Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: H2O2-induced different cell viability in M17, PC12 and MN9D cells as measured by MTT assay (A & B) and trypan blue dye exclusion assay (C). Each bar from Figs. represents data obtained from 5 separate experiments (N = 5 of independent cell culture preparations, which is the same in the following legends). *p < 0.05, **p < 0.01, ***p < 0.001, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: MTT Assay, Exclusion Assay, Cell Culture, Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: Effects of H2O2 treatment on DDRs in M17 and MN9D cells as demonstrated by measurements of γH2AX (A) and p53 (B). Cells were exposed to 100–400 μM H2O2 for 3 h. Top panels: autoradiograph obtained by western blotting. Button panel: the quantitative analysis of band densities in western blotting. Each bar from Figs. represents data obtained from 5–7 separate experiments (N = 5–7). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Autoradiography, Western Blot, Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: H2O2 treatment induces single-strand DNA breaks as determined by the Comet assay in M17 and MN9D cells. Cells were exposed to different concentrations of H2O2 for 3 h. The cells were processed for comet assays run under alkaline conditions to identify DNA SSBs. Tail moment (B), tail length (C) and % DNA in the tail (D) were analyzed to evaluate DNA damage. Each bar from Figs. represents data obtained from 5 separate experiments (N = 5). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, compared to corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Single Cell Gel Electrophoresis, Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: Effects of H2O2 treatment on ROS production in M17 and MN9D cells. Cells were exposed to different concentrations of for 3 h. ROS was measured by the DCFH2-DA assay. Each bar from Figs. represents data obtained from 6 separate experiments (N = 6). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to the corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: Effects of H2O2 treatment on Ctr1 (A) and OGG1 (B) protein levels in M17 and MN9D cells. Each bar represents data obtained from 5 separate experiments (N = 5) in (A), and 4 separate experiments (N = 4) in (B). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to the corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Control

Journal: Neuroscience

Article Title: Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability

doi: 10.1016/j.neuroscience.2019.09.036

Figure Lengend Snippet: Effects of H2O2 treatment on Cav1.2 (A) and Cav1.3 (B) protein levels in M17 and MN9D cells as determined by western blotting. Cells were exposed to different concentrations of H2O2 for 3 h. Each bar in Figs. represents data obtained from 6 to 7 separate experiments (N = 6–7). *p < 0.05, **p < 0.01, compared to the 0 group (control). †p < 0.05, ††p < 0.01, compared to the corresponding group in M17 cells.

Article Snippet: Cell cultures and drug exposure The growth medium for human neuroblastoma cell line SK-N-BE(2)-M17 (M17, ATCC, Cat#: CRL-2267) and the mouse hybrid dopaminergic cell line MN9D (transferred from Dr. Zigmond’s laboratory of University of Pittsburgh, MTA 0000434) was Dulbecco’s modified Eagle’s medium (DMEM), and for SH-SY5Y cells (ATCC, CRL-2266, Cat#: CRL-2266) was a 1:1 mix of DMEM and F12 media.

Techniques: Western Blot, Control

Journal: International journal of cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse.

doi: 10.1002/ijc.35164

Figure Lengend Snippet: FIGURE 2 Synergistic toxicity evoked by combined treatment with the CHK1 inhibitor PF477736 (PF) and the RAD51 inhibitor B02 in J82CisPt. (A) The viability of J82CisPt cells was analyzed after treatment with differently combined low and moderate toxic doses of CHK1i PF477736 and RAD51i B02 as indicated. Cell viability was measured after a 72 h treatment period using the AlamarBlue Assay. Based on the data obtained from three independent experiments each performed in quadruplicate, the combination indices (CIs) were calculated using CompuSyn software (CI < 0.9 indicating synergistic effects, CI ≈1 additive effects and CI > 1.2 antagonistic effects). (B) Protein expression and activation of different apoptosis-related factors were examined via Western Blot analyses using protein extracts of J82CisPt

Article Snippet: Cytostatics, DDR modifiers and other compounds were obtained from the following suppliers: Doxorubicin (Cellpharm [Bad Vilbel, Germany]), OH-Urea (Sigma [Steinheim, Germany]), 5-Fluorouracil (Medac [Wedel, Germany]), Carboplatin (TEVA [Ulm, Germany]), Oxaliplatin (Accord Healthcare [Munich, Germany]), hydrogen peroxide (H2O2) (Carl Roth GmbH [Karlsruhe, Germany]), checkpoint kinase 1 (CHK1) inhibitor PF477736 and LY2603618 (Sigma [Steinheim, Germany]), RAD51 inhibitor B02 and RI(dl)2 (Tocris Bioscience [Bristol, UK]), MRE11 inhibitor Mirin (Abcam [Cambridge, UK]), PARP inhibitor Niraparib (MedChemExpress [Monmouth Junction, NJ, USA]), HDAC inhibitors Entinostat (Selleckchem [Munich, Germany]) and Vorinostat (Sigma [Steinheim, Germany]).

Techniques: Alamar Blue Assay, Software, Expressing, Activation Assay, Western Blot

Journal: International journal of cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse.

doi: 10.1002/ijc.35164

Figure Lengend Snippet: FIGURE 3 S-phase arrest in J82CisPt cells following treatment with B02 and PF477736. (A, B) Inhibitors (10 μM B02 ± 1 μM PF477736) were added 24 h after seeding and cell cycle distribution was analyzed after a treatment period of 24 h (A) and 72 h (B) employing propidium iodide staining and flow cytometric analysis. Data are presented as mean + SD from n = 3 independent experiments. (C) The EdU incorporation of J82CisPt cells was analyzed after treatment with 10 μM B02 or/and 1 μM PF477736. EdU incorporation was analyzed after 24 h treatment period with an EdU pulse of 2 h. The graph shows the mean + SD of n = 3 independent experiments (1000–2000 nuclei analyzed per sample). The scale bars in the representative pictures correspond to 50 μm. ***p ≤.001; **p ≤.01; *p ≤.05; significant compared to control (*), to B02 mono- treatment (#) and to PF477736 mono-treatment (+). [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Cytostatics, DDR modifiers and other compounds were obtained from the following suppliers: Doxorubicin (Cellpharm [Bad Vilbel, Germany]), OH-Urea (Sigma [Steinheim, Germany]), 5-Fluorouracil (Medac [Wedel, Germany]), Carboplatin (TEVA [Ulm, Germany]), Oxaliplatin (Accord Healthcare [Munich, Germany]), hydrogen peroxide (H2O2) (Carl Roth GmbH [Karlsruhe, Germany]), checkpoint kinase 1 (CHK1) inhibitor PF477736 and LY2603618 (Sigma [Steinheim, Germany]), RAD51 inhibitor B02 and RI(dl)2 (Tocris Bioscience [Bristol, UK]), MRE11 inhibitor Mirin (Abcam [Cambridge, UK]), PARP inhibitor Niraparib (MedChemExpress [Monmouth Junction, NJ, USA]), HDAC inhibitors Entinostat (Selleckchem [Munich, Germany]) and Vorinostat (Sigma [Steinheim, Germany]).

