Journal: Cell Reports Medicine

Article Title: HIF-activated priming of TRAIL-induced cell death determines epigenetic vulnerability in kidney cancer

doi: 10.1016/j.xcrm.2026.102630

Figure Lengend Snippet: In vitro chemical screens identify SGI1027 and MS1129 specifically killing VHL -deficient ccRCC cells in an HIF-dependent manner (A) Scheme of cell viability screens with 2,645 FDA-approved drugs and bioactive compounds in isogenic ccRCC cells. (B) The cell viability ratio of all chemicals (5 μM) was ranked from the primary screen ( n = 1 biological replicate). (C) The IC50 ratio of 23 positive hits in the secondary screen ( n = 1 biological replicate). (D) Representative images of death of parental and HIF-1α/2α-DKO RCC10 cells treated with vehicle or SGI1027 for 3 days. BF, bright field. (E) Quantification of PI-positive cells in (D) ( n = 3 biological replicates). (F) Immunoblot analysis of HIF-1α and HIF-2α proteins in isogenic RCC10 cells ( n = 3 biological replicates). (G) The IC50 of SGI1027 in isogenic RCC10 and HIF-1α- and/or HIF-2α-ΚΟ cells ( n = 3 biological replicates). (H) Clonogenic growth of isogenic RCC10 and HIF-1α- and/or HIF-2α-ΚΟ cells treated with vehicle or SGI1027 for 9 days. (I) Quantification of crystal violet intensity in (H) ( n = 3 biological replicates). (J) The cell viability ratio of SGI1027 analogs (2 μM) in parental and HIF-1α/2α-DKO RCC10 cells ( n = 1 biological replicate). (K) Representative images of death of parental and HIF-1α/2α-DKO RCC10 cells treated with vehicle or MS1129 for 3 days. (L) Quantification of PI-positive cells in (K) ( n = 3 biological replicates). (M) The IC50s of SGI1027, MS1129, and MS1143 in isogenic RCC10 and RCC10-HIF-1α/2α-DKO cells ( n = 3 biological replicates). dDNMT, DNMT protein degrader. Data represent mean ± SEM. p value was determined by two-way ANOVA with Tukey’s test (E, I, and L). Scale bars, 100 μm in (D) and (K). See also and ; and .

Article Snippet: The supernatant was diluted in ChIP immunoprecipitation buffer (50 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail) and then subjected to immunoprecipitation overnight in the presence of Dynabeads (ThermoFisher) with anti-HIF-1α antibody (3 μg, home-made), anti-HIF-2α antibody (3 μg, home-made), anti-DNMT1 antibody (3 μg, 24206-1-AP, Proteintech), or anti-DNMT3A antibody (3 μg, 20954-1-AP, Proteintech), anti-DNMT3B antibody (3 μg, 26971-1-AP, Proteintech), or normal rabbit IgG (3 μg, 2729S, Cell Signaling Technology) at 4°C.

Techniques: In Vitro, Western Blot

Journal: Cell Reports Medicine

Article Title: HIF-activated priming of TRAIL-induced cell death determines epigenetic vulnerability in kidney cancer

