Journal: PLoS ONE

Article Title: Gemcitabine-induced Gli-dependent activation of hedgehog pathway resists to the treatment of urothelial carcinoma cells

doi: 10.1371/journal.pone.0254011

Figure Lengend Snippet: (A) NTUB1/NGR and (B) T24/T24_GR cells were treated with various doses of gemcitabine (0−0.4 μM) for 72 h, and were trypsinized and counted numbers by using a counting chamber to measure cellular viability. (C, D) Total RNA or (E-G) cell lysates were collected from NTUB1/NGR or T24/T24_GR cells to perform (C, D) quantitative real-time PCR or (E-G) Western blot, respectively. Gemcitabine resistance-related genes dCK and hENT1 were measured at (C, D) mRNA or (E) protein levels, respectively. Protein expression of phopho-AKT ser473 , phospho-GSK3β ser9 and Gli2 was also detected in (F) NTUB1/NGR and (G) T24/T24_GR cells, respectively.

Article Snippet: Then the PVDF membrane was incubated with primary antibodies dCK (Abcam; ab96599, Lot: GR3303586-1), hENT1 (Sigma-Aldrich; B5500117, Lot: GR3217413-6), pAKT Ser473 (Cell Signaling Technology; #4060, Lot: 25), AKT (Cell Signaling Technology; #9272, Lot: 27), pGSK3β Ser9 (Cell Signaling Technology; #9336, Lot: 13), GSK3α/β (Santa Cruz; SC-7291), and Gli2 (Invitrogen; #PA5-79314, Lot: VG3044693) at 4°C overnight for immunoblotting.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Gemcitabine-induced Gli-dependent activation of hedgehog pathway resists to the treatment of urothelial carcinoma cells

doi: 10.1371/journal.pone.0254011

Figure Lengend Snippet: The (A, B) pCS2MT-Gli2ΔN , (C, D) Gli2 specific shRNA plasmids, and were transfected in cells for 24 h, then the abilities of migration and invasion were detected as described in the “Materials and Methods” section. (E-G) NGR cells were treated with specific phospho-AKT inhibitor MK2206 for 24 h. The phopho-AKT ser473 , phospho-GSK3β ser9 and Gli2 protein were detected, and migration/invasion abilities were measured.

Article Snippet: Then the PVDF membrane was incubated with primary antibodies dCK (Abcam; ab96599, Lot: GR3303586-1), hENT1 (Sigma-Aldrich; B5500117, Lot: GR3217413-6), pAKT Ser473 (Cell Signaling Technology; #4060, Lot: 25), AKT (Cell Signaling Technology; #9272, Lot: 27), pGSK3β Ser9 (Cell Signaling Technology; #9336, Lot: 13), GSK3α/β (Santa Cruz; SC-7291), and Gli2 (Invitrogen; #PA5-79314, Lot: VG3044693) at 4°C overnight for immunoblotting.

Techniques: shRNA, Transfection, Migration

Journal: PLoS ONE

Article Title: Gemcitabine-induced Gli-dependent activation of hedgehog pathway resists to the treatment of urothelial carcinoma cells

doi: 10.1371/journal.pone.0254011

Figure Lengend Snippet: The possible mechanisms identified from the in vitro results of present study were summarized in this cartoon graph. Besides the involvement of aberrant gemcitabine metabolism, gemcitabine can activate AKT Ser473 phosphorylation to inactivate GSK3β kinase by phosphorylating its Ser9 residue. The inactivated GSK3β leads to stabilize Gli2 proteins to induce its downstream target genes expressions, which promotes migration/invasion abilities and resistance to gemcitabine.

Article Snippet: Then the PVDF membrane was incubated with primary antibodies dCK (Abcam; ab96599, Lot: GR3303586-1), hENT1 (Sigma-Aldrich; B5500117, Lot: GR3217413-6), pAKT Ser473 (Cell Signaling Technology; #4060, Lot: 25), AKT (Cell Signaling Technology; #9272, Lot: 27), pGSK3β Ser9 (Cell Signaling Technology; #9336, Lot: 13), GSK3α/β (Santa Cruz; SC-7291), and Gli2 (Invitrogen; #PA5-79314, Lot: VG3044693) at 4°C overnight for immunoblotting.

