Journal: iScience

Article Title: Therapy-induced normal tissue damage promotes breast cancer metastasis

doi: 10.1016/j.isci.2023.108503

Figure Lengend Snippet:

Article Snippet: Venetoclax , Selleckchem , Cat#S8048.

Techniques: Luciferase, Virus, Plasmid Preparation, Recombinant, Staining, Ab Array, Generated, Gene Expression, Software

Journal: bioRxiv

Article Title: RAS pathway activation drives clonal selection and monocytic differentiation in FLT3 and BCL2 inhibitor resistance

doi: 10.1101/2025.02.02.636108

Figure Lengend Snippet: A. Schematic depicting sample workflow for single-cell Dabseq. Ven/Gilt, venetoclax and gilteritinib; SCseq, single-cell sequencing B. Swimmer’s plot of 12 patient cohort treated on clinical trial. Each horizontal bar is a unique patient. Clinical responses were evaluated on the basis of guidelines adapted from the International Working Group for AML . Triangles indicate correlative samples. Bars to the left of the Y axis indicate treatment with TKI and/or venetoclax prior to trial enrollment. TKI, tyrosine kinase inhibitor; Ven, venetoclax; CR, complete response; CRi, complete response with incomplete hematologic recovery; MLFS, morphological leukemia-free state; PR, progressive disease. C. Oncoplot of the 12 patient cohort prior to Ven/Gilt therapy. Each column is a unique patient. Mutations are color-coded based on whether they were detected via bulk next-generation sequencing, single-cell DNA sequencing, or both. Only mutations covered by the SC amplicon panel are depicted. *Patient 7 did not have bulk sequencing available. D. Bar chart depicting diversity of clonal composition and size of FLT3- mutated clones prior to Ven/Gilt. Bars are color-coded based on FLT3 mutation(s) and co-mutations, and percentages reflect relative proportion of FLT3- mutated clones for individual patients. Dark gray sections represent clones with mutations detected aside from FLT3 and light gray sections represent sequenced cells without pathogenic mutations detected by the SC sequencing panel. E. Pairwise association of driver mutations identified via SC DNA sequencing across 48 clones in 12 patient cohort prior to Ven/Gilt therapy. For each mutation pair, cooccurrence is summarized as log odds ratio (OR), with positive values indicating cooccurrence and negative values mutual exclusivity. Statistical significance is indicated as *p, .05; **p, .01; ***p, .001. F. Correlation matrix of association between mutations and 9 cell-surface proteins in 11 patients prior to Ven/Gilt. The plot shows cooccurence (red) or exclusivity (blue) based on point-biserial estimate with statistician significance indicated as *p , .05; **p , .01; ***p , .001.

Article Snippet: Venetoclax/Gilteritinib (Ven/Gilt-R) and Venetoclax resistant (Ven-R) cells were generated by culturing quizartinib-selected NRAS G12C Molm14 cells in escalating doses of venetoclax (155 – 4454 nmol/L ) with or without gilteritinib (14 – 34 nmol/L) (both compounds from Selleckchem) .

Techniques: Sequencing, Next-Generation Sequencing, DNA Sequencing, Amplification, Clone Assay, Mutagenesis

Journal: bioRxiv

Article Title: RAS pathway activation drives clonal selection and monocytic differentiation in FLT3 and BCL2 inhibitor resistance

doi: 10.1101/2025.02.02.636108

Figure Lengend Snippet: A. Split violin plot comparing expression of select genes associated with venetoclax resistance in the literature in the immature vs monocytic leukemia populations at baseline and prior to Ven/Gilt therapy. B. Scatterplot depicting correlation between RAS signaling vs monocytic cell state in the immature and monocytic populations at baseline and relapse. Each point represents a single cell. C. Enrichment profile and ranking metric score for RAS signaling in the monocytic ( left) and immature ( right ) populations. In the monocytic population, RAS signaling is significantly positively enriched at relapse relative to pre-Ven/Gilt. E. Scatterplot depicting correlation between venetoclax resistance vs monocytic cell state in the immature and monocytic populations at baseline and relapse. Each point represents a single cell. F. Scatterplot depicting correlation between venetoclax resistance vs RAS signaling in the immature and monocytic populations at baseline and relapse. Each point represents a single cell.

Article Snippet: Venetoclax/Gilteritinib (Ven/Gilt-R) and Venetoclax resistant (Ven-R) cells were generated by culturing quizartinib-selected NRAS G12C Molm14 cells in escalating doses of venetoclax (155 – 4454 nmol/L ) with or without gilteritinib (14 – 34 nmol/L) (both compounds from Selleckchem) .