doi: 10.1016/j.xcrm.2026.102630

Figure Lengend Snippet: SGI1027 and MS1129 are DNMT protein degraders independent of VHL and HIF (A) Immunoblot analysis of DNMT1/3A/3B/3L proteins in ccRCC cell lines ( n = 2 biological replicates). (B) mRNA analysis of DNMT1 , DNMT3A , DNMT3B , and DNMT3L in RCC10 cells treated with vehicle or SGI1027 for 2 days ( n = 2 biological replicates). (C–F) Immunoblot analyses of DNMT1/3A/3B proteins in RCC10 (C, n = 3 biological replicates), 786-O (D, n = 3 biological replicates), RCC4 (E, n = 2 biological replicates), and UM-RC-2 (F, n = 2 biological replicates) cells treated with vehicle or SGI1027 for 2 days. (G) Immunoblot analysis of DNMT1/3A/3B proteins in RCC10 cells treated with vehicle or SGI1027 for indicated time ( n = 2 biological replicates). (H and I) Immunoblot analysis of DNMT1/3A/3B proteins in RCC10 cells treated with vehicle or SGI1027 for 36 h, followed by co-treatment with vehicle, MG132 (6 h, n = 2 biological replicates, H), or MG262 (2 h, n = 2 biological replicates, I). (J and K) Immunoblot analysis of DNMT1/3A/3B proteins in isogenic RCC10 (J)/786-O (K) cells treated with vehicle or SGI1027 for 2 days ( n = 2 biological replicates). (L) Immunoblot analysis of DNMT1/3A/3B proteins in RCC10 cells treated with vehicle or MS1129 for indicated time ( n = 2 biological replicates). (M and N) Immunoblot analysis of DNMT1/3A/3B proteins in RCC10 cells treated with vehicle or MS1129 for 36 h, followed by co-treatment with vehicle, MG132 (6 h, n = 2 biological replicates, M), or MG262 (2 h, n = 2 biological replicates, N). (O) Immunoblot analysis of DNMT1/3A/3B proteins in RCC10 cells treated with vehicle, MS1129, or MS1143 for 2 days ( n = 2 biological replicates). (P) Immunoblot analysis of DNMT1/3A/3B proteins in RCC10 cells treated with CHX and/or SGI1027 for indicated time ( n = 2 biological replicates). (Q) Immunoblot analysis of DNMT1/3A/3B proteins in parental and DNMT1/3A/3B-deficient cells ( n = 2 biological replicates). (R) Representative images of death of parental and DNMT1/3A/3B-deficient cells. Scale bars, 100 μm. (S) Quantification of PI-positive cells in (R) ( n = 3 biological replicates, mean ± SEM). p value was determined by unpaired 2-tailed Student’s t test. See also .

Article Snippet: The supernatant was diluted in ChIP immunoprecipitation buffer (50 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail) and then subjected to immunoprecipitation overnight in the presence of Dynabeads (ThermoFisher) with anti-HIF-1α antibody (3 μg, home-made), anti-HIF-2α antibody (3 μg, home-made), anti-DNMT1 antibody (3 μg, 24206-1-AP, Proteintech), or anti-DNMT3A antibody (3 μg, 20954-1-AP, Proteintech), anti-DNMT3B antibody (3 μg, 26971-1-AP, Proteintech), or normal rabbit IgG (3 μg, 2729S, Cell Signaling Technology) at 4°C.

Techniques: Western Blot

Journal: Cell Reports Medicine

Article Title: HIF-activated priming of TRAIL-induced cell death determines epigenetic vulnerability in kidney cancer

doi: 10.1016/j.xcrm.2026.102630

Figure Lengend Snippet: HIF-induced procaspase-10 predisposes VHL -deficient ccRCC to apoptosis upon dDNMT treatment (A) RT-qPCR analysis of CASP10 mRNA levels in isogenic RCC10 cells ( n = 3 biological replicates). (B) CASP10 mRNA levels in human VHL -WT and mutant ccRCC. Data were retrieved from TCGA, firehose Legacy in cBioPortal. (C) Immunoblot analysis of HIF-1α, HIF-2α, and procaspase-10 proteins in isogenic RCC10 cells. (D) Nucleotide sequence of the hypoxia response element ([HRE], in blue) at the promoter of the CASP10 gene (top). Genome browser snapshot of HIF-1α ChIP-seq peaks detected in hypoxic MDA-MB-231 cells (blue, n = 2 biological replicates), and HIF-2α ChIP-seq peaks detected 786-O cells (red, n = 2 biological replicates, GSE253325 ). (E) ChIP-qPCR assay showing enrichment of HIF-1α and HIF-2α at the promoter of CASP10 in RCC10 cells ( n = 3 biological replicates). (F) Representative images of death of RCC10 cells pre-treated with vehicle or CASP10i Z-AEVD-FMK for 30 min, followed by 3 days of co-treatment with SGI1027. (G) Quantification of PI-positive cells in (F) ( n = 3 biological replicates). (H) Representative images of death of RCC10 cells pre-treated with vehicle or CASP10i Z-AEVD-FMK for 30 min, followed by 3 days of co-treatment with MS1129. (I) Quantification of PI-positive cells in (H) ( n = 3 biological replicates). (J and K) Immunoblot analysis of procaspase-10, C-caspase-3, and C-caspase-7 proteins in RCC10 cells pre-treated with vehicle or CASP10i Z-AEVD-FMK for 30 min, followed by 2 days of co-treatment with SGI1027 (J, n = 2 biological replicates) or MS1129 (K, n = 2 biological replicates). Data represent mean ± SEM. p value was determined by one-way ANOVA with Dunnett’s test (A and E), unpaired 2-tailed Student’s t test (B), and one-way ANOVA with Tukey’s test (G and I). Scale bars: 100 μm in (F and H). See also .

Article Snippet: The supernatant was diluted in ChIP immunoprecipitation buffer (50 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail) and then subjected to immunoprecipitation overnight in the presence of Dynabeads (ThermoFisher) with anti-HIF-1α antibody (3 μg, home-made), anti-HIF-2α antibody (3 μg, home-made), anti-DNMT1 antibody (3 μg, 24206-1-AP, Proteintech), or anti-DNMT3A antibody (3 μg, 20954-1-AP, Proteintech), anti-DNMT3B antibody (3 μg, 26971-1-AP, Proteintech), or normal rabbit IgG (3 μg, 2729S, Cell Signaling Technology) at 4°C.

Techniques: Quantitative RT-PCR, Mutagenesis, Western Blot, Sequencing, ChIP-sequencing, ChIP-qPCR

Journal: Cell Reports Medicine

Article Title: HIF-activated priming of TRAIL-induced cell death determines epigenetic vulnerability in kidney cancer

doi: 10.1016/j.xcrm.2026.102630

Figure Lengend Snippet: dDNMT synergizes with TRAIL to enhance VHL -deficient ccRCC cell death (A) Representative images of death of RCC10 cells treated with vehicle, TRAIL (N114-281), SGI1027, or TRAIL (N114-281) plus SGI1027 for 3 days. (B) Quantification of PI-positive cells in (A) ( n = 3 biological replicates). (C) Representative images of death of RCC10 cells treated with vehicle, TRAIL (N114-281), MS1129, or TRAIL (N114-281) plus MS1129 for 3 days. (D) Quantification of PI-positive cells in (C) ( n = 2 biological replicates). (E) Representative images of death of 786-O cells treated with vehicle, TRAIL (N114-281), SGI1027, MS1129, TRAIL (N114-281) plus SGI1027, or TRAIL (N114-281) plus MS1129 for 3 days. (F) Quantification of PI-positive cells in (E) ( n = 3 biological replicates). (G) Tumor growth curves of 786-O tumors in mice treated with vehicle, TRAIL (N114-281), SGI1027, TRAIL (N114-281), plus SGI1027 for 10 days ( n = 5 biological replicates). (H) Tumor growth curves of 786-O tumors in mice treated with vehicle, MS1129, or TRAIL (N114-281) plus MS1129 for 10 days ( n = 5 biological replicates). (I) Representative cleaved caspase-3 (C-caspase-3) IHC in tumors from (G). (J) Quantification of C-caspase-3 IHC in (I) ( n = 4 biological replicates). (K) Representative C-caspase-3 IHC in tumors from (H). (L) Quantification of C-caspase-3 IHC in (K) ( n = 5 biological replicates). (M) A cell death priming model for VHL/HIF as gatekeepers activating TRAIL-induced apoptosis in ccRCC following dDNMT treatment. Data represent mean ± SEM. p value was determined by one-way ANOVA with Tukey’s test (B, D, F, J, and L), or two-way ANOVA with Tukey’s test (G and H). Scale bars, 100 μm in (A), (C), (E), (I), and (K). See also and .

Article Snippet: The supernatant was diluted in ChIP immunoprecipitation buffer (50 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail) and then subjected to immunoprecipitation overnight in the presence of Dynabeads (ThermoFisher) with anti-HIF-1α antibody (3 μg, home-made), anti-HIF-2α antibody (3 μg, home-made), anti-DNMT1 antibody (3 μg, 24206-1-AP, Proteintech), or anti-DNMT3A antibody (3 μg, 20954-1-AP, Proteintech), anti-DNMT3B antibody (3 μg, 26971-1-AP, Proteintech), or normal rabbit IgG (3 μg, 2729S, Cell Signaling Technology) at 4°C.

Techniques:

Journal: bioRxiv

Article Title: AAV2-Retro-Mediated Gene Transfer Selectively Targets Outer Retinal Cells Following Intravitreal Injection

doi: 10.64898/2026.03.10.710806

Figure Lengend Snippet: (A) Stitched 10X images of retinal cross-sections showing mGL expression at 14 dpi. (B) Higher-magnification view of a representative retinal region with mGL expression. (B’) Merged image from (B) highlighting layer-specific distribution (RPE, ONL, OPL, INL, IPL, and GCL), mGL, and DAPI across the layers. (C) Retinal flat-mount at 14 dpi after sequential injections, showing widespread mGL expression. (C’ and C’’) Higher magnification images of the boxed areas in (C). Scale bars: 200 µm (A, C); 50 µm (B’, C’, C’’). (D) Quantification of mGL- positive cells within the regions of high transduction in the ONL (i.e., region of interest, ROI). The percentage shown indicates the proportion of transduced cells among total DAPI-positive ONL cells. N= 3 animals. Scale bars : 200 µm (A, C); 50 µm (B’, D’).

Article Snippet: Samples were then incubated with anti-RBPMS antibody (1:2,000; Custom made at ProSci), anti-Cone arrestin (1:200; cat# AB15282; Sigma Aldrich), and anti- AP-2α antibody (1:50; cat# 3B5; DSHB) that were diluted in PBST blocking solution and incubated at 4 °C overnight.

Techniques: Expressing, Transduction

Journal: bioRxiv

Article Title: AAV2-Retro-Mediated Gene Transfer Selectively Targets Outer Retinal Cells Following Intravitreal Injection

doi: 10.64898/2026.03.10.710806

Figure Lengend Snippet: Retinal cross-sections show mGL expression (green) at 3 dpi (A) and 14 dpi (B). AP-2α immunoreactivity shown in magenta and RBPMS immunoreactivity in red. (A’ and B’) Higher-magnification images showing cells which appear to be co-labelled with mGL and AP-2α. INL, inner nuclear layer; GCL, ganglion cell layer. (C) Quantification shows that the percentage of mGL + cells that are positive for either RBPMS (in GCL and INL) or AP-2α at each time point. N=3 per group. Error bar, ± SEM Two-way ANOVA with Tukey’s multiple comparisons: * < 0.05, **** < 0.0001, ns = not significant. Scale bars : 50 µm (A, B); 5 µm (A’, B’).

Article Snippet: Samples were then incubated with anti-RBPMS antibody (1:2,000; Custom made at ProSci), anti-Cone arrestin (1:200; cat# AB15282; Sigma Aldrich), and anti- AP-2α antibody (1:50; cat# 3B5; DSHB) that were diluted in PBST blocking solution and incubated at 4 °C overnight.

Techniques: Expressing

Journal: Scientific Reports

Article Title: PRPF8-associated retinitis pigmentosa variant induces human neural retina-autonomous photoreceptor defects

doi: 10.1038/s41598-026-40376-y

Figure Lengend Snippet: Generation of 3D retinal organoids from PRPF8-WT and PRPF8-Y2334N hiPSC. a ) Schematic representation of the differentiation of hiPSC to 3D retinal organoids. Representative brightfield images illustrating the development of the neural retina during differentiation (Created in BioRender. Stanek, D. (2025) https://BioRender.com/2aaivcc ). b ) Comparative immunohistochemistry staining of 170-day-old retinal organoids, derived from PRPF8-WT and PRPF8-Y2334N hiPSC, showing the expression of indicated retinal markers. DNA was stained by DAPI (blue). Scale bar - 50 μm. c ) Western blot analysis of 170-day-old retinal organoids showing the protein levels of splicing factors PRPF8, PRPF6 and the retinal proteins recoverin and rhodopsin (see also ). Tubulin and GAPDH serve as loading controls. d ) Quantification of western blot results of at least three samples, each containing 10 retinal organoids from different differentiation batches of PRPF8-Y2334N hiPSC clone (black dots). Grey dots represent a sample of 10 retinal organoids differentiated from a different PRPF8-Y2334N_2 hiPSC clone. Horizontal lines indicate means and standard deviation. Statistical significance was determined using a two-tailed unpaired t-test.

Article Snippet: Sections were stained for retinal marker proteins overnight at 4 °C with the following primary antibodies: RHODOPSIN (Sigma-Aldrich, O4886, Lot#: 22180131, 1:200), RECOVERIN (Sigma-Aldrich, AB5585, Lot#: 3432603, 1:1000), CRALBP (Abcam, ab15051, Lot#: GR3284867-10 1:200), and AP-2α (Santa Cruz, sc-12726 Lot#: E1921, 1:100).

Techniques: Immunohistochemistry, Staining, Derivative Assay, Expressing, Western Blot, Standard Deviation, Two Tailed Test

Journal: Scientific Reports

Article Title: PRPF8-associated retinitis pigmentosa variant induces human neural retina-autonomous photoreceptor defects

doi: 10.1038/s41598-026-40376-y

Figure Lengend Snippet: Y2334N retinal organoids show impaired photoreceptor inner and outer segments. a ) Transmission electron microscopy (TEM) images of photoreceptors from 250-day-old retinal organoids. Outer segments were visible in the WT but not the Y2334N retinal organoids (upper panel). Scanning electron microscopy (SEM) images of 250-day-old retinal organoids revealed fewer inner/outer segments on the retinal organoid surface of Y2334N organoids (lower panel). Scale bars: Top panel - 2 μm, bottom panel - 50 μm. A Schematic representation of the photoreceptor structure was created in BioRender. Stanek, D. (2025) https://BioRender.com/8bgoqrx . IS - inner segment, CC - connecting cilia, OS - outer segment. b ) Analysis of the brush border thickness by light microscopy analysis. Bright field images of a representative neural retina from 250-day-old retinal organoids (upper panel). Machine-learning-supported WEKA segmentation analysis of the respective brightfield image (lower panel). The photoreceptor inner and outer segment layers are depicted in green. The top graph shows quantification of the brush border thickness, and the bottom graph shows quantification of inner segment coverage on the organoid surface. Three WT and PRPF8-Y2334N retinal organoids were used for the analysis. Data are represented as mean +/- standard error of the mean. Statistical significance was determined using a two-tailed unpaired t-test. Scale bar represents 50 µm.

Article Snippet: Sections were stained for retinal marker proteins overnight at 4 °C with the following primary antibodies: RHODOPSIN (Sigma-Aldrich, O4886, Lot#: 22180131, 1:200), RECOVERIN (Sigma-Aldrich, AB5585, Lot#: 3432603, 1:1000), CRALBP (Abcam, ab15051, Lot#: GR3284867-10 1:200), and AP-2α (Santa Cruz, sc-12726 Lot#: E1921, 1:100).

Techniques: Transmission Assay, Electron Microscopy, Light Microscopy, Two Tailed Test

Journal: Scientific Reports

Article Title: PRPF8-associated retinitis pigmentosa variant induces human neural retina-autonomous photoreceptor defects

doi: 10.1038/s41598-026-40376-y

Figure Lengend Snippet: 170-day-old PRPF8-Y2334N retinal organoids reveal limited changes in gene expression. a ) Differential expression analysis of PRPF8-WT vs. PRPF8-Y2334N retinal organoids. Genes were considered differentially expressed if FDR < 0.05 and fold change expression ≥ 2 (|log2 FC| ≥ 1). b ) Analysis of publicly available single-cell RNA sequencing data from the Single Cell Expression Atlas (E-MTAB-7316) reveals predominant SLITRK2 expression in Müller cells. c ) Differential intron retention analysis of Y2334N and WT retinal organoids. Introns were considered differentially retained if ΔPIR Y2334N-WT (difference in percentage intron retention) ≥ 0.05 and p < 0.05. d) The sashimi plot shows the indicated transcript and illustrates the intron with the largest difference in intron retention. The retained intron is highlighted in green. e ) Differential analysis of alternative exons (exon skipping). Transcripts were considered differentially spliced if ΔPSI Y2334N-WT (percentage spliced in) ≥ 0.05 and p < 0.05. f ) The sashimi plot shows the indicated transcript and illustrates the exon with the most substantial difference in alternative splicing. The alternatively spliced exon is highlighted in green.

Article Snippet: Sections were stained for retinal marker proteins overnight at 4 °C with the following primary antibodies: RHODOPSIN (Sigma-Aldrich, O4886, Lot#: 22180131, 1:200), RECOVERIN (Sigma-Aldrich, AB5585, Lot#: 3432603, 1:1000), CRALBP (Abcam, ab15051, Lot#: GR3284867-10 1:200), and AP-2α (Santa Cruz, sc-12726 Lot#: E1921, 1:100).

Techniques: Gene Expression, Quantitative Proteomics, Expressing, Single Cell, RNA Sequencing, Alternative Splicing