Techniques: Staining, Control

Journal: International journal of cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse.

doi: 10.1002/ijc.35164

Figure Lengend Snippet: FIGURE 4 Hampered replication occurring after combined treatment of J82CisPt with PF477736 and B02. (A) 24 h after seeding, J82CisPt cells were treated with either 10 μM B02, 1 μM PF477736 or both substances for 6 h. After the treatment, cells were incubated for 20 min with CldU, followed by 20 min incubation with IdU. The BrdU analogs were labeled by immunofluorescence, staining was analyzed microscopically and fiber lengths were measured using ImageJ. Data presented are from two independent experiments, whereby 200 fibers were measured for each sample. Each dot represents one analyzed fiber and the black lines show the mean ± SEM. The mean value is also given above the graphs. Upper panel, nascent DNA elongation, graphically displayed as IdU track lengths of bi-colored DNA fibers. Middle panel, table summarizing the evaluation of proportions of origins and terminations in the total fiber population (ns, not significant). Lower panel, as measure of DNA replication fork stalling fork asymmetry was determined from three-colored replication origins as the ratio of the longer red IdU fiber track length versus the shorter red IdU fiber track length departing from the same green CldU track. (B) Formation of RPA foci in the nuclei of J82CisPt cells was analyzed after 6 and 24 h treatment with 10 μM B02 or/and 1 μM PF477736 via immunocytochemical staining. Data are shown with each dot representing one analyzed nucleus and the black lines showing the mean ± SEM from three independent experiments, where in each case 50 nuclei were counted. The scale bars in the representative pictures correspond to 10 μm. ***p ≤.001; **p ≤.01; *p ≤.05; significant compared to control (*), to B02 mono-treatment (#) and to PF477736 mono-treatment (+). [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Cytostatics, DDR modifiers and other compounds were obtained from the following suppliers: Doxorubicin (Cellpharm [Bad Vilbel, Germany]), OH-Urea (Sigma [Steinheim, Germany]), 5-Fluorouracil (Medac [Wedel, Germany]), Carboplatin (TEVA [Ulm, Germany]), Oxaliplatin (Accord Healthcare [Munich, Germany]), hydrogen peroxide (H2O2) (Carl Roth GmbH [Karlsruhe, Germany]), checkpoint kinase 1 (CHK1) inhibitor PF477736 and LY2603618 (Sigma [Steinheim, Germany]), RAD51 inhibitor B02 and RI(dl)2 (Tocris Bioscience [Bristol, UK]), MRE11 inhibitor Mirin (Abcam [Cambridge, UK]), PARP inhibitor Niraparib (MedChemExpress [Monmouth Junction, NJ, USA]), HDAC inhibitors Entinostat (Selleckchem [Munich, Germany]) and Vorinostat (Sigma [Steinheim, Germany]).

Techniques: Incubation, Labeling, Immunofluorescence, Staining, Control

Journal: International journal of cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse.

doi: 10.1002/ijc.35164

Figure Lengend Snippet: FIGURE 5 S-phase-dependent formation of DNA damage and activation of DDR- and DNA repair-related mechanisms following treatment of J82CisPt with PF477736 and B02. (A) Protein expression and activation of different replication stress- and DDR-related factors was examined via Western Blot analyses with protein extracts of J82CisPt cells treated for 6 h or 24 h with 10 μM B02, 1 μM PF477736 or both substances. (B) Formation of DNA strand breaks was analyzed via alkaline Comet Assay after 24 h mono- and combination-treatment with 10 μM B02 and 1 μM PF477736. Tail intensity (% DNA in tail) is displayed as dot for every analyzed cell and the mean ± SEM calculated from n = 3; N = 50. ***p ≤.001; **p ≤.01; *p ≤.05; significant compared to control (*), to B02 mono-treatment (#) and to PF477736 mono-treatment (+). (C) To analyze in which cell cycle phase the damage predominantly occurs, a double staining with γH2AX antibody and propidium iodide was applied and examined by flow cytometry after a treatment period of 6 and 24 h in J82CisPt. Displayed representative images of the flow cytometrical analyses were generated using FlowJo software. (D) J82CisPt cells were co-treated with 10 μM B02 + 1 μM PF477736 for 24 h, afterwards immunocytochemical co-staining of γH2AX and RPA was performed to analyze the correlation of both markers. For γH2AX, the mean fluorescence intensity of the nuclei was measured and the number of RPA foci per nucleus were counted. Data are shown with each dot representing one analyzed nucleus and the black lines showing the mean ± SEM from two independent experiments, where in each case 50 nuclei were measured. The scale bar in the representative picture corresponds to 20 μm. ***p ≤.001; significant compared to nuclei with <10 RPA foci. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Cytostatics, DDR modifiers and other compounds were obtained from the following suppliers: Doxorubicin (Cellpharm [Bad Vilbel, Germany]), OH-Urea (Sigma [Steinheim, Germany]), 5-Fluorouracil (Medac [Wedel, Germany]), Carboplatin (TEVA [Ulm, Germany]), Oxaliplatin (Accord Healthcare [Munich, Germany]), hydrogen peroxide (H2O2) (Carl Roth GmbH [Karlsruhe, Germany]), checkpoint kinase 1 (CHK1) inhibitor PF477736 and LY2603618 (Sigma [Steinheim, Germany]), RAD51 inhibitor B02 and RI(dl)2 (Tocris Bioscience [Bristol, UK]), MRE11 inhibitor Mirin (Abcam [Cambridge, UK]), PARP inhibitor Niraparib (MedChemExpress [Monmouth Junction, NJ, USA]), HDAC inhibitors Entinostat (Selleckchem [Munich, Germany]) and Vorinostat (Sigma [Steinheim, Germany]).

Techniques: Activation Assay, Expressing, Western Blot, Alkaline Single Cell Gel Electrophoresis, Control, Double Staining, Flow Cytometry, Generated, Software, Staining, Fluorescence

Journal: International journal of cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse.

doi: 10.1002/ijc.35164

Figure Lengend Snippet: FIGURE 6 Combining B02 or PF477736 with other CHK1- or RAD51-inhibitors, respectively, likewise induces S-phase arrest, replication stress, and DNA damage in J82CisPt. J82CisPt cells were co-treated with 10 μM B02 + 1 μM LY2603618 (LY) (A) or 1 μM PF477736 (PF) + 30 μM RI(dl)2 (RI2) (B). Following 24 h treatment, propidium iodide-based cell cycle analysis was performed by flow cytometry with emphasis on the proportion of cells in S-phase. A total of 10,000 counts were measured for quantification. Induction of γH2AX and pRPA32 (S4, S8) was examined via Western Blot analyses with protein extracts of J82CisPt cells treated for 6 or 24 h with the corresponding combination or mono- treatments. [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Cytostatics, DDR modifiers and other compounds were obtained from the following suppliers: Doxorubicin (Cellpharm [Bad Vilbel, Germany]), OH-Urea (Sigma [Steinheim, Germany]), 5-Fluorouracil (Medac [Wedel, Germany]), Carboplatin (TEVA [Ulm, Germany]), Oxaliplatin (Accord Healthcare [Munich, Germany]), hydrogen peroxide (H2O2) (Carl Roth GmbH [Karlsruhe, Germany]), checkpoint kinase 1 (CHK1) inhibitor PF477736 and LY2603618 (Sigma [Steinheim, Germany]), RAD51 inhibitor B02 and RI(dl)2 (Tocris Bioscience [Bristol, UK]), MRE11 inhibitor Mirin (Abcam [Cambridge, UK]), PARP inhibitor Niraparib (MedChemExpress [Monmouth Junction, NJ, USA]), HDAC inhibitors Entinostat (Selleckchem [Munich, Germany]) and Vorinostat (Sigma [Steinheim, Germany]).

