Journal: bioRxiv

Article Title: Functionally distinct ALK and ROS1 fusions detected in infant-type hemispheric gliomas converge on STAT3 and SHP2 activation

doi: 10.1101/2025.05.27.656302

Figure Lengend Snippet: (A) Western blot analysis of CLIP1::ROS1-fusion expression and SHP2/MAPK and STAT3 signaling in CLIP1::ROS1-fusion iNHA; ΔTUB: abrogated microtubule interaction domain; GAPDH: loading control, p-ROS1 (Tyr2274) antibody used to validate fusion transgene activity, phospho-SHP2 (Tyr580), and p-ERK1/2 (Thr202/Tyr204) used to validate MAPK pathway activity and p-STAT3 (Tyr705), and p-STAT1 (Tyr701) for STAT activation. (B) Violin plots highlighting track mean speed from (C) for CCDC88A::ALK (left) and CLIP1::ROS1 (right); dots represent mean of biological replicates, significance calculated on mean values, significance calculated using unpaired two-tailed Student’s t-test, *: p-value ≤0.05. (C) Illustrative images of live cell tracking; inverted nuclear fluorescence, colored lines visualize tracks of individual cells within 12 hours; scalebar: 200µm. (D) Illustrative images of SIA assays; scalebar: 500µm. (E) SIA quantification of invading ALK- and ROS1-fusion iNHAs; left graph: number of invading cells, right graph: mean distance of invasion; one-way ANOVA (normally distributed) or Kruskal Wallis test (not normally distributed), post-hoc Dunn-Bonferroni test, *: p-value≤0.05, **: p-value <0.01,***:p-value<0.001, ****: p-value <0.0001. (F) SIA quantification of invading CCDC88A::ALK (first two graphs) or CLIP1::ROS1 (last two graphs) iNHAs treated with indicated STAT3i (Stattic) concentrations; first and third graph: number of invading cells, second and fourth graph: mean distance of invasion; one-way ANOVA (normally distributed) or Kruskal Wallis test (not normally distributed), post-hoc Dunn-Bonferroni test, *: p-value≤0.05, **: p-value <0.01, ****: p-value <0.0001.

Article Snippet: Antibodies used in this study: mouse-α-ALK (CST #3791), rabbit-α-p-ALK(Y1507) (CST #14678), mouse-α-ROS1 (CST #3266), rabbit-α-p-ROS1(Y2274) (CST #3078), mouse-α-SHP2 (Abcam #ab76285), rabbit-α-p-SHP2(Y580) (CST #5431), mouse-α-MEK1/2 (CST #4694), rabbit-α-p-MEK1/2 (S217/221) (CST #9154), mouse-α-ERK1/2 (CST #4696), rabbit-α-p-ERK1/2(T202,Y204), rabbit-α-GAB1 (CST #3232), rabbit-α-p-GAB1(Y627) (CST #3233), mouse-α-STAT3 (CST #9139), rabbit-α-p-STAT3(Y705) (CST #9145), rabbit-α-p-STAT3(Y705) ( in vitro kinase only; Abcam #ab267373), mouse-α-STAT1 (CST #9176), rabbit-α-p-STAT1(Y701) (CST #9167), rabbit-α-SHC1 (CST #2432), rabbit-α-p-SHC1(Y239/240) (CST #2434), rabbit-α-SHC3 (Proteintech #12436-1-AP), mouse-α-GAPDH-HRP (Proteintech #HRP-60004), rabbit-α-β-TUBULIN-HRP (CST #5346), anti-rabbit IgG-HRP (CST #7074), and anti-mouse IgG-HRP (CST #7076).