Techniques: In Vitro, Phospho-proteomics, Residue, Migration

Journal: Frontiers in Immunology

Article Title: HDAC3 inhibition as a therapeutic strategy in T-cell acute lymphoblastic leukemia via the TYK2-STAT1-BCL2 signaling pathway

doi: 10.3389/fimmu.2026.1752727

Figure Lengend Snippet: Chidamide and a TYK2 inhibitor synergistically inhibited the growth of T-ALL cells both in vitro and in vivo . Jurkat and Molt-4 cells were incubated with 1 μM chidamide or 4 μM deucravacitinib as a monotherapy or with the combination of chidamide and deucravacitinib for 36 h. (A) CCK-8 assays were used to assess cell proliferation. (B) Flow cytometry was used to analyze cell apoptosis. Jurkat cells (1 × 10 7 cells) were implanted into NOD/SCID mice. The mice were randomly divided into four groups (six mice in each group). The mice in the chidamide-treated group were intragastrically administered with chidamide three times a week for 2 weeks. The mice in the deucravacitinib-treated group were intragastrically administered deucravacitinib twice daily for 2 weeks. The mice in the control group were treated with both PBS and normal saline as a control. (C) The volume of each tumor was measured every 3 days. The tumor volume was calculated using the formula: V = 0.5 × length × width2. (D) A visual analysis of tumors harvested from mice. (E, F) The measurement of xenograft tumor volume and weight. (G) Representative expression of p-STAT1, BCL2 in tumor sections by immunohistochemistry. Data represent three independent experiments and are expressed as mean values ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, NS: P > 0.05).

Article Snippet: When tumors approached 50 mm3, mice were randomly assigned to 4 groups (n=6 mice per group, biological replicates): control (PBS + normal saline), chidamide (5 mg/kg, intragastrically, 3 times/week for 2 weeks), deucravacitinib (a highly selective inhibitor of tyrosine kinase 2, MedChemExpress, China, BMS-986165) (30 mg/kg, intragastrically, twice daily for 2 weeks), or combination therapy.

Techniques: In Vitro, In Vivo, Incubation, CCK-8 Assay, Flow Cytometry, Control, Saline, Expressing, Immunohistochemistry

Journal: eLife

Article Title: Srs2 promotes synthesis-dependent strand annealing by disrupting DNA polymerase δ-extending D-loops

doi: 10.7554/eLife.22195

Figure Lengend Snippet: ( A ) D-loop disruption assay. Fully homologous ssDNA with 607 nucleotides was used as substrates in B and C. Rad51 (0.2 µM) was incubated with 1 nM ssDNA substrate (Rad51/nt = 1:3) for 10 min at 30°C. RPA (33 nM) was added for another 10 min incubation before the addition of 84 nM Rad54 and 7 nM supercoiled plasmid dsDNA (21 µM bp). ( B ) Time course of Srs2 titration into pre-formed D-loops produced by Rad51 and Rad54. Srs2 (0 or 5 nM) was added and incubated for various time at 30°C. Samples were taken at 0, 5, 15, and 25 min and quenched by SDS/Proteinase K treatment. ( C ) Quantitation of normalized total D-loop yield in ( B ) and additional titrations. Absolute initial D-loop yields were between 50% and 75%. ( D ) Srs2-K41S is deficient in ATP hydrolysis. Rates of ATP hydrolysis were determined for 5 nM wild type Srs2 (WT) or catalytic-deficient Srs2-K41S (K41S) in D-loop reaction buffer containing either 0 or 100 mM NaCl, with or without 10 µM φX174 ssDNA as cofactor. ( E ) Time course of D-loop disruption by 5 nM of either wild type Srs2 (WT) or catalytic-deficient Srs2-K41S (K41S). Plotted are means ± standard deviation from n = 3. Absolute initial D-loop yields were between 44% and 72%. ( F ) Induced sister chromatid recombination assay. The normalized (wild type = 100%) induced recombination frequencies are shown at 0.24 µg/mL 4-NQO as means ± standard deviations from three experiments for wild type, srs2△ , and srs2-K41A (see ). The frequency of spontaneous G418 R cells was subtracted from the induced recombination frequencies. The full 4-NQO dose response is shown in . DOI: http://dx.doi.org/10.7554/eLife.22195.002 10.7554/eLife.22195.003 Figure 1—source data 1. Source data for . DOI: http://dx.doi.org/10.7554/eLife.22195.003 10.7554/eLife.22195.004 Figure 1—source data 2. Source data for . DOI: http://dx.doi.org/10.7554/eLife.22195.004 10.7554/eLife.22195.005 Figure 1—source data 1. Source data for . DOI: http://dx.doi.org/10.7554/eLife.22195.005 10.7554/eLife.22195.006 Figure 1—source data 4. Source data for and . DOI: http://dx.doi.org/10.7554/eLife.22195.006