Techniques: Cell Cycle Assay, Flow Cytometry, Western Blot

Journal: Cell

Article Title: TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes.

doi: 10.1016/j.cell.2012.06.039

Figure Lengend Snippet: Figure 6. Deregulation of TRIP12 and UBR5 Alters the Dynamics of DNA Repair (A) U-2-OS cells were transfected with the indi- cated siRNA for 72 hr and irradiated. WCE were analyzed by immunoblotting. (B) U-2-OS cells were irradiated (2 Gy), and 1 hr later, they were immunostained with an antibody to RPA (the FITC channel); nuclear DNA was coun- terstained by DAPI. Cell-cycle distribution was determined for each individual cell by quantifying the total DAPI and chromatin-bound RPA intensity per nucleus. (C) U-2-OS cells were transfected with the indi- cated siRNAs, irradiated, immunostained with antibodies to RPA and RAD51, sorted according to cell cycle as in (B), and subjected to an automated analysis of RAD51 focus formation. At least 500 cells were analyzed for each condition; represen- tative images are shown. (D) U-2-OS cells were transfected with siRNAs for 72 hr and irradiated. Where indicated, cells were treated for 2 hr with ATM or DNA-PK inhibitors prior to irradiation. WCE were analyzed by immuno- blotting (left). In parallel, cells were treated with the indicated siRNAs and inhibitors as indicated, irra- diated (2 Gy), and after 4 hr, were subjected to an automated single cell analysis for the number of MDC1 nuclear foci (right). (E) U-2-OS cells were treated with the indicated siRNAs and inhibitors, irradiated, and analyzed as in (D). Scale bar, 10 mm. See also Figure S7.

Article Snippet: Rabbit polyclonal antibodies included the following: RNF168 (Stewart et al., 2009; provided by Daniel Durocher), RNF8 (rabbit antiserum, provided by Xiaochun Yu), 53BP1 (sc-22760, Santa Cruz), MDC1 (ab11171, Abcam), TRIP12 (A301-814A, Bethyl), UBR5/EDD (A300-573A, Bethyl), PARP1 (sc-7150, Santa Cruz), RPA32 phospho Ser33 (A300-246A, Bethyl), RPA32-phospho S4/S8 (A300245A, Bethyl), CHK1 (sc-8404, Santa Cruz), CHK1 phospho S345 (#2341, Cell Signaling), pCHK1-S317 (#2344, Cell Signaling), KAP1 (A300-274A, Bethyl), KAP1 phospho S824 (ab70369, Abcam), Herc2 (Bekker-Jensen et al., 2010), Smc1 (ab9262, Abcam), Rap80 (A300-763A, Bethyl), RAD51 (sc-8394, Santa Cruz), H2AX (NB100-638 Novus Biologicals).

Techniques: Transfection, Irradiation, Western Blot, Single-cell Analysis

Journal: The Journal of Biological Chemistry

Article Title: Tripartite Motif-containing 33 (TRIM33) Protein Functions in the Poly(ADP-ribose) Polymerase (PARP)-dependent DNA Damage Response through Interaction with Amplified in Liver Cancer 1 (ALC1) Protein *

doi: 10.1074/jbc.M113.459164

Figure Lengend Snippet: TRIM33 interacts with ALC1 upon induction of DNA damage. A, HEK293 cells expressing either vector or FLAG-ALC1 were treated with either vehicle or bleomycin, subjected to immunoprecipitation using FLAG beads, and then processed by LC-MS/MS. The relative peptide counts for each condition are shown for ALC1, PARP1, APLF, histone H2B, Ku70, and TRIM33. As shown, TRIM33 peptides were identified only in the bleomycin-treated samples. B, endogenous interaction of TRIM33 and ALC1 upon hydroxyurea (HU) and bleomycin treatment. DNase-treated nuclear extracts from untreated (Un) cells or cells treated with HU (3 mm, 3 h) or bleomycin (300uM, 1 h) were immunoprecipitated with anti-TRIM33 antibody. Immunoprecipitates were processed for Western blotting (WB) using antibodies to TRIM33 and ALC1 (top two panels, IP). Aliquots of nuclear extract were also directly processed for Western blotting with these antibodies (bottom two panels, Inputs).

Article Snippet: Antibodies The antibodies used were rabbit anti-TRIM33 (Bethyl Laboratories), mouse anti-ALC1 and mouse anti-XRCC1 antibodies (Abcam), mouse anti γH2AX antibody and mouse anti-P21 antibody (Millipore), rabbit anti-γH2AX antibody (AbD Serotec), mouse anti-PAR antibody (Trevigen), rabbit anti-PARP1 antibody (Enzo Life Sciences), rabbit antibodies against total and phosphorylated Chk2 (Cell Signaling Technology), and mouse anti-tubulin antibody (Sigma).

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Tripartite Motif-containing 33 (TRIM33) Protein Functions in the Poly(ADP-ribose) Polymerase (PARP)-dependent DNA Damage Response through Interaction with Amplified in Liver Cancer 1 (ALC1) Protein *

doi: 10.1074/jbc.M113.459164

Figure Lengend Snippet: Localization of TRIM33 to DNA breaks is dependent upon PARP and ALC1. A, PAR (top two panels) and TRIM33 (bottom two panels) were localized by IF in untreated (Un) and in cells pretreated with 1 μm PARPi (Pi) ABT-888 for 1 h. B, quantitation of PAR and TRIM33 colocalization with γH2AX at sites of laser scissors. *, p < 0.005. C, Parp1+/+ or Parp1−/− mouse embryonic fibroblasts were treated with laser scissors, and γH2AX and TRIM33 were localized by IF. Images are shown at identical magnification. D, quantitation of PAR and TRIM33 colocalization with γH2AX at sites of laser scissors. *, p < 0.005. E, APLF, WtALC1, C1 (ALC1 macro domain only), and TRIM33 proteins were dot-blotted onto a nitrocellulose membrane and incubated with 32P-labeled PAR. F, TRIM33 localization to DNA breaks is ALC1-dependent. U2OS cells stably expressing control sh, ALC1sh, or cells expressing ALC1 sh were reconstituted with WT ALC1, KR (kinase dead) and DA (PAR binding mutant) were analyzed. All cells were subjected to UV laser scissor-induced DNA breaks. After 10 min, cell were fixed, and IF was performed using antibodies to γH2AX and TRIM33. G, Western blot analyses showing levels of ALC1 and TRIM33 in U2OS cells expressing control and ALC1 shRNA and different constructs of ALC1 in ALC1sh cells. H, quantitation of relative intensity of TRIM33 at sites of DNA damage. Each data point is mean ± S.D. of at least 20 cells. *, p < 0.005.

