SEM. p-values were calculated through two-tailed Student’s t-tests (B, E) and one-way ANOVA test (H, I).

Article Snippet: Scale bars: 10 μm. (E-F) the number of GFP + RFP + LC3 (autophagosome) and GFP − RFP + LC3 (autolysosome) was calculated from multicell images.

Techniques: Western Blot, Immunoprecipitation, Control, Expressing, Co-Immunoprecipitation Assay, Transfection, Confocal Microscopy, Staining, Two Tailed Test

Journal: Autophagy

Article Title: USP4 depletion-driven RAB7A ubiquitylation impairs autophagosome-lysosome fusion and aggravates periodontitis

doi: 10.1080/15548627.2024.2429371

Figure Lengend Snippet: USP4 promotes autophagosome-lysosome fusion by deubiquitination of RAB7A. (A) the whole lysates were extracted from THP-1 cells with or without P. g . stimulation. Lysosomal interacting proteins were immunoprecipitated and were subject to mass spectrometry. The data revealed multiple USP4 peptides in lysosome-bound protein samples, indicating the interaction between RAB7A and USP4. (B, C) Western blot analysis of Co-ip from THP-1 cells transfected and treated as indicated. (B) THP-1 cells were transfected with siRNA to knock down the USP gene. Ubiquitin-conjugated proteins were pulled down using anti-ub antibody, followed by Western blot analysis using anti-RAB7A antibody. (C) THP-1 cells were transfected with USP4 overexpression lentivirus and used GTP-RAB7A and USP4 antibodies to pull down GTP-RAB7A-binding and USP4-binding proteins to measure RAB7A (GTP-RAB7A and USP4 bounding RAB7A. (D) Representative confocal images of RAB7A with lysosome marker LAMP1. Scale bars: 10 μm. (E) LAMP1 fluorescent intensity distribution is expressed as distance along a straight line from the center of the nucleus to the peripheral area. Fluorescence intensities were quantified along a straight line extending from the center of the cell’s nucleus (designated as fractional distance = 0) to the plasma membrane (designated as fractional distance = 1.0). (F) GFP-RFP-LC3 THP-1 cells were transfected with USP4 siRNA and USP4 overexpressed lentivirus to detect autophagy flux. Representative images were captured to display the colocalization of GFP-LC3 and RFP-LC3 after being treated with P. g . (MOI = 100, 8 h). Red dots represent autolysosomes and yellow dots autophagosomes. Scale bars: 10 μm. (G-H) the number of GFP + RFP + LC3 (autophagosome) and to GFP − RFP + LC3 (autolysosome) was calculated from multicell images. Data were presented as mean ± SEM. p-values were calculated through one-way ANOVA test.

Article Snippet: Scale bars: 10 μm. (E-F) the number of GFP + RFP + LC3 (autophagosome) and GFP − RFP + LC3 (autolysosome) was calculated from multicell images.

Techniques: Immunoprecipitation, Mass Spectrometry, Western Blot, Co-Immunoprecipitation Assay, Transfection, Knockdown, Ubiquitin Proteomics, Over Expression, Binding Assay, Marker, Fluorescence, Clinical Proteomics, Membrane

Journal: Autophagy

Article Title: USP4 depletion-driven RAB7A ubiquitylation impairs autophagosome-lysosome fusion and aggravates periodontitis

doi: 10.1080/15548627.2024.2429371

Figure Lengend Snippet: Model of RAB7A ubiquitination-mediated impaired autophagosome-lysosome fusion in periodontitis pathogenesis. Research Paperhe periodontitis condition (right panel): in the absence of USP4, lack of RAB7A deubiquitination (GDP-RAB7A form) results in impaired autophagosome-lysosome fusion. The impaired autophagy leads to increased IL1B maturation and release, which contributes to periodontitis pathogenesis.

Article Snippet: Scale bars: 10 μm. (E-F) the number of GFP + RFP + LC3 (autophagosome) and GFP − RFP + LC3 (autolysosome) was calculated from multicell images.

Techniques: Ubiquitin Proteomics