Article Snippet: The protein bands were visualized through immunoblots since GST-Rad54 (anti-GST; GE healthcare #27-4577-01, lot# 9610802) and Srs2 (anti-Srs2 (yC-18); Santa Cruz #sc11991, lot# C0905) were very similar in size.

Techniques: Incubation, Plasmid Preparation, Titration, Produced, Quantitation Assay, Standard Deviation, Recombination Assay

Journal: eLife

Article Title: Srs2 promotes synthesis-dependent strand annealing by disrupting DNA polymerase δ-extending D-loops

doi: 10.7554/eLife.22195

Figure Lengend Snippet: ( A ) Extended D-loop disruption assay. Fully homologous 100mer ssDNA was used as substrates. 0.2 µM Rad51 was incubated with 6 nM ssDNA substrates (Rad51/nt = 1:3) for 10 min at 30°C. RPA (165 nM) was added for another 10 min incubation before the addition of 84 nM Rad54 and 7 nM supercoiled plasmid dsDNA (21 µM bp). After 2 min, 20 nM RFC and 20 nM PCNA were added and incubated for another two min at 30°C before the addition of 7 nM DNA polymerase δ. After 15 min, different amount of Srs2 or storage buffer were added and samples at 25 min were taken and stopped by SDS/Proteinase K treatment. ( B ) Titration of Srs2 (0, 2.5, 7.5, or 25 nM final concentration) in extended D-loop disruption. ( C ) Quantitation of extended D-loops in B ). Plotted are means ± standard deviation from n = 3. ( D ) Extended D-loop disruption assay using α- 32 P-dCTP incorporation. Experimental setup and protein concentrations are identical as in A ), except that α- 32 P-dCTP was included in the buffer and 100mer ssDNA was not radiolabeled. ( E ) Time course of extended D-loop disruption by Srs2, in the presence of PCNA and SUMO-PCNA. After 15 min, Srs2 (25 nM final concentration) or storage buffer were added and samples at 0, 5, 15, and 25 min were taken and stopped by SDS/Proteinase K treatment. DOI: http://dx.doi.org/10.7554/eLife.22195.017 10.7554/eLife.22195.018 Figure 5—source data 1. Source data for . DOI: http://dx.doi.org/10.7554/eLife.22195.018

Article Snippet: The protein bands were visualized through immunoblots since GST-Rad54 (anti-GST; GE healthcare #27-4577-01, lot# 9610802) and Srs2 (anti-Srs2 (yC-18); Santa Cruz #sc11991, lot# C0905) were very similar in size.