Article Snippet: Antibodies The antibodies used were rabbit anti-TRIM33 (Bethyl Laboratories), mouse anti-ALC1 and mouse anti-XRCC1 antibodies (Abcam), mouse anti γH2AX antibody and mouse anti-P21 antibody (Millipore), rabbit anti-γH2AX antibody (AbD Serotec), mouse anti-PAR antibody (Trevigen), rabbit anti-PARP1 antibody (Enzo Life Sciences), rabbit antibodies against total and phosphorylated Chk2 (Cell Signaling Technology), and mouse anti-tubulin antibody (Sigma).

Techniques: Quantitation Assay, Incubation, Labeling, Stable Transfection, Expressing, Binding Assay, Mutagenesis, Western Blot, shRNA, Construct

Journal: The Journal of Biological Chemistry

Article Title: Tripartite Motif-containing 33 (TRIM33) Protein Functions in the Poly(ADP-ribose) Polymerase (PARP)-dependent DNA Damage Response through Interaction with Amplified in Liver Cancer 1 (ALC1) Protein *

doi: 10.1074/jbc.M113.459164

Figure Lengend Snippet: TRIM33 dynamically interacts with ALC1 in a PARP-dependent manner. A, HeLa cells were untreated (Un) or treated with IR or UV light, with or without PARP inhibitor (PARPi), and DNase-treated nuclear extracts were prepared at 5 and 10 min. TRIM33 IP was performed, followed by Western blotting (WB) using the indicated antibodies. IP, immunoprecipitation; No Ab, no antibody. B, quantitation of ALC1 interaction with TRIM33. The plot shows the ratio of the signal of ALC1 coimmunoprecipitation to ALC1 input. C, The FLAG WtALC1, ALC1K77R, and ALC1D723A mutants were expressed in 293 cells and subjected to UV irradiation. Protein extracts were prepared after 5 min and immunoprecipitated with anti-FLAG antibodies, followed by Western blotting using antibody against TRIM33 and FLAG. D, PAR-bound ALC1 does not bind TRIM33 in vitro. WtALC1, KR (ATPase dead) and DA (PAR binding mutant) ALC1 mutants and TRIM33 proteins were dot-blotted onto a nitrocellulose membrane and incubated with PAR, washed, and then incubated with purified TRIM33. Membranes were then processed for Western blot analysis with antibodies to PAR (top panel) or antibody to TRIM33 (bottom panel).

Article Snippet: Antibodies The antibodies used were rabbit anti-TRIM33 (Bethyl Laboratories), mouse anti-ALC1 and mouse anti-XRCC1 antibodies (Abcam), mouse anti γH2AX antibody and mouse anti-P21 antibody (Millipore), rabbit anti-γH2AX antibody (AbD Serotec), mouse anti-PAR antibody (Trevigen), rabbit anti-PARP1 antibody (Enzo Life Sciences), rabbit antibodies against total and phosphorylated Chk2 (Cell Signaling Technology), and mouse anti-tubulin antibody (Sigma).

Techniques: Western Blot, Immunoprecipitation, Quantitation Assay, Irradiation, In Vitro, Binding Assay, Mutagenesis, Incubation, Purification

Journal: The Journal of Biological Chemistry

Article Title: Tripartite Motif-containing 33 (TRIM33) Protein Functions in the Poly(ADP-ribose) Polymerase (PARP)-dependent DNA Damage Response through Interaction with Amplified in Liver Cancer 1 (ALC1) Protein *

doi: 10.1074/jbc.M113.459164

Figure Lengend Snippet: TRIM33 knockdown induces delayed dissociation of ALC1 but does not affect PAR dissociation from sites of DNA damage. A, HeLa cells expressing either control shRNA, TRIM33 shRNA, TRIM33shRNA + WtTRIM33, or the TRIM33shRNA + TRIM33CA mutant were subjected to UV laser scissors and stained for γH2AX (green) and ALC1 (red) at the time points indicated. B, Western blot analysis showing levels of TRIM33 in cells treated with control shRNA (lane 1), TRIM33 shRNA (lane 2), TRIM33sh RNA + WtTRIM33 (lane 3), and TRIM33sh RNA + TRIM33CA (lane 4). C, quantification of ALC1 localization to DNA damage in TRIM33 knockdown, WtTRIM33, or TRIM33CA reconstituted cells. Each data point is mean ± S.D. of at least 20 cells. D, TRIM33 knockdown does not affect ALC1 protein levels. Shown is a Western blot analysis showing levels of ALC1, TRIM33, and tubulin in control sh (1), TRIM33sh and TRIM33sh (2) cells reconstituted with either TRIM33Wt (3) or TRIM33CA (4). Protein extracts were collected from untreated or UV-treated cells, as indicated, and probed by Western blot analysis using the antibodies indicated. E, HeLa cells expressing either control shRNA or TRIM33 sh2 RNA were treated with laser scissors and stained by IF for γH2AX (green) and PAR (red). F, quantification of PAR intensity was performed 5 and 45 min after induction of DNA damage. Data are plotted as the mean ± S.D. of at least 20 cells. *, p < 0.005 for control versus TRIM33 shRNA-treated cells. G, Western blot analysis showing TRIM33 knockdown. HeLa cells transfected with control or TRIM33 shRNA and proteins levels were detected by Western blot analysis by probing with antibodies against TRIM33 and tubulin.

Article Snippet: Antibodies The antibodies used were rabbit anti-TRIM33 (Bethyl Laboratories), mouse anti-ALC1 and mouse anti-XRCC1 antibodies (Abcam), mouse anti γH2AX antibody and mouse anti-P21 antibody (Millipore), rabbit anti-γH2AX antibody (AbD Serotec), mouse anti-PAR antibody (Trevigen), rabbit anti-PARP1 antibody (Enzo Life Sciences), rabbit antibodies against total and phosphorylated Chk2 (Cell Signaling Technology), and mouse anti-tubulin antibody (Sigma).