Techniques: Western Blot, Expressing, Control, Activity Assay, Activation Assay, Two Tailed Test, Cell Tracking Assay, Fluorescence

Journal: bioRxiv

Article Title: Functionally distinct ALK and ROS1 fusions detected in infant-type hemispheric gliomas converge on STAT3 and SHP2 activation

doi: 10.1101/2025.05.27.656302

Figure Lengend Snippet: (A) Affinity purification MS/MS identifying direct interactors of ALK- and ROS1-fusions used in this study; size: −log 10 BFDR, color gradient: log 2 EFC high (red) to low (grey). (B) Immunoprecipitation validating SHC1/3 as direct interactors of ALK-fusion (top two blot) and SHP2 as direct interactor of ROS1-fusion (bottom three blots),respectively; GAPDH: loading control, p-ALK (Tyr1507), -ROS1 (Tyr2274) antibody used to validate KD mutants, p-SHC1 (Tyr239/240), and p-SHP2 (Tyr580) antibodies used to validate activity of interactors; dashed lines: marker lane. (C) Western blot analysis of MAPK signaling in CCDC88::ALK and CLIP1::ROS1 models. GAPDH: loading control, p-ALK (Tyr1507), -ROS1 (Tyr2274) antibody used to validate KD mutants, p-SHP2 (Tyr580), p-GAB1 (Tyr642), p-MEK1/2 (Ser217/221), and p-ERK1/2 (Thr202/Tyr204) used to validate MAPK pathway activity, p-STAT3 (Tyr705), and p-STAT1 (Tyr701) used to validate STAT activation. (D) Western blot analysis of RNAi effect in PPP1CB::ALK models. GAPDH: loading control, p-ALK (Tyr1507) antibody used to validate retained ALK activity, p-SHP2 (Tyr580) and p-GAB1 (Tyr642), used to validate shPTPN11 , p-STAT3 (Tyr705), used to validate shSTAT3 . (E) Kaplan-Meier survival curves showing tumor induced mortality upon orthotopic intracranial injection of shRNA inhibited PPP1CB::ALK cells in NSG mice, groups are represented by individual curves, with a n=8 mice per group,; grey: PPP1CB::ALK shCtrl , dark petrol: PPP1CB::ALK shSTAT3 , light petrol: PPP1CB::ALK shPTPN11 ; statistical significance determined by log-rank test, **: p-value<0.01, *:p-value<0.05.

Article Snippet: Antibodies used in this study: mouse-α-ALK (CST #3791), rabbit-α-p-ALK(Y1507) (CST #14678), mouse-α-ROS1 (CST #3266), rabbit-α-p-ROS1(Y2274) (CST #3078), mouse-α-SHP2 (Abcam #ab76285), rabbit-α-p-SHP2(Y580) (CST #5431), mouse-α-MEK1/2 (CST #4694), rabbit-α-p-MEK1/2 (S217/221) (CST #9154), mouse-α-ERK1/2 (CST #4696), rabbit-α-p-ERK1/2(T202,Y204), rabbit-α-GAB1 (CST #3232), rabbit-α-p-GAB1(Y627) (CST #3233), mouse-α-STAT3 (CST #9139), rabbit-α-p-STAT3(Y705) (CST #9145), rabbit-α-p-STAT3(Y705) ( in vitro kinase only; Abcam #ab267373), mouse-α-STAT1 (CST #9176), rabbit-α-p-STAT1(Y701) (CST #9167), rabbit-α-SHC1 (CST #2432), rabbit-α-p-SHC1(Y239/240) (CST #2434), rabbit-α-SHC3 (Proteintech #12436-1-AP), mouse-α-GAPDH-HRP (Proteintech #HRP-60004), rabbit-α-β-TUBULIN-HRP (CST #5346), anti-rabbit IgG-HRP (CST #7074), and anti-mouse IgG-HRP (CST #7076).

Techniques: Affinity Purification, Tandem Mass Spectroscopy, Immunoprecipitation, Control, Activity Assay, Marker, Western Blot, Activation Assay, Injection, shRNA

