Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Therapeutic mRNA delivery of CRISPR-Cas9 to the trabecular meshwork reverses ocular hypertension in myocilin glaucoma

doi: 10.1016/j.omtm.2025.101614

Figure Lengend Snippet: Lipoplex encapsulating Cas9-mRNA and gRNA targeting MYOC reduces intracellular accumulation of mutant myocilin protein in cultured TM cells GTM3 cells stably expressing mutant MYOC were transfected with lipoplexes containing either Cas9 mRNA + scrambled gRNA (Control) or Cas9 mRNA + MYOC -targeting gRNA (Treated). Immunostaining for (A) KDEL or (B) Protein disulfide isomerase (PDI) revealed a reduction in intracellular accumulation of mutant myocilin protein in the ER and its associated ER stress in treated cells compared to controls. N = 3. (C) Western blot analysis of MYOC and GRP78 in cell lysates from TM cells expressing mutant MYOC treated with either Cas9 mRNA + scrambled gRNA (Control) or Cas9 mRNA + MYOC -targeting gRNA (Treated). (D) Densitometric analysis of the Western blots revealed a significant reduction in MYOC protein levels in CRISPR-Cas9-treated cells compared to controls. GRP78, an ER stress marker, was reduced significantly upon Cas9 mRNA + MYOC -targeting gRNA treatment compared to controls, ( n = 7 MYOC, n = 4 KDEL, ∗∗∗∗ p < 0.0001).

Article Snippet: Fixed cells were blocked with 10% goat serum in 0.1% Triton X-100 for 2 h. Primary antibodies against MYOC (Catalog #60357, Proteintech) and KDEL (Catalog #NBP1-97469, Novus) were incubated overnight, followed by four washes with 1x PBS.

Techniques: Mutagenesis, Cell Culture, Stable Transfection, Expressing, Transfection, Control, Immunostaining, Western Blot, CRISPR, Marker

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Therapeutic mRNA delivery of CRISPR-Cas9 to the trabecular meshwork reverses ocular hypertension in myocilin glaucoma

doi: 10.1016/j.omtm.2025.101614

Figure Lengend Snippet: Cas9 mRNA and gRNA targeting MYOC rescues the glaucomatous pathology induced by mutant MYOC in the TM of Tg.CreMYOC Y437H mice (A) Tg.CreMYOC Y437H mice were injected with lipoplex-Cre mRNA along with Cas9 mRNA+scrambled gRNA or gRNA targeting MYOC . IOP was measured weekly. Tg.CreMYOC Y437H mice injected with Cre and Cas9+scrambled gRNA developed a significant IOP elevation 3 weeks post-injection and sustained IOP elevation throughout the study. Tg.CreMYOC Y437H mice injected with lipoplex loaded with Cre and Cas9 mRNA+gRNA targeting MYOC did not show changes in IOPs, indicating rescue of ocular hypertension in Tg.CreMYOC Y437H mice. Two-way ANOVA with repeated measurements and Bonferroni post-hoc analysis were performed. Data represented as mean ± SEM; ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Representative immunostaining for MYOC and KDEL (B) and its quantification (C) demonstrated that Cas9 mRNA+g MYOC significantly reduces MYOC and its co-localization with the ER marker KDEL in the TM of Tg.CreMYOC Y437H mice compared to control-treated mice. Quantification revealed a 54% reduction in MYOC levels and a 55% reduction in KDEL levels following Cas9/gRNA treatment ( n = 4, ∗∗∗∗ p < 0.0001).

Article Snippet: Fixed cells were blocked with 10% goat serum in 0.1% Triton X-100 for 2 h. Primary antibodies against MYOC (Catalog #60357, Proteintech) and KDEL (Catalog #NBP1-97469, Novus) were incubated overnight, followed by four washes with 1x PBS.

Techniques: Mutagenesis, Injection, Immunostaining, Marker, Control

Journal: bioRxiv

Article Title: ncBAF, a chromatin remodeler, enhances PXR-mediated transcriptional activation in the human and mouse liver

doi: 10.1101/2023.02.03.527063

Figure Lengend Snippet: Effects of iBRD9 on the induction of CYP3A in mouse liver. C57BL/6J or hCYP3A-MAC/hPXR male mice (n = 6) were intraperitoneally treated with 50 mg/kg PCN or 10 mg/kg rifampicin for four consecutive days and intraperitoneally treated with 10 mg/kg iBRD9 every other day. Cyp3a11, Cyp3a25, CYP3A4, and Gapdh mRNA (A, D), and Cyp3a, CYP3A4, and KDEL protein (B, E) levels were evaluated by real-time RT□PCR and Western blotting, respectively. (C, F) Triazolam α- and 4-hydroxylase activities were evaluated as marker activity for Cyp3a and CYP3A4. Each column represents the mean ± SD (n = 6). n refers to biological repeats. ** P < 0.01 and *** P < 0.001, compared with vehicle, † P < 0.05 and †† P < 0.01, compared with iBRD9 (−). The experiments were repeated two times with similar results.