Techniques: Expressing

Journal: bioRxiv

Article Title: RAS pathway activation drives clonal selection and monocytic differentiation in FLT3 and BCL2 inhibitor resistance

doi: 10.1101/2025.02.02.636108

Figure Lengend Snippet: B. Heatmap of normalized gene expression of select genes associated with Venetoclax resistance in the non-resistant (NR), Ven-R, and Ven/Gilt-R MOLM-14 ( FLT3- ITD) and NRAS G12C co-mutant cell lines. C. Heatmap of normalized gene expression of select genes associated with monocytic differentiation in the non-resistant (NR), Ven-R, and Ven/Gilt-R MOLM-14 ( FLT3- ITD) and NRAS G12C co-mutant cell lines. D. Dose response curves representing relative proliferation of Ven-R, Ven/Gilt-R, and parental non-resistant NRAS G12C mutant MOLM-14 ( FLT3 -ITD+) cells with and without the addition of trametinib after 48 hours of increasing doses of venetoclax (top) and gilteritinib (bottom). Experiments done in 3 technical replicates and error bars represent standard deviation E. Dose response curves representing caspase 3/7 expression of Ven-R, Ven/Gilt-R, and parental non-resistant NRAS G12C mutant MOLM-14 ( FLT3 -ITD+) cells with and without the addition of trametinib after 24 hours of increasing doses of venetoclax (top) and gilteritinib (bottom). Experiments done in 3 technical replicates and error bars represent standard deviation. F. Bar plot of normalized enrichment scores (NES) from Gene Set Expression Analysis (GSEA) comparing venetoclax/gilteritinib-resistant (Ven/Gilt-R) vs non-resistant FLT3-ITD/NRAS co-mutant cells (turquoise) and venetoclax-resistant (Ven-R) vs non-resistant FLT3-ITD/NRAS co-mutant cells (red). Positive scores indicate enrichment in the resistant cells. Statistical significance is indicated as ***q <0.001, ** q <0.01, *q <0.05. G. Enrichment profile and ranking metric score for the RAS signaling ( top) and monocyte-like AML ( bottom ) gene sets in the Ven/Gilt-R ( left) and Ven-R (right) cells vs the non-resistant cells.

Article Snippet: Venetoclax/Gilteritinib (Ven/Gilt-R) and Venetoclax resistant (Ven-R) cells were generated by culturing quizartinib-selected NRAS G12C Molm14 cells in escalating doses of venetoclax (155 – 4454 nmol/L ) with or without gilteritinib (14 – 34 nmol/L) (both compounds from Selleckchem) .

Techniques: Gene Expression, Mutagenesis, Standard Deviation, Expressing

Journal: Journal of Virology

Article Title: HIV-1 Nef activates proviral DNA transcription by recruiting Src kinase to phosphorylate host protein Nef-associated factor 1 to compromise its viral restrictive function

doi: 10.1128/jvi.00280-25

Figure Lengend Snippet: Nef recruits Src and promotes HIV-1 proviral DNA transcription by stimulating PI3K/AKT/mTOCR1 pathways. ( A, B ) Nef promotes HIV-1 replication. PHA-P-activated CD4 + T cells (10 6 cells) were infected with replication-competent HIV-1 NL4-3 (WT) or Nef-deficient (ΔNef) mutant virus (50 ng p24 gag amounts of viruses) ( A ), or with pseudotyped single-cycle infectious HIV-H131(ΔNef)/VSV-G or HIV-H132 (Nef)/VSV-G (5 ng p24 gag amounts of viruses) ( B ), for 5 days. Viral replication was detected by measuring the production of cell-associated gag mRNA. ( C ) Nef increases LTR-driven gene expression. Nef-expressing plasmid pCDH-CMV-MCS-EF1-Puro-Nef, HIV-1- tat expressing plasmid pRK-Flag/tat, and a luciferase reporter driven by the full-length LTR promoter derived from HIV-1 NL4-3 were co-transfected into HEK293T cells, and β-Gal-expressing vector was used to normalize transfection efficiency. At 24 h post-transfection, cells were harvested, and the reporter gene expressions were assessed. ( D, E ) Nef promotes the transcription of HIV-1 proviral DNA. C11 cells were transduced with lentiviruses (5 ng p24 gag amounts of viruses) containing HIV-1 Nef- or mutant-expressing plasmid or vector control for 2 days; the transcription of HIV-1 proviral DNA was measured by detecting GFP expression ( D ) or measuring the production of cell-associated gag mRNA ( E ). ( F ) The illumination of Nef motif and mutants. ( G through J ) Nef activates the PI3K/AKT/mTORC1 pathway. C11 cells were transfected with pCDH-CMV-MCS-EF1-Puro-Nef (0.2, 1, or 2 ng plasmids in “G”; 1 ng plasmids in “H” and “I"), mutant-expressing plasmid, or a vector control for 2 days. The expression and phosphorylation of PI3K p85 subunit, AKT, mTORC1, P70S6K S6, and CDK9 were detected by western blotting. One representative from at least five repeats is shown. The Image J software was used to calculate the gray intensity of western blotting strips, and the relative values were labeled below ( G through I ). ** P < 0.01 and *** P < 0.001 are considered significant differences determined by an unpaired t test.