Journal: bioRxiv

Article Title: Functionally distinct ALK and ROS1 fusions detected in infant-type hemispheric gliomas converge on STAT3 and SHP2 activation

doi: 10.1101/2025.05.27.656302

Figure Lengend Snippet: (A) In vitro kinase assay validating SHP2 and STAT3 as substrates of ALK- and ROS1-fusions; GAPDH: loading control, phospho-ALK,-ROS1 antibody used to validate KD mutants, phospho-SHP2 (Tyr580) or phospho-STAT3 (Tyr705) validate ALK- and ROS1-fusion kinase specificity towards SHP2 or STAT3, respectively. (B) Western blot analysis of MAPK signaling in ALK- and ROS1-fusion models. GAPDH: loading control, phospho-ALK (Tyr1507), -ROS1 (Tyr2274) antibody used to validate KD mutants, phospho-SHP2 (Tyr580), phospho-GAB1 (Tyr642), phospho-MEK1/2 (Ser217/221), and phospho-ERK1/2 (Thr202/Tyr204) used to validate MAPK pathway activity, phospho-STAT3 (Tyr705), and phospho-STAT1 (Tyr701) used to validate STAT activation. (C) Subcellular fractionation of CLIP1::ROS1 samples validating increased STAT3 activity; phospho-ROS1 (Tyr2274) antibody used to validate KD mutant and phospho-STAT3 (Tyr705) used to validate pathway activity, β-TUB: cytoplasmic marker, H3: nuclear marker. (D) Western blots analyzing the effect of RTK inhibition (Entrectinib 500nM, 4hours) on MAPK and STAT signaling in ALK- and ROS1-fusion models. GAPDH: loading control, phospho-ALK (Tyr1507), -ROS1 (Tyr2274) antibody used to validate inhibition, phospho-SHP2 (Tyr580), phospho-GAB1 (Tyr642), phospho-MEK1/2 (Ser217/221), and phospho-ERK1/2 (Thr202/Tyr204) used to validate MAPK pathway inhibition and phospho-STAT3 (Tyr705), and phospho-STAT1 (Tyr701) for STAT inhibition.

Article Snippet: Antibodies used in this study: mouse-α-ALK (CST #3791), rabbit-α-p-ALK(Y1507) (CST #14678), mouse-α-ROS1 (CST #3266), rabbit-α-p-ROS1(Y2274) (CST #3078), mouse-α-SHP2 (Abcam #ab76285), rabbit-α-p-SHP2(Y580) (CST #5431), mouse-α-MEK1/2 (CST #4694), rabbit-α-p-MEK1/2 (S217/221) (CST #9154), mouse-α-ERK1/2 (CST #4696), rabbit-α-p-ERK1/2(T202,Y204), rabbit-α-GAB1 (CST #3232), rabbit-α-p-GAB1(Y627) (CST #3233), mouse-α-STAT3 (CST #9139), rabbit-α-p-STAT3(Y705) (CST #9145), rabbit-α-p-STAT3(Y705) ( in vitro kinase only; Abcam #ab267373), mouse-α-STAT1 (CST #9176), rabbit-α-p-STAT1(Y701) (CST #9167), rabbit-α-SHC1 (CST #2432), rabbit-α-p-SHC1(Y239/240) (CST #2434), rabbit-α-SHC3 (Proteintech #12436-1-AP), mouse-α-GAPDH-HRP (Proteintech #HRP-60004), rabbit-α-β-TUBULIN-HRP (CST #5346), anti-rabbit IgG-HRP (CST #7074), and anti-mouse IgG-HRP (CST #7076).

Techniques: In Vitro, Kinase Assay, Control, Western Blot, Activity Assay, Activation Assay, Fractionation, Mutagenesis, Marker, Inhibition

Journal: International journal of molecular sciences

Article Title: IRF5 Mediates Artery Inflammation in Salt-Sensitive Hypertension by Regulating STAT1 and STAT2 Phosphorylation to Increase ESM1 Transcription: Insights from Bioinformatics and Mechanistic Analysis.

doi: 10.3390/ijms26083722

Figure Lengend Snippet: Figure 5. IRF5 and downstream target expression levels between NS and DOCA groups. (A) Western blot analysis showing the expression levels of IRF5 and total STAT1/2 proteins in aortic

Article Snippet: For antigen retrieval, the sections were placed in citrate solution and heated at 37 ◦C for 10 min. After retrieval, the slides were rinsed three times with PBS (pH 7.4) for 5 min each in the shaker and then incubated overnight at 4 ◦C with primary antibodies: rabbit monoclonal IRF5 (#ab181553, 1:200, Abcam, Cambridge, UK), mouse monoclonal STAT1 (#sc-464, 1:100, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and mouse monoclonal STAT2 (#sc-166201, 1:100, Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques: Expressing, Western Blot

