Journal: bioRxiv

Article Title: rDNA breaks activate dsRNA pattern recognition through sense-antisense transcription

doi: 10.64898/2026.05.26.728043

Figure Lengend Snippet: (A) Representative images of γH2AX immunostaining with following I-PpoI- or AsiSI-mediated DNA double-strand breaks. Scale bar, 20 μm. (B) Immunoblots confirming DNA damage and immune activation induced by I-PpoI or AsiSI. (C, D) Immunofluorescence of nucleolar RNA synthesis visualized by EU-click labeling with representative images and quantification. For EU analysis, at least 150 cells were quantified per experiment. Three independent experiments were performed, and one representative was displayed. Bars represent mean ± SEM. Statistical significance was determined by an unpaired two-tailed Student’s t -test. **** p < 0.0001. Scale bar, 20 μm. (E, F) Immunoblots of whole-cell lysates showing STAT1 phosphorylation and ISG56 expression 48 h after I-PpoI-mediated rDNA breaks in the presence of ATM, ATR or DNA-PK inhibition in hTERT RPE-1 p53 KO cells. (G) (Top) Schematic of the human rDNA showing the 47S pre-rRNA transcription unit and the positions of sgRNAs targeting the 5’ETS, 18S, ITS2, and 28S regions, along with the I-PpoI recognition site. (Bottom) Immunoblots of whole-cell lysates following transfection of rDNA-targeting sgRNAs in Cas9-expressing hTERT RPE-1 p53 KO cells. (H) Immunoblots of whole-cell lysates assessing the dependence of the cytosolic RNA sensors MDA5 and RIG-I on I-PpoI-induced immune activation in the presence or absence of ATM inhibition.

Article Snippet: hTERT RPE-1 p53 KO cell line was generated by transfecting pSpCas9(BB)-2A-GFP(PX458) vector targeting p53 into hTERT RPE-1 cell line (ATCC).

Techniques: Immunostaining, Western Blot, Activation Assay, Immunofluorescence, Labeling, Two Tailed Test, Phospho-proteomics, Expressing, Inhibition, Transfection

Journal: bioRxiv

Article Title: rDNA breaks activate dsRNA pattern recognition through sense-antisense transcription

doi: 10.64898/2026.05.26.728043

Figure Lengend Snippet: (A) Schematic diagram of proximity ligation assay (PLA) between biotin-labeled nascent RNA and the cytosolic RNA pattern recognition receptors MDA5 and RIG-I following rDNA breaks. Nascent RNA was labeled with 5’-ethynyl uridine (EU) and biotinylated by click chemistry prior to PLA detection. ( B ) Experimental scheme for detecting cytosolic nascent RNA sensing by MDA5 or RIG-I. (C) Immunoblots of p-STAT1 and ISG56 following I-PpoI induction, camptothecin (CPT), or etoposide treatment. (D) Representative images of PLA following EU labeling and biotin click reaction after DNA damage. (Top) PLA signals indicate proximity between biotin-labeled nascent RNA (EU) and MDA5 or RIG-I. (Bottom) Nucleolar RNA synthesis visualized by EU-click labeling. Scale bar, 20 μm. (E-G) Quantification of cytosolic PLA signals EU-labeled nascent RNA and MDA5 (E), RIG-I (F), and nucleolar EU signal intensity ( G ). ( H ) Immunoblots of p-STAT1 and ISG56 following I-PpoI-mediated rDNA breaks in the presence or absence of RNA polymerase I inhibitor BMH-21. (I) Representative images of cytosolic PLA foci between biotin-labeled nascent RNA (EU) and MDA5 or RIG-I following BMH-21 treatment. Scale bar, 20 μm. (J, K) Quantification of cytosolic PLA signals between biotinylated EU-labeled nascent RNA and MDA5 (J) or RIG-I (K). ( C-K ) Experiments were performed in hTERT RPE-1 p53 KO cells with inducible expression of I-PpoI. ( E, F, J, K ) Each dot represents the mean value of at least 50 cells. ( G ) For EU analysis, at least 150 cells were quantified per experiment. Three independent experiments were performed, and one representative result is displayed. ( E-G, J, K ) Bars represent mean ± SEM. Statistical significance was determined by unpaired two-tailed Student’s t -test. **** p < 0.0001, *** p <0.001, ** p < 0.01.

Article Snippet: hTERT RPE-1 p53 KO cell line was generated by transfecting pSpCas9(BB)-2A-GFP(PX458) vector targeting p53 into hTERT RPE-1 cell line (ATCC).

Techniques: Proximity Ligation Assay, Labeling, Western Blot, Expressing, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Age-dependent induction of ER stress in retinal pigment epithelium impairs phagocytosis via ADAM17-dependent MERTK shedding