Techniques: Incubation, Plasmid Preparation, Titration, Concentration Assay, Quantitation Assay, Standard Deviation

Journal: eLife

Article Title: Srs2 promotes synthesis-dependent strand annealing by disrupting DNA polymerase δ-extending D-loops

doi: 10.7554/eLife.22195

Figure Lengend Snippet: ( A ) Extended D-loop disruption assay using α- 32 P-dCTP incorporation. Fully unlabeled and homologous 100mer ssDNA was used as substrates, and α- 32 P-dCTP was included in the buffer. 0.2 µM Rad51 was incubated with 6 nM ssDNA substrates (Rad51/nt = 1:3) for 10 min at 30°C. RPA (165 nM) was added for another 10 min incubation before the addition of 84 nM Rad54 and 7 nM supercoiled plasmid dsDNA (21 µM bp). After 2 min, 20 nM RFC and 20 nM PCNA, PCNA-SUMO, or PCNA-Ubi, were added and temperature was lowered to 25°C. DNA polymerase δ (7 nM) was added after two more min and incubated for 6 min at 25°C. Reactions with PCNA, SUMO-PCNA, and Ubi-PCNA were individually split into Srs2 or buffer only control. Final concentrations of Srs2 are 0, 2.5, 7.5, or 25 nM. Samples at 25 min were taken and stopped by SDS/Proteinase K treatment. ( B ) Titration of Srs2 in extended D-loop disruption. ( C ) Quantitation of normalized extended D-loops in ( B ). The intensities of the extended D-loops were normalized against the signal of the extended D-loop produced with wild type PCNA without Srs2 at 0 min in each repeat. Plotted are means ± standard deviation from n = 3. DOI: http://dx.doi.org/10.7554/eLife.22195.021 10.7554/eLife.22195.022 Figure 6—source data 1. Source data for . DOI: http://dx.doi.org/10.7554/eLife.22195.022

Article Snippet: The protein bands were visualized through immunoblots since GST-Rad54 (anti-GST; GE healthcare #27-4577-01, lot# 9610802) and Srs2 (anti-Srs2 (yC-18); Santa Cruz #sc11991, lot# C0905) were very similar in size.

Techniques: Incubation, Plasmid Preparation, Titration, Quantitation Assay, Produced, Standard Deviation

Journal: eLife

Article Title: Srs2 promotes synthesis-dependent strand annealing by disrupting DNA polymerase δ-extending D-loops

doi: 10.7554/eLife.22195

Figure Lengend Snippet: ( A ) Extended D-loop disruption assay using α- 32 P-dCTP incorporation. Fully unlabeled and homologous 100mer ssDNA was used as substrates, and α- 32 P-dCTP was included in the buffer. 0.2 µM Rad51 was incubated with 6 nM ssDNA substrates (Rad51/nt = 1:3) for 10 min at 30°C. RPA (165 nM) was added for another 10 min incubation before the addition of 84 nM Rad54 and 7 nM supercoiled plasmid dsDNA (21 µM bp). After 2 min, 20 nM RFC and 20 nM PCNA, SUMO-PCNA, or Ubi-PCNA, were added and temperature was lowered to 25°C. DNA polymerase δ (7 nM) was added after two more min and incubated for 6 min at 25°C. Reactions with PCNA, SUMO-PCNA, and Ubi-PCNA were individually split into Srs2 or buffer only control. Samples at 0, 5, 15, and 25 min were taken and stopped by SDS/Proteinase K treatment. ( B ) Time course of extended D-loop disruption by Srs2. DOI: http://dx.doi.org/10.7554/eLife.22195.023

Article Snippet: The protein bands were visualized through immunoblots since GST-Rad54 (anti-GST; GE healthcare #27-4577-01, lot# 9610802) and Srs2 (anti-Srs2 (yC-18); Santa Cruz #sc11991, lot# C0905) were very similar in size.

Techniques: Incubation, Plasmid Preparation

Journal: eLife

Article Title: Srs2 promotes synthesis-dependent strand annealing by disrupting DNA polymerase δ-extending D-loops

