Journal: Scientific Reports

Article Title: Meiotic progression in multinuclear mouse spermatocytes without the spindle pole clustering motor protein KIFC1 or cytokinesis forms single-cell late-stage spermatids

doi: 10.1038/s41598-025-20463-2

Figure Lengend Snippet: The microtubule motor clustering protein KIFC1 is not detected in polynuclear metaphase-I primary spermatocytes. Panel 1. Human MCF7 diploid ( A-D ) and polyploid ( E-H ) cancer cells showing robust spindle microtubule detection with KIFC1 (A, E: green, arrows, spindle poles; inset, microtubules [red] and DNA [blue]). B, F: blue, DNA; C and D: overlays. Volume rendering of microtubules (red), KIFC1 (green), and DNA (blue). Right: Relative fluorescent intensity line tracings and surface intensity plot (upper right) of the MCF7 control (upper graphs) or polyploid (lower graphs) spindles showing microtubules (red) and KIFC1 (green) spindle poles overlap (arrows, sp1, sp2). Scale bars = 5 μm. Panel II. GFP-CETN2-expressing polyploid meiosis-I spermatocytes do not detect KIFC1. ( A , C , E ) Centrioles (green, arrows), spindle microtubules (red) and DNA (blue) in di-nuclear (A, C) and tri-nuclear (E)-derived metaphase-I spindles. C, E: arrowheads, apposed spindle poles. B , D , F : Centrioles (green, arrows), spindle microtubules (red) but not KIFC1 (blue) detection of spindle microtubules (D, F: arrowheads, apposed poles). Right: fluorescent line intensity traces through the polyploid spindles show no KIFC1 detection (blue line) with spindle pole microtubules (red line; spindle poles, sp1 and sp2, arrows). Upper right: surface intensity plot confirming no KIFC1 detection in the di-nuclear meiotic spermatocyte four spindle poles ( A-B; sp1→sp4). Scale bars = 5 μm. Panel III. The microtubule crosslinking motor protein KIF11 (Eg5; B, E, H: red, arrows) and the microtubule spindle pole binding protein NuMA (C, F, I: blue, arrows) are present in CB6F1 or C57BL mouse polyploid meiotic spindle pole microtubules. A, D, G: microtubules (green) and DNA (blue). Scale bars = 5 μm. Panel IV. Single cell RNA-seq analysis of KIFC1 and TACC3 mRNA expression in male adult Stay-Put enriched spermatocytes from Hermann et al. . A: 10x Genomic tSNE plot profiling of selected adult mouse spermatogenic cells (key: color code). B and C: tSNE plots of KIFC1 (B) and TACC3 (C) expression on cell cluster showing minimal mRNA KIFC1 detection. Right: log2 expression color bar. D: Violin plots of KIFCI (upper) and TACC3 (lower) mRNA expression depicting limited KIFC1 distribution and density in various spermatocyte populations, compared to TACC3 mRNA.

Article Snippet: Here, a rabbit polyclonal KIFC1 antibody validated in Western blots of mammalian testis tissues (mice, human) and cell lines (Proteintech group; https://www.ptglab.com ) was applied to methanol fixed human MCF7 cancer cells strongly labeling spindle microtubules, including in multipolar spindles (Fig. , panel 1, left).

Techniques: Control, Expressing, Derivative Assay, Binding Assay, RNA Sequencing

Journal: Signal Transduction and Targeted Therapy

Article Title: DNA/RNA-binding protein KIN17 supports esophageal cancer progression via resolving noncanonical STING activation induced by R-loop

doi: 10.1038/s41392-025-02344-2

Figure Lengend Snippet: High expression of KIN in esophageal squamous cell carcinoma. a Log2FC of differentially expressed genes between DDR-high group and DDR-low group (DDR-high group versus DDR-low group) using data from HRA003107 dataset. b Reactome pathway analysis of differentially expressed genes between DDR-high group and DDR-low group using data from HRA003107 dataset. c ESTIMATE score in DDR dependent subgroup using data from HRA003107 dataset. Red represents DDR-high group and blue represents DDR-low group. d Heatmap shows distribution of subtypes of immune cells using data from HRA003107 dataset. e , f Differentially expressed DDR proteins between tumor and normal tissues ( e ). Top 10 proteins were showed ( f ). g KIN expression in TCGA dataset. h – j GSEA comparing the gene expression profiles between KIN-high group and KIN-low group using data from HRA003107 dataset. (KIN-low group versus KIN-high group). Statistical significance is indicated as **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05