Article Snippet: Endogenous Naf1 was detected with a mouse mAb at a dilution of 1:1,000 , and the following other antibodies were used: anti-Flag tag mouse (M20008; Abmart Inc., Shanghai, China), anti-GAPDH (clone 3B3) ( M20006 ; Abmart Inc., Shanghai, China), anti-Src Rabbit mAb (2109, Cell Signaling Technology), anti-phosphorylation-Src family (Tyr416) rabbit mAb (6943, Cell Signaling Technology), anti-PI3K kinase p85 subunit (4292; Cell Signaling Technology), anti-PI3K phosphorylation p85 Tyr458/p55 Tyr 199 (4228; Cell Signaling Technology), anti-AKT (pan) (C67E7) (4691S; Cell Signaling Technology), anti-AKT phosphorylation Thr308 (D25E6) (13038; Cell Signaling Technology), anti-mTORC1 (ab32028; Abcam), anti-mTORC1 phosphorylation S2448 (ab109268; Abcam), anti-p70 S6 kinase (9202; Cell Signaling Technology), anti-p70 S6 kinase phosphor Thr389 (9205; Cell Signaling Technology), anti-S6 ribosomal protein(5G10) (2217; Cell Signaling Technology), anti-S6 ribosomal protein phosphorylation Ser235/236 (D57.2.2E) (4858; Cell Signaling Technology), anti-myc tag (ab9106; Abcam), anti-Cyclin-dependent protein kinase 9 (anti-CDK9)(2316; Cell Signaling Technology), anti-phosphorylation-CDK9 (Thr186) (2549; Cell Signaling Technology), and anti-Cyclin T1 (81464; Cell Signaling Technology).

Techniques: Infection, Mutagenesis, Virus, Gene Expression, Expressing, Plasmid Preparation, Luciferase, Derivative Assay, Transfection, Transduction, Control, Phospho-proteomics, Western Blot, Software, Labeling

Journal: Journal of Virology

Article Title: HIV-1 Nef activates proviral DNA transcription by recruiting Src kinase to phosphorylate host protein Nef-associated factor 1 to compromise its viral restrictive function

doi: 10.1128/jvi.00280-25

Figure Lengend Snippet: Naf1 is required for Nef-induced transcription of HIV proviral DNA. ( A ) C11 cells were transduced with lentiviruses containing Naf1 shRNA or the scramble control for 48 h and further transduced with pCDH-CMV-MCS-EF1-Puro-Nef or a vector control for an additional 48 h. HIV proviral DNA transcription was measured by detecting GFP expression, and the protein expression and Nef-induced phosphorylation of PI3K p85 subunit, AKT, mTORC1, and CDK9 were detected by western blotting (B). ( C–E ) Naf1 suppresses the PI3K/AKT/mTOCR1 pathway. C11 cells were transfected with pCMV-Tag3B-myc-Naf1 (0.2, 1, or 2 ng plasmids) or a vector control ( C ), or transduced with lentiviruses containing Naf1 shRNA or the scramble control ( D ), for 48 h. Western blotting was performed to detect the protein expressions and the phosphorylation levels of p85, AKT, mTORC1, P70S6K, and CDK9 ( C, D ). ( E ) LTR-driven gene expression. HEK293T cells were transduced with lentiviruses containing Naf1 shRNA or scramble control for 48 h, then were further transfected with pCDH-CMV-MCS-EF1-Puro-Nef, pRK-Flag/tat, and a luciferase reporter driven by HIV-1 NL4-3 -LTR for 24 h, and the reporter gene expressions were assessed. ( F ) Viral replication. PHA-P-activated CD4 + T cells (10 6 cells) were transduced with lentiviruses containing Naf1 shRNA or scramble control for 2 days, then were further infected with HIV-1 NL4-3 (WT) or Nef-deficient (ΔNef) mutant virus (50 ng p24 gag amounts of viruses) for 5 days. Viral replication was detected by measuring the p24 gag in cell supernatant. One representative from four repeats is shown. The Image J software was used to calculate the gray intensity of western blotting strips, and the relative values were labeled below ( B–D ). ** P < 0.01 and *** P < 0.001 are considered significant differences determined by an unpaired t test.