doi: 10.7554/eLife.22195

Figure Lengend Snippet: ( A ) Extended D-loop disruption assay. Fully homologous 100mer ssDNA was used as substrates. Rad51 (0.2 µM) was incubated with 6 nM ssDNA substrates (Rad51/nt = 1:3) for 10 min at 30°C. RPA (165 nM) was added for another 10 min incubation before the addition of 84 nM Rad54 and 7 nM supercoiled plasmid dsDNA (21 µM bp). After 2 min, 20 nM RFC and 20 nM PCNA, SUMO-PCNA, or Ubi-PCNA, were added and temperature was lowered to 25°C. DNA polymerase δ (7 nM) was added after two more min and incubated for 6 min at 25°C. Reactions with PCNA, SUMO-PCNA, and Ubi-PCNA were individually split into Srs2 or buffer only control. ( B ) Time course of extended D-loop disruption by 7.5 nM Srs2. Samples were taken at 0, 5, 15, and 25 min and stopped by SDS/Proteinase K treatment. ( C ) Quantitation of total D-loops in ( C ), unextended D-loops in ( D ), extended D-loops in ( E ), and ratio of extended D-loops/unextended D-loops in ( F ). Plotted are means ± standard deviation from n = 3. DOI: http://dx.doi.org/10.7554/eLife.22195.024 10.7554/eLife.22195.025 Figure 7—source data 1. Source data for and . DOI: http://dx.doi.org/10.7554/eLife.22195.025

Article Snippet: The protein bands were visualized through immunoblots since GST-Rad54 (anti-GST; GE healthcare #27-4577-01, lot# 9610802) and Srs2 (anti-Srs2 (yC-18); Santa Cruz #sc11991, lot# C0905) were very similar in size.

Techniques: Incubation, Plasmid Preparation, Quantitation Assay, Standard Deviation

Journal: eLife

Article Title: Srs2 promotes synthesis-dependent strand annealing by disrupting DNA polymerase δ-extending D-loops

doi: 10.7554/eLife.22195

Figure Lengend Snippet: After DNA resection and Rad51 filament formation, Rad54 assists Rad51 to search for homology and form D-loops. During heteroduplex formation, Rad54 removes Rad51 at the 3' invading end to prepare the loading of DNA polymerase through RFC and PCNA. DNA synthesis is initiated by DNA polymerase δ. SUMO-PCNA actively recruits Srs2 to the D-loop. Srs2 potentially enforces SDSA through three distinct mechanisms: 1) Srs2 competes with DNA polymerase for PCNA controlling the length of new DNA synthesis . 2) Srs2 translocates in a 3' → 5' direction to dissociate the hDNA incorporated in the D-loop (this work). After the newly extended 3' invading end is dissociated it is available to anneal with the second end to generate non-crossover products. 3) Srs2 may also dissociate Rad51 from the second end to allow single-strand annealing in analogy to RECQ5 ( ; ). DOI: http://dx.doi.org/10.7554/eLife.22195.031

Article Snippet: The protein bands were visualized through immunoblots since GST-Rad54 (anti-GST; GE healthcare #27-4577-01, lot# 9610802) and Srs2 (anti-Srs2 (yC-18); Santa Cruz #sc11991, lot# C0905) were very similar in size.

Techniques: DNA Synthesis

Journal: eLife

Article Title: Srs2 promotes synthesis-dependent strand annealing by disrupting DNA polymerase δ-extending D-loops

doi: 10.7554/eLife.22195

Figure Lengend Snippet: Numerous proteins have been implicated genetically and biochemically in targeting the D-loop intermediate for disruption as discussed in the introduction. In the budding yeast alone, the DNA helicases Srs2, Sgs1, and Mph1 ( ; ; ; ) (this study), the motor protein Rad54 ( ; ), as well as Top3-Rmi1 were shown to disrupt D-loops in vitro. We highlight possible reasons for the involvement of multiple proteins in D-loop disruption. First, before extension by DNA polymerase, disruption of a nascent D-loop is an inherently anti-recombination activity, whereas disruption of a D-loop extended by DNA polymerases limits crossover and favors SDSA. What distinguished these D-loops is their potential length, although little is known about this in vivo, and the proteins bound to the intermediate. While the genetic signatures of anti-recombination and pro-SDSA are different, the biochemical reaction of disrupting nascent or extending D-loops is highly similar and protein interactions involving also posttranslational modifications between the various D-loop disrupting enzymes may only impart preference rather than absolute specificity. Second, D-loops are dynamic entities, likely with varying length and structure, where for example the 3’ end may be embedded in the heteroduplex or transiently extruded. As we show for Srs2 , such structural variations may influence the activity of the enzymes involved. Finally, D-loops occur not only in the context of DSB repair but also in the context of gap repair. The D-loop structure resulting from gap invasion differs, as there are no free ends available to allow true intertwining. We tried to model this reaction using invading DNA molecules with duplex heterology on both ends and show that Srs2 can disrupt such pairing intermediate, albeit with lower efficiency than regular D-loops. It will be interesting to test the activity of other D-loop disrupting proteins on such substrates. We propose that multiple enzymes with individual preferences for specific D-loop substrates are acting in vivo in a partially overlapping fashion to control anti-recombination, crossover, and SDSA. This view is consistent with genetic data and the observations that mutations in SRS2, SGS1, and MPH1 engage in synthetic lethal/negative interactions in budding yeast ( ; ; ; ). DOI: http://dx.doi.org/10.7554/eLife.22195.032