Journal: International journal of molecular sciences

Article Title: IRF5 Mediates Artery Inflammation in Salt-Sensitive Hypertension by Regulating STAT1 and STAT2 Phosphorylation to Increase ESM1 Transcription: Insights from Bioinformatics and Mechanistic Analysis.

doi: 10.3390/ijms26083722

Figure Lengend Snippet: Figure 7. ESM1 promoter reporters are transactivated by STAT1::STAT2. (A) A diagram shows the relative positions of full-length (FL) and fragments of ESM1 promoter reporters. (B) Responses of the FL reporter, and (C) the individual fragments of ESM1 promoter to STAT1 and STAT2 or the HDD mutants were investigated. (D) Reporter assays of the P3 fragment of the ESM1 promoter containing two mutated Binding elements (BEs) as indicated. (E) A schematic illustrates the relative positions of qPCR probes to putative BEs for ChIP-qPCR experiments. (F) Antibody-pulled-down chromatins were analyzed by qPCR. Rb, rabbit. TSS, transcription start site. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant.

Article Snippet: For antigen retrieval, the sections were placed in citrate solution and heated at 37 ◦C for 10 min. After retrieval, the slides were rinsed three times with PBS (pH 7.4) for 5 min each in the shaker and then incubated overnight at 4 ◦C with primary antibodies: rabbit monoclonal IRF5 (#ab181553, 1:200, Abcam, Cambridge, UK), mouse monoclonal STAT1 (#sc-464, 1:100, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and mouse monoclonal STAT2 (#sc-166201, 1:100, Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques: Binding Assay, ChIP-qPCR

Journal: International journal of molecular sciences

Article Title: IRF5 Mediates Artery Inflammation in Salt-Sensitive Hypertension by Regulating STAT1 and STAT2 Phosphorylation to Increase ESM1 Transcription: Insights from Bioinformatics and Mechanistic Analysis.

doi: 10.3390/ijms26083722

Figure Lengend Snippet: Figure 8. Central illustration. High salt stimulation upregulates IRF5 expression, which enhances the phosphorylation and dimerization of STAT1 and STAT2. The activated STAT1/STAT2 complex translocates into the nucleus, binds to the ESM1 promoter region, and promotes ESM1 transcription. Elevated ESM1 expression contributes to vascular remodeling and the development of salt-sensitive hypertension, as illustrated by the transition from a physiological to a pathological vascular pheno- type. P, phosphorylation.

Article Snippet: For antigen retrieval, the sections were placed in citrate solution and heated at 37 ◦C for 10 min. After retrieval, the slides were rinsed three times with PBS (pH 7.4) for 5 min each in the shaker and then incubated overnight at 4 ◦C with primary antibodies: rabbit monoclonal IRF5 (#ab181553, 1:200, Abcam, Cambridge, UK), mouse monoclonal STAT1 (#sc-464, 1:100, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and mouse monoclonal STAT2 (#sc-166201, 1:100, Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques: Expressing, Phospho-proteomics

Journal: Science Advances

Article Title: Targeting HMGB2 acts as dual immunomodulator by bolstering CD8 + T cell function and inhibiting tumor growth in hepatocellular carcinoma