Article Snippet: Rabbit anti-human GAPDH polyclonal antibody (NB100-56875) and mouse anti-KDEL monoclonal antibody (NBP1-97469) were purchased from Novus Biologicals (Centennial, CO).

Techniques: Western Blot, Marker, Activity Assay

Journal: PLoS ONE

Article Title: β-AR Blockers Suppresses ER Stress in Cardiac Hypertrophy and Heart Failure

doi: 10.1371/journal.pone.0027294

Figure Lengend Snippet: (A) ER chaperone GRP78 and spliced XBP-1 were increased in AAC rats (1or 4 or 8 weeks after operation), and metoprolol treatment (30 mg/kg/d for 8 weeks) reduced the epression of GRP78 and spliced XBP-1. Proteins were normalized to β-actin. (B) GRP78 was increased in AAC rats (4 weeks), and propranolol treatment (30 mg/kg/d for 4 weeks) reduced the epression of GRP78. Protein was normalized to β-actin. (C) Immunohistochemical analyses of rats’ hearts and number of GRP78, KDEL and CHOP-positive cells per mm 2 . Scale bar, 40 µm. For (A) to (C), *P<0.05 vs. sham, #P<0.05 vs. AAC. (D) GRP78 and spliced XBP-1 was increased in Iso rats, and metoprolol treatment (30 mg/kg/d for 2 weeks) reduced the epression of GRP78 and spliced XBP-1. Proteins were normalized to β-actin. *P<0.05 vs. control, #P<0.05 vs. Iso.

Article Snippet: Antibodies for phosphorylated PERK, PERK, phosphorylated eIF2α, JNK1, phospho-c-Jun (p-Ser73), GRP78, CHOP (GADD153), KDEL receptor, XBP1, phosphorylated CaMKIIδ, CaMKIIδ, β-actin and PKI (6–22) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Immunohistochemical staining, Control

Journal: Molecules and Cells

Article Title: OASL1 Traps Viral RNAs in Stress Granules to Promote Antiviral Responses

doi: 10.14348/molcells.2018.2293

Figure Lengend Snippet: (A) OASL1 associated with cytoplasmic speckles specifically upon poly(I:C) treatment. NIH-3T3 cells expressing EGFP-OASL1 are shown following stimulation with various TLRs (indicated at the top) for 6 h (top panel) or 12 h (lower panel). (B) OASL2 did not associate with speckles upon poly(I:C) stimulation. EGFP-OASL2–overexpressing NIH-3T3 cells were stimulated with poly(I:C) for 6 h. (C) OASL1-containing speckles do not overlap with secretory vesicles. EGFP-OASL1–overexpressing NIH-3T3 cells were stained with specific antibodies against the indicated markers (KDEL, RCAS, LAMP1, and PMP70; shown in red). (D) OASL1-containing speckles colocalized with SG markers. EGFP-OASL1–expressing cells were stained with the indicated SG markers (TIAR, S6, eIF4E, and G3BP1) after stimulation with poly(I:C) for 6 h. (E) Actin and microtubules are required for formation of OASL1-containing speckles. EGFP-OASL1–xpressing cells were stained for β-actin after pre-treatment with Cytochalasin D or colchicine treatment followed by poly(I:C) stimulation. Nuclei were stained with DAPI (blue). Scale bars correspond to 10 μm. Images are representative of at least two independent experiments.

Article Snippet: Blocking was performed with 3% bovine serum albumin (BSA) in PBST (blocking solution) for 1 h at RT, and the cells were then incubated with a primary antibody [rabbit α-KDEL (NOVUS Biologicals), rabbit α-RCAS (Cell Signaling), rabbit α-LAMP1 (Sigma), rabbit α-PMP70 (Abcam), goat α-TIAR (Santa Cruz Biotechnology), rabbit α-eIF4E (Cell Signaling), rabbit α-S6 (Cell Signaling), rabbit α-G3BP1 (Santa Cruz Biotechnology), rabbit α-MDA5 (Abcam), MitoTracker ® Red (Invitrogen), rabbit α-LC3b (MBL International), or rabbit α-p62 (MBL International)] for 1 h at RT or overnight at 4°C.

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: KDELR3 overexpression as a novel prognostic and diagnostic biomarker in glioma: comprehensive bioinformatic analysis insights

doi: 10.1038/s41598-024-80991-1

Figure Lengend Snippet: The expression of KDELR3 in different types of cancer, including glioma. ( A ) The expression of KDELR3 in different types of cancer cells was obtained from the CCLE database. ( B ) The expression of KDELR3 in different types of cancer was obtained from Tumor Immune Estimation Resource database. * p < 0.05; *** p < 0.001.