Article Snippet: The protein bands were visualized through immunoblots since GST-Rad54 (anti-GST; GE healthcare #27-4577-01, lot# 9610802) and Srs2 (anti-Srs2 (yC-18); Santa Cruz #sc11991, lot# C0905) were very similar in size.

Techniques: In Vitro, Activity Assay, In Vivo

Journal: Asaio Journal

Article Title: A Versatile Hybrid Mock Circulation for Hydraulic Investigations of Active and Passive Cardiovascular Implants

doi: 10.1097/MAT.0000000000000851

Figure Lengend Snippet: Electric analog of the numerical models of the cardiovascular system used in this study. Path 1 (P1) corresponds to the BiVAD test case, path 2 (P2) to the TAH test case, and path 3 (P3) to the TCPC test case. Gray lines indicate the interfaces of the respective implant to the numerical model. The additional control mechanisms for heart rate and maximum elastance are only used for the Fontan circulation based on Granegger et al. BiVAD, biventricular assist device; TAH, total artificial heart; TCPC, total cavopulmonary connection.

Article Snippet: The Donovan mock circulation, which is the best-known conventional system, was developed in 1975 and was recently used to evaluate the SynCardia TAH in vitro .

Techniques:

Journal: Asaio Journal

Article Title: A Versatile Hybrid Mock Circulation for Hydraulic Investigations of Active and Passive Cardiovascular Implants

doi: 10.1097/MAT.0000000000000851

Figure Lengend Snippet: Left: Picture of the HMC during the BiVAD and TAH configuration experiments with the two HeartWare HVADs installed. Right: Picture of the HMC during the Fontan experiments with the three-dimensional-printed TCPC. AoP, aortic pressure; BiVAD, biventricular assist device; HMC, hybrid mock circulation; IVCP, inferior vena cava pressure; LAP, left atrial pressure; LPAP, left pulmonary arterial pressure; LVP, left ventricular pressure; PAP, pulmonary arterial pressure; RAP, right atrial pressure; RPAP, right pulmonary arterial pressure; RVP, right ventricular pressure; SVCP, superior vena cava pressure; TAH, total artificial heart; TCPC, total cavopulmonary connection.

Article Snippet: The Donovan mock circulation, which is the best-known conventional system, was developed in 1975 and was recently used to evaluate the SynCardia TAH in vitro .

Techniques:

Journal: Asaio Journal

Article Title: A Versatile Hybrid Mock Circulation for Hydraulic Investigations of Active and Passive Cardiovascular Implants

doi: 10.1097/MAT.0000000000000851

Figure Lengend Snippet: In-vitro performance of a TAH configuration consisting of two HeartWare HVADs and operating either at a constant speed or with physiologic control during an increase of PVR. The signals of the pump speeds and flows as well as of the LAP, the AoP, the RAP, and the PAP are depicted. AoP, aortic pressure; LAP, left atrial pressure; LVAD, left ventricular assist device; PAP, pulmonary arterial pressure; PVR, pulmonary vascular resistance; RAP, right atrial pressure; RVAD, right ventricular assist device; TAH, total artificial heart.

Article Snippet: The Donovan mock circulation, which is the best-known conventional system, was developed in 1975 and was recently used to evaluate the SynCardia TAH in vitro .

Techniques: In Vitro