doi: 10.1126/sciadv.ads8597

Figure Lengend Snippet: ( A ) Representative images of harvested Hepa1-6 subcutaneous HCC tumors. Scale bar, 1 cm ( B ) Tumor growth curves of Hepa1-6 subcutaneous tumors in BALB/C nude mice and C57BL/6J mice. ( C ) Tumor weights of subcutaneous Hepa1-6 tumors in BALB/C nude mice and C57BL/6J mice ( n = 6). ( D ) Difference of tumor volumes between shCtrl and shHmgb2 subcutaneous tumors in BALB/C nude mice and C57BL/6J mice ( n = 6). ( E ) Differential GO pathways in Hepa1-6 shHmgb2 cells. JAK, Janus kinase. ( F ) GSEA analysis shows top pathway enriched in Hepa1-6 shHmgb2 cells. ( G ) Western blotting experiment shows STAT1 pathway changes in Hepa1-6 cells and Huh7 cells. Cells were treated with IFN-γ (10 ng/ml) or fludarabine (10 μM) for 24 hours. ( H ) Annexin V apoptosis analysis for Hepa1-6 cells treated with vehicle and IFN-γ (10 or 20 ng/ml). PI, propidium iodide. ( I ) Quantification for proportions of apoptotic cells after treatment of IFN-γ. ( J ) T cell killing assay with Hepa1-6 shCtrl and shHmgb2 cells and wild-type (WT) CD8 + T cells ( n = 5). RLU, relative light unit. ( K ) Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining and quantification of Hepa1-6 subcutaneous tumors ( n = 4). Scale bar, 25 μm. HPF, high power field. ( L ) Representative immunohistochemistry images of CD8, IFN-γ, and CXCL10 staining in Hepa1-6 subcutaneous tumors. Scale bars, 20 μm. ( M ) Quantification of CD8, IFN-γ, and CXCL10 staining in Hepa1-6 subcutaneous tumors ( n = 4). Data are presented as the means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001. Two-way ANOVA test for (B) Student’s t test for (C), (D), (I), (J), (K), and (M).

Article Snippet: Anti-human/mouse STAT1 , TP53449 , Abmart, China.

Techniques: Western Blot, End Labeling, TUNEL Assay, Staining, Immunohistochemistry

Journal: Science Advances

Article Title: Targeting HMGB2 acts as dual immunomodulator by bolstering CD8 + T cell function and inhibiting tumor growth in hepatocellular carcinoma

doi: 10.1126/sciadv.ads8597

Figure Lengend Snippet: ( A ) ChIP assay shows the interaction of HMGB2 with Stat1 chromatin ( n = 3). ( B ) Colocalization of HMGB2, STAT1 and TRIM 24 in HCC subcutaneous tumor tissue. Scale bar, 10 μm. ( C ) CoIP assay shows the interaction of HMGB2 and TRIM24. ( D ) ChIP-PCR shows that TRIM24 modulates the transcriptional level of Stat1 ( n = 3). ( E ) Luciferase reporter assay shows that the Trim24 / Hmgb2 signal modulates the transcriptional level of Stat1 ( n = 3). OE, overexpression. ( F ) Representative images of the orthotopic HCC models. ( G ) Tumor weights of different groups from the orthotopic HCC models ( n = 6). ( H ) Overall survival of different treatment groups from the orthotopic HCC models. ( I ) Flow cytometry of intratumoral CD3 + CD8 + T cells as in (G). ( J ) Flow cytometry of intratumoral effector markers as in (G). ( K ) Quantification of intratumoral IFN-γ + CD8 + T cells and GranB + CD8 + T cells in different treatment groups ( n = 5). Data are presented as the means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Student’s t test for (A) and (D). One-way ANOVA test for (E), (G), and (K).

Article Snippet: Anti-human/mouse STAT1 , TP53449 , Abmart, China.

Techniques: Co-Immunoprecipitation Assay, Luciferase, Reporter Assay, Over Expression, Flow Cytometry

Journal: Science Advances

Article Title: Targeting HMGB2 acts as dual immunomodulator by bolstering CD8 + T cell function and inhibiting tumor growth in hepatocellular carcinoma

doi: 10.1126/sciadv.ads8597

Figure Lengend Snippet: Markers and article numbers of antibodies. PE, phycoerythrin; FITC, fluorescein isothiocyanate; APC, antigen-presenting cell; HRP, horseradish peroxidase; mAb, monoclonal antibody.

Article Snippet: Anti-human/mouse STAT1 , TP53449 , Abmart, China.

Techniques: Ubiquitin Proteomics, Purification, In Vivo

Journal: Science Advances

Article Title: Targeting HMGB2 acts as dual immunomodulator by bolstering CD8 + T cell function and inhibiting tumor growth in hepatocellular carcinoma

doi: 10.1126/sciadv.ads8597

Figure Lengend Snippet: Names and sequence of primers. F, forward; R, reverse.

Article Snippet: Anti-human/mouse STAT1 , TP53449 , Abmart, China.

Techniques: Sequencing