Techniques: Expressing, shRNA, Mutagenesis, Staining, Western Blot, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Tripartite Motif-containing 33 (TRIM33) Protein Functions in the Poly(ADP-ribose) Polymerase (PARP)-dependent DNA Damage Response through Interaction with Amplified in Liver Cancer 1 (ALC1) Protein *

doi: 10.1074/jbc.M113.459164

Figure Lengend Snippet: TRIM33 rescues ALC1 overexpression sensitivity to DNA-damaging agents. A, ALC1 overexpression results in delayed dissociation of ALC1 from DNA damage. FLP-In ALC1 and FLP-In ALC1 + WtTRIM33 cells were exposed to UV laser scissor-induced DNA damage, and cells were fixed at the indicated time points. Cells were stained by IF for γH2AX (green) and ALC1 (red). B, Western blot analysis showing levels of TRIM33 and ALC1 in ALC1-overexpressing cells. C, quantification of ALC1 intensity was performed at the indicated time points after induction of DNA damage. Data are plotted as the mean ± S.D. of at least 20 cells. D, effect of TRIM33 on sensitivity of ALC1-overexpressing cells to bleomycin. U2OS cells stably expressing either empty vector (FLP-In-FLAG), WtALC1 (FLP-In-ALC1), WtALC1 (FLP-In-ALC1) + WtTRIM33, and WtALC1 (FLP-In-ALC1) + TRIM33CA were treated with increasing concentrations of bleomycin for 3 days. Relative cell counts at day 3 were measured by MTS assay and were plotted normalized to untreated cells. Data are mean ± S.D. of three experiments. E, induction of DNA breaks in cells (n > 200 cells) of indicated genotypes after treatment with increasing amounts of phleomycin were measured by alkaline comet assay. Data are plotted as the mean ± S.D. of at least three experiments.

Article Snippet: Antibodies The antibodies used were rabbit anti-TRIM33 (Bethyl Laboratories), mouse anti-ALC1 and mouse anti-XRCC1 antibodies (Abcam), mouse anti γH2AX antibody and mouse anti-P21 antibody (Millipore), rabbit anti-γH2AX antibody (AbD Serotec), mouse anti-PAR antibody (Trevigen), rabbit anti-PARP1 antibody (Enzo Life Sciences), rabbit antibodies against total and phosphorylated Chk2 (Cell Signaling Technology), and mouse anti-tubulin antibody (Sigma).

Techniques: Over Expression, Staining, Western Blot, Stable Transfection, Expressing, Plasmid Preparation, MTS Assay, Alkaline Single Cell Gel Electrophoresis

Journal: Hepatology Communications

Article Title: MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells

doi: 10.1002/hep4.1862

Figure Lengend Snippet: Hepatic MTH1+CD3+ T cells correlated with disease activity in AIH. (A) Representative confocal staining of CD3 (red), MTH1 (green), and DAPI (for nuclei in blue) (magnification ×400) in the liver of patients with AIH. Scale bar, 20 μm. (B) Number of MTH1+CD3+ T cells in portal areas was positively correlated with degree of hepatic inflammation but showed no clear link with fibrosis stages in AIH. (C) Number of MTH1+CD3+ T cells in portal areas had a significant positive correlation with levels of serum ALT and AST in patients with AIH. (D) Number of MTH1+CD3+ T cells in portal areas was positively correlated with levels of serum ALP and GGT. Bars reflect the mean ± SEM. Abbreviations: DAPI, 4 0, 6‐diamidino‐2‐phenylindole; hpf, high‐power field.

Article Snippet: Confocal laser scanning microscopy was used for the detection of costaining markers, as described. ( ) Briefly, after antigen retrieval, liver samples were incubated with donkey serum for 30 minutes before being incubated with primary antibodies MTH1 (ab200832; Abcam), CD3 (60181‐1‐Ig; Proteintech), CD4 (ab67001; Abcam), and CD8 (ab17147; Abcam) at 4°C overnight.

Techniques: Activity Assay, Staining

Journal: Hepatology Communications

Article Title: MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells

doi: 10.1002/hep4.1862

Figure Lengend Snippet: Karonudib significantly inhibited T‐cell proliferation in human T cells in vitro . Isolated human CD3+ T cells were cultured with/without anti‐CD3/CD28 beads for 72 hours. Representative western blot analyses of MTH1 72 hours after anti‐CD3/CD28 beads stimulation. (B,C) Isolated T cells were activated with anti‐CD3/CD28 beads with or without 2 μM karonudib for 72 hours. The percentage of CD25+ and CD69+ T cells was determined on day 3 by flow cytometry. (D) Analysis of T‐cell number treated with karonudib for 72 hours after anti‐CD3/CD28 beads stimulation. (E) Statistical analysis of the T‐cell proliferation assay treated with/without 2 μM karonudib for 72 hours from HCs and patients with AIH who were treatment naive. (F) Representative western blot analyses of P53, P21, P27, CDK2, and cyclin E 72 hours after anti‐CD3/CD28 beads stimulation. The GAPDH blot was used as a loading control. Data are from one experimental representative of at least three independent experiments and represent triplicate wells. Bars reflect the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase.

Article Snippet: Confocal laser scanning microscopy was used for the detection of costaining markers, as described. ( ) Briefly, after antigen retrieval, liver samples were incubated with donkey serum for 30 minutes before being incubated with primary antibodies MTH1 (ab200832; Abcam), CD3 (60181‐1‐Ig; Proteintech), CD4 (ab67001; Abcam), and CD8 (ab17147; Abcam) at 4°C overnight.

Techniques: In Vitro, Isolation, Cell Culture, Western Blot, Flow Cytometry, Proliferation Assay, Control

Journal: Hepatology Communications

Article Title: MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells

doi: 10.1002/hep4.1862

Figure Lengend Snippet: Karonudib rendered activated T cells more susceptible to DNA damage in vitro . (A) Representative fields corresponding to each treatment were photographed. Isolated human T cells were activated with/without anti‐CD3/CD28 beads in the presence of DMSO or 2 μM karonudib for 72 hours. The alkaline comet assay was conducted and nucleoids were visualized by epifluorescence microscopy using a fluorescein isothiocyanate filter. (B) Quantification of comet tail moment. Values represent mean ± SEM from three independent experiments (100 comets per experiment). (C) Levels of cleaved‐PARP and phosphorylated histone H2AX (γ‐H2AX) were determined by immunoblot analysis. The GAPDH blot was used as a loading control. (D,E) Intracellular flow cytometry assessment of annexin V and propidium iodide expression in anti‐CD3/CD28 beads‐stimulated CD4+ and CD8+ T lymphocytes treated with 2 μM karonudib or DMSO after 72 hours. Data are from one experimental representative of at least three independent experiments. Bars reflect the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase.

Article Snippet: Confocal laser scanning microscopy was used for the detection of costaining markers, as described. ( ) Briefly, after antigen retrieval, liver samples were incubated with donkey serum for 30 minutes before being incubated with primary antibodies MTH1 (ab200832; Abcam), CD3 (60181‐1‐Ig; Proteintech), CD4 (ab67001; Abcam), and CD8 (ab17147; Abcam) at 4°C overnight.