doi: 10.1016/j.jbc.2026.111397

Figure Lengend Snippet: Pharmacological induction of ER stress attenuates phagocytic activity in cultured RPE cells. A , schematic diagram of the phagocytosis assay using fluorescein isothiocyanate (FITC)- and pHrodo succinimidyl ester (pHrodo)-conjugated photoreceptor outer segments (POS). B , a representative image of engulfed FITC-POS ( green ) and Hoechst 33,342 ( blue ) with plasma membrane staining 6 h after FITC-POS treatment. The plasma membrane ( gray ) was visualized by PlasMem Bright Red. Scale bar = 10 μm. C , a representative image of pHrodo signal ( yellow ), LAMP1 (magenta) at 24 h after pHrodo-POS treatment. Scale bar = 10 μm. D–F , Tunicamycin (Tm)-induced short-term ER stress reduces phagocytic activity in ARPE-19 and human primary RPE (hRPE) cells. D , experimental timeline for the assays shown in ( E ) and ( F ). E , quantification of fluorescence intensity for FITC-POS and pHrodo-POS in ARPE-19. Data are presented as mean ± SEM (n = 6). Each point represents one independent sample prepared from separate wells. ### p < 0.001 vs. control (Cont) group (Dunnett’s test). F , quantitative data of fluorescence intensity for pHrodo-POS in hRPE cells. Data are presented as mean ± SEM (n = 6). Each point represents one independent sample prepared from separate wells. ## p < 0.01, ### p < 0.001 vs. Cont group (Dunnett’s test). G , quantification of phagocytized pHrodo-POS after co-treatment with thapsigargin (Tg). Data are presented as mean ± SEM (n = 6). Each point represents one independent sample prepared from separate wells. # p < 0.05 vs. Cont group (Student's t test). H , cell death rate following Tm or Tg treatment for 6 h. Data are presented as mean ± SEM (n = 6). Each point represents one independent sample prepared from separate wells. N.S. > 0.05 vs. Cont group (Dunnett’s test). I – K , long-term ER stress reduces phagocytic activity in ARPE -19 and hRPE. I , experimental timelines for assays shown in ( J ) and ( K ). Quantitative data of fluorescence intensity of pHrodo-POS in ARPE-19 ( J ) and hRPE ( K ). Data are presented as mean ± SEM (n = 6). Each point represents one independent sample prepared from separate wells. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Cont group (Dunnett’s test).

Article Snippet: The human-derived RPE cell line, ARPE-19, was purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Activity Assay, Cell Culture, Phagocytosis Assay, Clinical Proteomics, Membrane, Staining, Fluorescence, Control

Journal: The Journal of Biological Chemistry

Article Title: Age-dependent induction of ER stress in retinal pigment epithelium impairs phagocytosis via ADAM17-dependent MERTK shedding

doi: 10.1016/j.jbc.2026.111397

Figure Lengend Snippet: Maturation of ADAM17 mediated by Ca 2+ release from inositol - 1,4,5-trisphosphate receptors contributes to MERTK shedding and dysfunction of POS uptake. A , time-dependent change of the mature form of ADAM17 (matADAM17) in ARPE-19 cells after Tm treatment at 10 μg/ml. Data are presented as mean ± SEM (n = 4). Each point represents one independent sample prepared from separate wells. # p < 0.05 vs. Cont group (Welch's t test). B , expression level of matADAM17 in ARPE-19 after Tm treatment at 1 μg/ml for 54 h. Data are presented as mean ± SEM (n = 6). Each point represents one independent sample prepared from separate wells. ### p < 0.001 vs. Cont group (Welch’s t test). C , localization of ADAM17 after Tm treatment at 10 μg/ml for 3 h. Representative images of ADAM17 ( yellow ), Golgin-97 ( magenta ), and Hoechst 33,342 ( blue ). Scale bar = 10 μm. Quantitative data of fluorescence intensity of ADAM17 colocalized with Golgin-97 after 1 and 3 h after Tm treatment. Data are presented as mean ± SEM (Cont; n = 97 cells, Tm 1 h; n = 103 cells, Tm 3 h; n = 104 cells). ### p < 0.001 vs. Cont group (Dunnett's T3 test). D – F , expression level of matADAM17 after Tm treatment at 10 μg/ml in the presence of decanoyl-Arg-Val-Lys-Arg-chloromethylketone (CMK) (100 μM, D), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) (100 μM, E ), or 2-aminoethoxydiphenyl borate (2-APB) (100 μM, F ). Data are presented as mean ± SEM (n = 6). Each point represents one independent sample prepared from separate wells. ## p < 0.01, ### p < 0.001 vs. Cont group; ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. Tm-only treated group (Games–Howell test). G , schematic diagram of ER stress-induced ADAM17 maturation and MERTK shedding. H and I , effect of ADAM17 small interfering RNA (siRNA) treatment on Tm-induced MERTK downregulation in ARPE-19. H , representative immunoblots of MERTK (extracellular domain), ADAM17, and β-actin. I , quantitative data for MERTK (extracellular domain). Data are presented as mean ± SEM (n = 6). Each point represents one independent sample prepared from separate wells. ## p < 0.01 vs. control siRNA (siCont) single-treated group; ∗ p < 0.05 vs. siCont and Tm co-treated group (Student's t test). J and K , effect of ADAM17 siRNA on Tm-induced dysfunction of POS uptake in ARPE-19. J , schematic protocol of the POS uptake assay and ( K ) quantitative data of fluorescence intensity of FITC-POS internalized in RPE cells. Data are presented as mean ± SEM (n = 8). Each point represents one independent well. # p < 0.05 vs. siCont single-treated group; †† p < 0.01 vs. siCont and chloroquine co-treated group; ∗ p < 0.05 vs. siCont, chloroquine, and Tm co-treated group (Kruskal–Wallis test followed by post hoc Bonferroni test).

Article Snippet: The human-derived RPE cell line, ARPE-19, was purchased from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Fluorescence, Small Interfering RNA, Western Blot, Control