Article Snippet: The sources of antibodies against the following proteins or post-translational modifications were used: KIN (12313-1-AP), DHX9 (17721-1-AP), RAD51 (14961-1-AP), HA-tag (81290-1-RR), and Flag-tag (80010-1-RR) from Proteintech. p-γH2AX (#97148), p-STING (#50907), STING (#13647), p-NFκB (#3033), NFκB (#8242), p-IRF3 (#29047), and IRF3 (#11904) from CST, S9.6 (ENH001) from Kerafast.

Techniques: Expressing, Gene Expression

Journal: Signal Transduction and Targeted Therapy

Article Title: DNA/RNA-binding protein KIN17 supports esophageal cancer progression via resolving noncanonical STING activation induced by R-loop

doi: 10.1038/s41392-025-02344-2

Figure Lengend Snippet: Knockdown of KIN inhibits esophageal squamous cell carcinoma progress. a KIN protein expression in KYSE30 cells transfected with shNC or shKIN virus. b Growth curves of KYSE30 cell line transfected with shNC or shKIN virus. c Representative images (left) and quantification (right) of the colony-forming ability of KYSE30 cell line transfected with shNC or shKIN virus ( n = 3). d Representative images (left) and statistic (right) of the colony-forming ability of mEC25 cell line transfected with shNC or shKIN virus ( n = 3). e , f Representative charts ( e ) and quantification ( f ) of flow cytometry results for propidium iodide and annexin V staining in the indicated cell lines after treatment of 5 μg/ml DDP for 12 h. Error bars represent SD obtained from three independent experiments ( n = 3). g Immunoblotting analysis of apoptosis proteins in KIN overexpressed KYSE30 after treatment of 5 μg/ml DDP for 12 h. h Tumor growth curves and representative image of shNC and shKIN KYSE30 tumors treated with DDP or PBS ( n = 6). i Tumor growth curves and representative image of shNC and shKIN mEC25 tumors treated with DDP or PBS ( n = 6). Statistical significance is indicated as **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05

Article Snippet: The sources of antibodies against the following proteins or post-translational modifications were used: KIN (12313-1-AP), DHX9 (17721-1-AP), RAD51 (14961-1-AP), HA-tag (81290-1-RR), and Flag-tag (80010-1-RR) from Proteintech. p-γH2AX (#97148), p-STING (#50907), STING (#13647), p-NFκB (#3033), NFκB (#8242), p-IRF3 (#29047), and IRF3 (#11904) from CST, S9.6 (ENH001) from Kerafast.

Techniques: Knockdown, Expressing, Transfection, Virus, Flow Cytometry, Staining, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: DNA/RNA-binding protein KIN17 supports esophageal cancer progression via resolving noncanonical STING activation induced by R-loop

doi: 10.1038/s41392-025-02344-2

Figure Lengend Snippet: KIN forms a complex with DHX9. a , b Coomassie blue staining of proteins co-precipitated by anti-Flag beads from lysates of Flag-KIN or empty vector expressed KYSE30 ( a ) and KYSE510 ( b ). c , d Differential protein bands of the immune-precipitates from KYSE30 or KYSE510 cells extracts were analyzed by mass spectrometry ( c ). Intersected proteins were showed ( d ). e Protein expression in Flag co-precipitate effluent from Flag-KIN expressed HEK293T. f Protein expression in HA-DHX9 co-precipitate effluent from HA-DHX9 expressed HEK293T. g Proteins precipitated by S9.6 antibody. h Proteins precipitated by S9.6 antibody after addition of RNaseH. i , j S9.6 IP analysis of the association of DHX9 and KIN mutants with the R-loop in HEK293T cells. k , l Flag-IP analysis of the association of DHX9 with the indicated FLAG-tagged KIN mutants in HEK293T cells

Article Snippet: The sources of antibodies against the following proteins or post-translational modifications were used: KIN (12313-1-AP), DHX9 (17721-1-AP), RAD51 (14961-1-AP), HA-tag (81290-1-RR), and Flag-tag (80010-1-RR) from Proteintech. p-γH2AX (#97148), p-STING (#50907), STING (#13647), p-NFκB (#3033), NFκB (#8242), p-IRF3 (#29047), and IRF3 (#11904) from CST, S9.6 (ENH001) from Kerafast.

Techniques: Staining, Plasmid Preparation, Mass Spectrometry, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: DNA/RNA-binding protein KIN17 supports esophageal cancer progression via resolving noncanonical STING activation induced by R-loop

doi: 10.1038/s41392-025-02344-2

Figure Lengend Snippet: KIN recruits DHX9 to R-loop site. a Protein expression in shNC and shKIN KYSE30 cells treated with PBS or 5 μg/ml DDP for 2 h. b , c Fluorescence image ( b ) and quantification ( c ) of DHX9 in shNC and shKIN KYSE30 cells treated with 5 μg/ml DDP for 2 h ( n > 50). Scale bars, 10 μm. d Proteins precipitated by S9.6 antibody in shNC and shKIN KYSE30. e , f Fluorescence image ( e ) and quantification ( f ) of R-loops in shNC and shKIN KYSE30 cells ( n > 50). Scale bars, 10 μm. g Protein expression in shNC and shKIN KYSE30 cells treated with PBS or 5 μg/ml DDP for 2 h. h , i Fluorescence image ( h ) and quantification ( i ) of RAD51 in shNC and shKIN KYSE 30 cells treated with 5 μg/ml DDP for 2 h ( n > 50). Scale bars, 10 μm. Statistical significance is indicated as **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05

Article Snippet: The sources of antibodies against the following proteins or post-translational modifications were used: KIN (12313-1-AP), DHX9 (17721-1-AP), RAD51 (14961-1-AP), HA-tag (81290-1-RR), and Flag-tag (80010-1-RR) from Proteintech. p-γH2AX (#97148), p-STING (#50907), STING (#13647), p-NFκB (#3033), NFκB (#8242), p-IRF3 (#29047), and IRF3 (#11904) from CST, S9.6 (ENH001) from Kerafast.

Techniques: Expressing, Fluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: DNA/RNA-binding protein KIN17 supports esophageal cancer progression via resolving noncanonical STING activation induced by R-loop

doi: 10.1038/s41392-025-02344-2

Figure Lengend Snippet: Defect of KIN induces interferon response via NFκB. a , b GSEA comparing the gene expression profiles between KIN high and low group, data from TCGA ( a ) and data from HRA003107 ( b ). c Differentially expressed cytokine/chemokine in shNC and shKIN KYSE30 cells ( n = 3). d Differentially expressed cytokine/chemokine in KYSE30 cells expressed KIN or empty vector ( n = 3). e IFN-β protein expression in shNC and shKIN KYSE30 cells. f STING activation via IRF3 detected by WB in shNC and shKIN KYSE30 cells. g STING activation via NFκB detected by WB in shNC and shKIN KYSE30 cells. Grey values of p-NFκB were showed under the band of p-NFκB. h STING activation via NFκB detected by WB in KYSE30 cells expressed KIN or empty vector treated with 5 μg/ml DDP for 2 h. i Tumor growth curves and representative image of shNC and shKIN mEC25 tumors treated with anti-PD-1 antibody or isotype ( n = 6). j Tumor growth curves and representative image of mEC25 tumors expressing KIN or empty vector treated with anti-PD-1 antibody or isotype ( n = 6). k Percentages of infiltrating CD8 + GZMB + T cells, CD8 + IFN-γ + T cells and CD8 + TNF-α + T cells in shNC and shKIN mEC25 tumors ( n = 6) were analyzed by flow cytometry. l Percentages of infiltrating CD8 + GZMB + T cells, CD8 + IFN-γ + T cells and CD8 + TNF-α + T cells in mEC25 tumors expressing KIN or empty vector ( n = 6) were analyzed by flow cytometry. Statistical significance is indicated as **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05

Article Snippet: The sources of antibodies against the following proteins or post-translational modifications were used: KIN (12313-1-AP), DHX9 (17721-1-AP), RAD51 (14961-1-AP), HA-tag (81290-1-RR), and Flag-tag (80010-1-RR) from Proteintech. p-γH2AX (#97148), p-STING (#50907), STING (#13647), p-NFκB (#3033), NFκB (#8242), p-IRF3 (#29047), and IRF3 (#11904) from CST, S9.6 (ENH001) from Kerafast.

Techniques: Gene Expression, Plasmid Preparation, Expressing, Activation Assay, Flow Cytometry