Techniques: In Vitro, Isolation, Alkaline Single Cell Gel Electrophoresis, Epifluorescence Microscopy, Western Blot, Control, Flow Cytometry, Expressing

Journal: Cell Death and Differentiation

Article Title: Glycoprotein PTGDS promotes tumorigenesis of diffuse large B-cell lymphoma by MYH9-mediated regulation of Wnt–β-catenin–STAT3 signaling

doi: 10.1038/s41418-021-00880-2

Figure Lengend Snippet: A The expression level of PTGDS mRNA was reduced by adriamycin (ADR) and bendamustine (BEN). B and C The treatment of AT56 (50 μM) enhanced the cytotoxicity of ADR and BEN in LY1 and LY3 cells. D and E AT56 sensitized DLBCL cells to ADR and BEN in cell apoptosis. F Representative images and quantification of the tail of DLBCL cells treated with AT65 (75 and 125 µM) in the comet assay. Bar = 40 μm. G and H Western blotting and immunofluorescent images indicated that AT56 (75 μM) increased the expression of p-H2AX in DLBCL cells. Bar = 40 μm. Data are shown as the mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Adriamycin, bendamustine, and WP1066 were purchased from Selleck Chemicals (TX, USA).

Techniques: Expressing, Single Cell Gel Electrophoresis, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: Impact of the modulation of TYRO3 expression on the radiosensitivity of bladder cancer cell lines. ( a – c ) Representative clonogenic survival curves of RT112 and 5637 cell lines ( upper panel). 48 h after transfection with siLUC (control, red), siTYRO3#4 (blue), siTYRO3#801 (green) or diluted concentration of siRNA#1 (black) cells were exposed to increased doses of gamma-rays. In each case, downregulation was confirmed by western blot at 48 h after transfection. The corresponding D 10 values were calculated after fitting the experimental data to the classical linear-quadratic equation ( lower panel); ( d ) Representative survival curves of UM-UC-3 control (empty plasmid, red) and TYRO3 over-expressed cell lines (TYRO3 encoding- plasmid, purple) ( upper panel). The upregulation was confirmed by western blot. The corresponding D 10 values were calculated after fitting the experimental data to the classical linear-quadratic equation ( lower panel); ( e ) Phosphorylated TYRO3 shown in western blot ( lower panel) after immunoprecipitation of the cell extracts with phospho-Tyrosine antibodies and its relative expression in 5637 cells quantified using Image-J software and normalized to that of total TYRO3 ( upper panel). Data represents mean ± SD of 3 independent experiments. Unpaired t-test analysis: * p < 0.05; ** p < 0.005, ns: not significant.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Expressing, Transfection, Control, Concentration Assay, Western Blot, Plasmid Preparation, Immunoprecipitation, Software

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 modulation and its impact on Ionizing Radiation-Induced Foci and DNA damage. γH2AX foci visualized after 24 h of 2 Gy irradiation in TYRO3 downregulated RT112 ( a ) or 5637 ( c ) cells (scale bar 5 microns); Quantification of cells containing more than 10 γH2AX foci at 24 h after 2 Gy of irradiation in the downregulated RT112 ( b ) and 5637 ( d ) cell lines; ( e ) γH2AX foci visualized after 24 h of 2 Gy irradiation in TYRO3 overexpressing UM-UC-3 cell line (Scale bar 5 microns); ( f ) Quantification of cells containing more than 10 γH2AX foci at 30 min and 24 h after 2 Gy of irradiation in TYRO3 overexpressing cell lines. The data shown above is from three different experiments and error bars represent the SD. Unpaired t -test analysis: * p < 0.05; ** p < 0.005; *** p < 0.0005; **** p < 0.0005, ns: non-significant. Representative images of the alkaline Comet assay performed on TYRO3 downregulated RT112 ( g ) and 5637 ( h ) cells and the resulting Olive tail moments analysis in TYRO3 downregulated RT112 ( i ) and 5637 ( j ) cells irradiated at 6 Gy. The data shown here is from three independent experiments analyzing 200 nucleus per condition per experiment, horizontal bars represent the median values. Kruskal-Wallis nonparametric tests with Multiple comparisons were used: * p < 0.05; ** p < 0.005, ns: non-significant; ( k ) western blot of DNA damage repair proteins; TYRO3 was downregulated, irradiated at 6 Gy and the lysates were prepared and analyzed 0.5–24 h after.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Alkaline Single Cell Gel Electrophoresis, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 modulation and its impact on DNA damage response pathways. ( a ) Volcano plot showing the fold change (Log2 Ratio) versus negative log of the p-value of differentially expressed genes after Nanostring analysis between BCa (RT112 and 5637) 6 Gy irradiated cells versus BCa 6 Gy irradiated cells after complete TYRO3 knock-down (siTYRO3#4 and siTYRO3#801). Significant: p -value < 0.05 ( b ) Log2 ratio of the significantly expressed genes ( p < 0.05) after Nanostring analysis between the two groups. The name of the up- or downregulated genes are listed on the left. On the right are the corresponding Nanostring gene annotations. ( c ) Protein-protein association network of the up and downregulated genes assessed using the STRING database.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 downregulation affects cell cycle following irradiation. Analysis of the cell cycle distribution 24 h after 6 Gy irradiation on TYRO3-downregulated RT112 ( a ) and 5637 ( b ) cells. Comparison of percentage (%) of cells in G2/M in RT112 ( c ) and 5637 ( d ) cells. Data shown is from three different experiments and error bars represent the SD. Unpaired t-test analysis: * p < 0.05; ** p < 0.005; *** p < 0.0005, ns: non-significant. ( e ) western blot of cell cycle proteins 30 min and up to 24 h after irradiation.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Comparison, Western Blot

Journal: eLife

Article Title: Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers

doi: 10.7554/eLife.74342

Figure Lengend Snippet:

Article Snippet: Antibody , Anti- PERK (Rabbit monoclonal) , Cell Signaling Technology , 3192 , WB (1:1000).

Techniques: Cloning, CRISPR, Knock-Out, Expressing, Recombinant, Plasmid Preparation, Transfection, Sequencing, Software, Diffusion-based Assay, Single Particle

Journal: Journal for Immunotherapy of Cancer

Article Title: Dual regulation of CXCR6+CD8+ T cells modulates cytotoxic and exhaustion-associated programs during prostate cancer progression

doi: 10.1136/jitc-2025-014276

Figure Lengend Snippet: M1-like macrophages secrete CXCL16 and support CXCR6 + CD8 + T-cell recruitment but are progressively lost during PCa progression. ( A ) UMAP plot of tumor-infiltrating myeloid cells from PCa tissues, identifying five distinct clusters, including an IL1B + macrophage subset. ( B ) Dot plot showing average expression and detection frequency of selected marker genes across macrophage and dendritic cell (DC) clusters. ( C ) Violin plots illustrating the expression of key pro-inflammatory ( IL1B , TLR2 , CD86 ), anti-inflammatory ( CD163 , MRC1 ), and chemokine ( CXCL16 ) genes across myeloid subsets. ( D ) AUCell-based quantification of M1 and M2 gene signatures across clusters; IL1B + macrophages exhibit the highest M1 signature score. Kruskal-Wallis test, ****p<0.0001. ( E ) CellChat network visualizing outgoing macrophage-derived signals to CD8 + T-cell subsets; IL1B + macrophages prominently interact with CXCR6 + TEff-like CD8 + T cells. ( F ) Bubble plot visualizing the results of ligand–receptor interaction analysis; CXCL16–CXCR6 axis ranks among the strongest predicted signals. ( G ) Gating strategy for the identification of murine bone marrow-derived macrophages (BMDMs) induced with M-CSF. ( H ) Flow cytometry of BMDMs polarized to M1 (IFN-γ+LPS) or M2 (IL-4) states, assessed by CD80 and CD206 expression. ( I ) Confocal images of THP-1-derived macrophages stained for CD68 after PMA induction. ( J ) Flow cytometry of THP-1-derived macrophages polarized to M1 (IFN-γ+LPS) or M2 (IL-4) states, assessed by MHC-II and CD206 expression. ( K ) Immunoblots showing higher CXCL16 levels in M1-polarized BMDMs compared with their M2 counterparts. ( L ) ELISA quantification of secreted CXCL16 in the supernatants of M1-polarized and M2-polarized THP-1-derived macrophages. Mann-Whitney U test, **p<0.01. ( M ) Immunoblot analysis demonstrating elevated levels of CXCL16 in M1-polarized THP-1-derived macrophages compared with M2-polarized cells. (N, O). A total of 5×10⁶ TRAMP-C1 cells suspended in 100 µL PBS were subcutaneously implanted into the right flank of 5–6-week-old male WT C57BL/6J mice (n=5 per group). Tumors were harvested at day 35 (early stage) and day 49 (advanced stage) post-inoculation. Flow cytometric analysis of TAMs revealed a significant reduction in the ratio of MHCII + CD206⁻ (M1-like) to MHCII⁻ CD206 + (M2-like) macrophages during tumor progression. Mann-Whitney U test, **p<0.01. ( P ) Multiplex immunohistochemistry of human PCa tissues (GS=3+4 vs GS=5+5) demonstrated spatial proximity between CXCL16 + M1-like macrophages (HLA-DRA + ) and CXCR6 + CD8 + T cells in lower-grade (GS=3+4) tumors, which was largely diminished in high-grade (GS=5+5) lesions. Black arrows indicate matched regions across serial tissue sections. Scale bars: upper panels, 100 µm; lower panels, 40 µm. AUCell, area under the recovery curve; GS, Gleason Score; M-CSF, macrophage colony-stimulating factor; PCa, prostate cancer; PBS, phosphate-buffered saline; TAMs, tumor-associated macrophages.

Article Snippet: Sections were then incubated overnight at 4°C with primary antibodies against CD8 (Abcam, ab17147; 1:200), CXCR6 (Abcam, ab8023; 1:100), CXCL16 (ProteinTech Group, 60123-1-Ig; 1:100), CD68 (Abcam, ab125212; 1:200), iNOS (Abcam, ab3523; 1:200), HLA-DRA (ProteinTech Group, 17221-1-AP; 1:100), CD163 (Abcam, ab182422; 1:200), KLF2 (ABclonal, A16480; 1:200), FOXO1 (ProteinTech Group, 18592-1-AP; 1:100), Granzyme B (Thermo Fisher, MA1-80734; 1:100), α-SMA (Abcam, ab5694; 1:100) and AR (Abcam, ab133273; 1:100).

Techniques: Expressing, Marker, Derivative Assay, Flow Cytometry, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Multiplex Assay, Immunohistochemistry, Saline

Journal: Journal for Immunotherapy of Cancer

Article Title: Dual regulation of CXCR6+CD8+ T cells modulates cytotoxic and exhaustion-associated programs during prostate cancer progression

doi: 10.1136/jitc-2025-014276

Figure Lengend Snippet: IL-10–STAT3–FOXO1 signaling reprograms CXCR6 + CD8 + T cells toward a dysfunctional state. ( A ) Dot plot showing the expression of IL10RA , STAT3 , STAT4 , CXCR6 , and related markers across CD8 + T cell subsets in scRNA-seq data. ( B ) IL-10 pathway activity scores across CD8 + T cell clusters. Kruskal-Wallis test, ****p<0.0001. (C, D) Murine splenic CD8 + T cells cultured in a medium containing anti-CD3 (5 µg/mL), anti-CD28 (5 µg/mL), and IL-2 (10 ng/mL) for 48 hours. Following initial activation, cells were maintained in fresh medium supplemented with IL-2 (10 ng/mL) for an additional 7 days. On day 9, cells were treated with 20 ng/mL murine IL-10, IL-15, or STAT3 inhibitor Stattic (2 µM) for 24 hours. The protein expression of STAT3, p-STATS, FOXO1, KLF2, and CXCR6 was assessed by Western blot analysis. (E, F) Flow cytometry of mouse spleen-derived CD8 + T cells shows preferential expression of IL-10R on CXCR6 + CD8 + T cells, with upregulation observed following TCR stimulation (anti-CD3/CD28+IL-2, day 10), indicating heightened susceptibility to IL-10-mediated signaling. (G–I) Flow cytometry of human peripheral blood mononuclear cell (PBMC) CD8 + T cells from healthy donors similarly demonstrates enhanced IL-10R expression on CXCR6 + CD8 + T cells and its induction on TCR stimulation. ( J ) PCA of bulk RNA-seq. Prostate tissues from Pb-Cre; Pten flox/flox ( T ) and WT mice (n=3/group) were profiled by bulk RNA-seq. PCA separated T (blue) from WT (red) chiefly along PC1 (87.24% variance) and PC2 (5.64%). ( K ) Sample-to-sample distance heatmap. Distance matrix based on transformed expression values shows tight clustering of biological replicates within genotype and clear segregation between T and WT. ( L ) Volcano plot. Differential expression analysis between T and WT (cut-offs |log2FC|≥1.5, FDR<0.05). Points are colored by direction (Up=red; Down=blue). Dashed lines indicate thresholds. Il10 , Mrc1 , Cd163 , and Cxcr6 are highlighted in purple; other selected genes are labeled as indicated. Y-axis shows –log 10 (adjusted p). ( M ) Multiplex immunofluorescence (human prostate). Representative fields from human prostate specimens (n=9). Channels: DAPI (nuclei), CD68 (pan-macrophage), HLA-DRA (M1-like macrophage), CD163 (M2-like macrophage), and IL-10. IL-10 signal is enriched in tumor regions and co-localizes with CD68 + CD163 + macrophages. Scale bar: 20 µm. DAPI, 4′,6-diamidino-2-phenylindole; FDR, false discovery rate; PCA, principal component analysis; scRNA-seq, single-cell RNA sequencing; WT, wild-type.

Article Snippet: Sections were then incubated overnight at 4°C with primary antibodies against CD8 (Abcam, ab17147; 1:200), CXCR6 (Abcam, ab8023; 1:100), CXCL16 (ProteinTech Group, 60123-1-Ig; 1:100), CD68 (Abcam, ab125212; 1:200), iNOS (Abcam, ab3523; 1:200), HLA-DRA (ProteinTech Group, 17221-1-AP; 1:100), CD163 (Abcam, ab182422; 1:200), KLF2 (ABclonal, A16480; 1:200), FOXO1 (ProteinTech Group, 18592-1-AP; 1:100), Granzyme B (Thermo Fisher, MA1-80734; 1:100), α-SMA (Abcam, ab5694; 1:100) and AR (Abcam, ab133273; 1:100).

Techniques: Expressing, Activity Assay, Cell Culture, Activation Assay, Western Blot, Flow Cytometry, Derivative Assay, RNA Sequencing, Transformation Assay, Quantitative Proteomics, Labeling, Multiplex Assay, Immunofluorescence, Single Cell

Journal: bioRxiv

Article Title: Tumor nutrient stress gives rise to a drug tolerant cell state in pancreatic cancer

doi: 10.1101/2025.09.04.673818

Figure Lengend Snippet: (A) (Left) Immunoblot confirmation of Bax and Bak1 double knockout ( Bax/Bak1 DKO) in mPDAC1-RPMI cells. (Right) Cell viability measured by IncuCyte cell viability assay of mPDAC1-RPMI NTC and Bax/Bak1 DKO cultures treated with the indicated concentration of gemcitabine (n = 3) for 72 hours. (B) Diagram of cell death by chemotherapy. Initial chemotherapeutic response requires exposure mediated by cellular transporters and damage from on-target activity such as DNA double stranded breaks, indicated by the presence of γH2AX. Following cellular damage, a pro-death cellular response is generated, which may feature the upregulated expression of pro-apoptotic BH3-only proteins. A sufficiently strong death signal, in the case of apoptotic cell death, results in mitochondrial outer membrane permeabilization (MOMP). This event results in a commitment to cell death leading to cellular disassembly mediated by the activation of executioner caspases, which finally leads to loss of plasma membrane integrity. (C) (Right) Alkaline comet assay of mPDAC1-RPMI and mPDAC1-TIFM cultures treated for 18hrs with 150nM Gemcitabine (RPMI n = 58, TIFM n = 64) or DMSO vehicle (RPMI n = 61, TIFM n = 52). (Left) Representative images of comets following treatment with gemcitabine or vehicle. (D) Heatmap of C2 MSigDB gene sets with differential enrichment from GSEA analysis of mPDAC1 RPMI cultures treated for 24 hours with 125nM Gemcitabine versus DMSO vehicle control. All gene sets displayed a statistically significant enrichment of less than 0.05 after p-value adjustment by Benjamini-Hochberg correction. Right column displaying enrichment scores for the corresponding gene set following GSEA analysis of mPDAC1 TIFM cultures treated with gemcitabine under identical conditions. Values in heatmap displayed reflect normalized enrichment score (NES). (E) ULM enrichment scores predicting enhanced transcription factor activity for the top 5 differentially enriched TFs in mPDAC1-RPMI cells in response to gemcitabine treatment, with the enrichment scores for mPDAC1-TIFM cells displayed in parallel. (F) Immunoblots for γH2AX, BIM, and Cleaved Caspase-3 in mPDAC1-RPMI and mPDAC1-TIFM cells treated with 150nM Gemcitabine over time at the indicated timepoints. (G) Red fluorescence intensity of Omi-mCherry reporter expressing mPDAC1-RPMI and mPDAC1-TIFM cells treated with either gemcitabine or vehicle. (H) Percent of Omi-mCherry low cells (post-MOMP) in mPDAC cells cultured under the indicated medium and drug treatment conditions (n = 4). (I) Immunoblots for BAX in mitochondrial and cytosolic isolates from mPDAC1-RPMI and mPDAC1-TIFM cells treated with 250 nM gemcitabine over time at the indicated timepoints. (J) Relative mitochondrial depolarization of mPDAC1-RPMI and mPDAC1-TIFM cells in response to treatment with recombinant BIM peptide at the indicated concentrations (n = 3). (K) Immunoblots for BCL2 family members in the indicated mPDAC cell line and culture condition. (L) Immunoblots for BCL-XL, MCL-1, BIM and BAX following immunoprecipitation of BCL-XL or MCL-1 in mPDAC1-RPMI cell lysates. “Iso” sample reflects pulldown with isotype control rabbit IgG antibody. (M) Immunoblots for BCL-XL and BAX following immunoprecipitation for BCL-XL in mPDAC1-TIFM or mPDAC1-RPMI cell lysates following treatment with gemcitabine (125 nM) and A1331852 (100nM) as indicated for 24 hours. (N) Cell viability of mPDAC1-TIFM cells measured by IncuCyte cell viability assay following treatment with the indicated compounds for 72 hours (Gemcitabine, 150 nM), (A1331852 1 µM), (S63845 1 µM), (Venetoclax 1 µM), (Navitoclax 1 µM). Individual datapoints reflect the mean viability of 3 replicates for a single cell line (mPDAC1-4), as indicated in the figure legend (n = 4 cell lines). (O) (Left) Immunoblot confirmation of Bcl-xL knockout in mPDAC1-TIFM cells. (Right) Cell viability of Bcl-xL knockout or NTC mPDAC1-TIFM cells measured by IncuCyte cell viability assay, following treatment with the indicated concentrations of gemcitabine for 72 hours (n = 3). (P) Immunoblots for γH2AX, BIM, and Cleaved Caspase-3 in Bcl-xL knockout or NTC mPDAC1-TIFM cells following treatment with 150 nM Gemcitabine at the indicated timepoints. For A,J,O, the area under the curve (AUC) was calculated and unpaired t-test was performed to determine significance of differences in AUC for the indicated cultures. For C, H, N, unpaired t-tests were performed to determine significance of differences between sample conditions. For E, all gene sets displayed for the mPDAC1-RPMI sample had significant enrichment with a p-value of less than p=0.05 after multiple tests correction. For J, all transcription factors displayed had significant enrichment with a p-value of less than p=0.0001 after multiple tests correction. * p≤0.05, ** p≤0.01, and **** p≤0.0001.

Article Snippet: In vitro cell viability assays were performed using the following compounds: Gemcitabine hydrochloride (Sigma, G6523), SN-38 (Cayman, 15632), 5-Fluorouracil (Cayman, 14416), Palbociclib (Cayman, 16273), Paclitaxel (Cayman, 10461), Oxaliplatin (Cayman, 13106), Trametinib (Cayman, 16292), RMC-7977 (MedChem Express, HY-156498), S63845 (Selleck Chem, S8383), Venetoclax (Selleck Chem, S8048), Navitoclax (Selleck Chem, S1001), A-1331852 (Selleck Chem, S7801).

Techniques: Western Blot, Double Knockout, Viability Assay, Concentration Assay, Activity Assay, Generated, Expressing, Membrane, Activation Assay, Clinical Proteomics, Alkaline Single Cell Gel Electrophoresis, Control, Fluorescence, Cell Culture, Recombinant, Immunoprecipitation, Knock-Out