Article Snippet: Endogenous Naf1 was detected with a mouse mAb at a dilution of 1:1,000 , and the following other antibodies were used: anti-Flag tag mouse (M20008; Abmart Inc., Shanghai, China), anti-GAPDH (clone 3B3) ( M20006 ; Abmart Inc., Shanghai, China), anti-Src Rabbit mAb (2109, Cell Signaling Technology), anti-phosphorylation-Src family (Tyr416) rabbit mAb (6943, Cell Signaling Technology), anti-PI3K kinase p85 subunit (4292; Cell Signaling Technology), anti-PI3K phosphorylation p85 Tyr458/p55 Tyr 199 (4228; Cell Signaling Technology), anti-AKT (pan) (C67E7) (4691S; Cell Signaling Technology), anti-AKT phosphorylation Thr308 (D25E6) (13038; Cell Signaling Technology), anti-mTORC1 (ab32028; Abcam), anti-mTORC1 phosphorylation S2448 (ab109268; Abcam), anti-p70 S6 kinase (9202; Cell Signaling Technology), anti-p70 S6 kinase phosphor Thr389 (9205; Cell Signaling Technology), anti-S6 ribosomal protein(5G10) (2217; Cell Signaling Technology), anti-S6 ribosomal protein phosphorylation Ser235/236 (D57.2.2E) (4858; Cell Signaling Technology), anti-myc tag (ab9106; Abcam), anti-Cyclin-dependent protein kinase 9 (anti-CDK9)(2316; Cell Signaling Technology), anti-phosphorylation-CDK9 (Thr186) (2549; Cell Signaling Technology), and anti-Cyclin T1 (81464; Cell Signaling Technology).

Techniques: Transduction, shRNA, Control, Plasmid Preparation, Expressing, Phospho-proteomics, Western Blot, Transfection, Gene Expression, Luciferase, Infection, Mutagenesis, Virus, Software, Labeling

Journal: Journal of Virology

Article Title: HIV-1 Nef activates proviral DNA transcription by recruiting Src kinase to phosphorylate host protein Nef-associated factor 1 to compromise its viral restrictive function

doi: 10.1128/jvi.00280-25

Figure Lengend Snippet: Naf1-Y552 phosphorylation activates PI3K/AKT/mTORC1 signaling and compromises Naf1's inhibitor roles in HIV proviral DNA transcription. HEK293T cells were co-transfected with pCMV-Tag3B-myc-Naf1 (or Naf1 mutant plasmids) and p85 plasmid. Immunoprecipitations were performed with anti-myc antibody, and p85 was immunoblotted with a specific antibody ( A ), and western blotting was performed to detect the expression and phosphorylation levels of Naf1, p85 subunit, AKT, mTORC1, and CDK9 ( B ). ( C ) Naf1-Y552D induces transcription of HIV proviral DNA. C11 cells were transduced with lentiviruses containing WT Naf1-expressing plasmid pCDH-CMV-MCS-EF1-Puro/Naf1, Y552D, or Y552F mutant-expressing plasmid. The transcription of HIV proviral DNA was measured either by detecting GFP expression or quantifying the production of cell-associated gag mRNA. The results from five repeats ( n = 5) are summarized. Mean fluorescence intensity (MFI) was calculated. ( D ) The association of Cyclin T1 with HIV-1 5`-LTR in C11 or ACH2 cells was determined by a cross-linked ChIP assay. ( E ) Nef compromises Naf1's inhibitory roles of HIV-1 replication. Jurkat CD4 + T cells were transfected with pCMV-Tag3B-myc-Naf1 or vector and then infected with HIV-1 NL4-3 or ΔNef mutant virus for 48 h. Viral replication was detected by measuring the production of cellular gag mRNA. ( F ) Nef compromises Naf1's inhibitory roles of LTR activation. HEK293T cells were transfected with pCMV-Tag3B-myc-Naf1, pCDH-CMV-MCS-EF1-Puro-Nef, pRK-Flag/tat, and a luciferase reporter driven by HIV-1 NL4-3 -LTR, for 24 h, and the reporter gene expressions were assessed. One representative from four repeats is shown. * P < 0.05, ** P < 0.01, and *** P < 0.001 denote the significant difference determined by an unpaired t test.