Article Snippet: The Anti-KDELR3 polyclonal antibody (1:50) was sourced from Boster Inc., China (Catalog No. A14682-1).

Techniques: Expressing

Journal: Scientific Reports

Article Title: KDELR3 overexpression as a novel prognostic and diagnostic biomarker in glioma: comprehensive bioinformatic analysis insights

doi: 10.1038/s41598-024-80991-1

Figure Lengend Snippet: Expression of KDELR3 in gliomas. ( A − F ) Expression levels of KDELR3 in normal tissues, WHO grade, Age, IDH status, 1p19q codeletion, Primary therapy outcome (ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: The Anti-KDELR3 polyclonal antibody (1:50) was sourced from Boster Inc., China (Catalog No. A14682-1).

Techniques: Expressing

Journal: Scientific Reports

Article Title: KDELR3 overexpression as a novel prognostic and diagnostic biomarker in glioma: comprehensive bioinformatic analysis insights

doi: 10.1038/s41598-024-80991-1

Figure Lengend Snippet: Survival analysis of KDELR3 in gliomas. ( A ) OS Kaplan-Meier analysis of KDELR3 in gliomas. ( B ) DSS Kaplan-Meier analysis of KDELR3 in gliomas. ( C ) PFI Kaplan-Meier analysis of KDELR3 in gliomas. ( D − M ) Kaplan-Meier analysis of overall survival of KDELR3 in WHO grade 2 and 3, Age>60, Age ≤ 60 , Female, Male, IDH Mut, 1p19q codel, 1p19q non-codel, primary therapy outcome PD gliomas.

Article Snippet: The Anti-KDELR3 polyclonal antibody (1:50) was sourced from Boster Inc., China (Catalog No. A14682-1).

Techniques:

Journal: Scientific Reports

Article Title: KDELR3 overexpression as a novel prognostic and diagnostic biomarker in glioma: comprehensive bioinformatic analysis insights

doi: 10.1038/s41598-024-80991-1

Figure Lengend Snippet: Correlation analysis between KDELR3 expression and immune cell infiltration. ( A ) Correlation between KDELR3 expression and different immune cell infiltration in gliomas KDELR3 expression. ( B ) Correlation between KDELR3 expression and abundance of tumor-infiltrating immune cells in gliomas.

Article Snippet: The Anti-KDELR3 polyclonal antibody (1:50) was sourced from Boster Inc., China (Catalog No. A14682-1).

Techniques: Expressing

Journal: Scientific Reports

Article Title: KDELR3 overexpression as a novel prognostic and diagnostic biomarker in glioma: comprehensive bioinformatic analysis insights

doi: 10.1038/s41598-024-80991-1

Figure Lengend Snippet: Functional enrichment analysis of related genes of KDELR3 in gliomas.

Article Snippet: The Anti-KDELR3 polyclonal antibody (1:50) was sourced from Boster Inc., China (Catalog No. A14682-1).

Techniques: Functional Assay

Journal: Scientific Reports

Article Title: KDELR3 overexpression as a novel prognostic and diagnostic biomarker in glioma: comprehensive bioinformatic analysis insights

doi: 10.1038/s41598-024-80991-1

Figure Lengend Snippet: KDELR3 PPI network in gliomas.

Article Snippet: The Anti-KDELR3 polyclonal antibody (1:50) was sourced from Boster Inc., China (Catalog No. A14682-1).

Techniques:

Journal: Scientific Reports

Article Title: KDELR3 overexpression as a novel prognostic and diagnostic biomarker in glioma: comprehensive bioinformatic analysis insights

doi: 10.1038/s41598-024-80991-1

Figure Lengend Snippet: Correlation between KDELR3 expression and clinical characteristics of gliomas ( A ) was significantly associated with high expression of KDELR3 in gliomas in TCGA. ( B ) was Time dependent ROC curve.

Article Snippet: The Anti-KDELR3 polyclonal antibody (1:50) was sourced from Boster Inc., China (Catalog No. A14682-1).

Techniques: Expressing

Journal: Scientific Reports

Article Title: KDELR3 overexpression as a novel prognostic and diagnostic biomarker in glioma: comprehensive bioinformatic analysis insights

doi: 10.1038/s41598-024-80991-1

Figure Lengend Snippet: ( A ) was significantly associated with high expression of KDELR3 in glioma in clinical samples ( B ) and poor OS with high expression of KDELR3 in clinical samples. ( C ) Expression levels of KDELR3 in WHO grade.

Article Snippet: The Anti-KDELR3 polyclonal antibody (1:50) was sourced from Boster Inc., China (Catalog No. A14682-1).

Techniques: Expressing