Article Snippet: Endogenous Naf1 was detected with a mouse mAb at a dilution of 1:1,000 , and the following other antibodies were used: anti-Flag tag mouse (M20008; Abmart Inc., Shanghai, China), anti-GAPDH (clone 3B3) ( M20006 ; Abmart Inc., Shanghai, China), anti-Src Rabbit mAb (2109, Cell Signaling Technology), anti-phosphorylation-Src family (Tyr416) rabbit mAb (6943, Cell Signaling Technology), anti-PI3K kinase p85 subunit (4292; Cell Signaling Technology), anti-PI3K phosphorylation p85 Tyr458/p55 Tyr 199 (4228; Cell Signaling Technology), anti-AKT (pan) (C67E7) (4691S; Cell Signaling Technology), anti-AKT phosphorylation Thr308 (D25E6) (13038; Cell Signaling Technology), anti-mTORC1 (ab32028; Abcam), anti-mTORC1 phosphorylation S2448 (ab109268; Abcam), anti-p70 S6 kinase (9202; Cell Signaling Technology), anti-p70 S6 kinase phosphor Thr389 (9205; Cell Signaling Technology), anti-S6 ribosomal protein(5G10) (2217; Cell Signaling Technology), anti-S6 ribosomal protein phosphorylation Ser235/236 (D57.2.2E) (4858; Cell Signaling Technology), anti-myc tag (ab9106; Abcam), anti-Cyclin-dependent protein kinase 9 (anti-CDK9)(2316; Cell Signaling Technology), anti-phosphorylation-CDK9 (Thr186) (2549; Cell Signaling Technology), and anti-Cyclin T1 (81464; Cell Signaling Technology).

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Plasmid Preparation, Western Blot, Expressing, Transduction, Fluorescence, Infection, Virus, Activation Assay, Luciferase

Journal: Journal of the Anus, Rectum and Colon

Article Title: Evaluation of the Amounts of Sennosides A and B in Rhubarb-containing Kampo Medicines to Create a Ranking of Kampo Medicines for Appropriate Selection of Laxatives

doi: 10.23922/jarc.2020-102

Figure Lengend Snippet: Comparison of the Estimated and Analysed Strength Scores of Tsumura’s Medicinal Rhubarb-containing Formulas Based on Their Daiokanzoto.

Article Snippet: 1.99 , Tsumura-134 , Keishikashakuyakudaioto.

Techniques: Comparison

Journal: Surgical Endoscopy

Article Title: Evaluation of a novel home-based laparoscopic and core surgical skills programme (Monash Online Surgical Training)

doi: 10.1007/s00464-023-10669-8

Figure Lengend Snippet: The eoSim SurgTrac Core laparoscopic simulation bench trainer (eoSurgical Ltd, Scotland, UK) used with an iPad Tablet (Apple Inc, Cupertino, California, USA)

Article Snippet: Fig. 2 Example of the feedback template for the assessment task and metrics on SurgTrac, version 1.9.9, eoSurgical, Scotland UK

Techniques:

Journal: Surgical Endoscopy

Article Title: Evaluation of a novel home-based laparoscopic and core surgical skills programme (Monash Online Surgical Training)

doi: 10.1007/s00464-023-10669-8

Figure Lengend Snippet: Example of the feedback template for the assessment task and metrics on SurgTrac, version 1.9.9, eoSurgical, Scotland UK

Article Snippet: Fig. 2 Example of the feedback template for the assessment task and metrics on SurgTrac, version 1.9.9, eoSurgical, Scotland UK

Techniques:

Journal: Surgical Endoscopy

Article Title: Evaluation of a novel home-based laparoscopic and core surgical skills programme (Monash Online Surgical Training)

doi: 10.1007/s00464-023-10669-8

Figure Lengend Snippet: Major themes obtained from the thematic analysis of participants written feedback, with illustrative direct participant comment

Article Snippet: Fig. 2 Example of the feedback template for the assessment task and metrics on SurgTrac, version 1.9.9, eoSurgical, Scotland UK

Techniques: