Journal: Molecular Oncology

Article Title: Raphin‐1 mediates the survival and sensitivity to radiation of pediatric‐type diffuse high‐grade glioma via phosphorylated eukaryotic initiation factor 2α‐dependent and ‐independent processes

doi: 10.1002/1878-0261.70081

Figure Lengend Snippet: The effect of HA15, a specific inhibitor of BiP, on raphin‐1's downstream targets. (A, B) SU‐DIPG‐VI and KNS‐42 cells were plated in triplicates and treated with HA15 as described in Section . Values are mean survival (%) or cell death (%) ± SD of two independent experiments. The significance of the differences between treatments and control were determined using an unpaired Student t ‐test—** P < 0.005. (C, D) SU‐DIPG‐VI and KNS‐42 cells were treated with the indicated concentrations of HA15 for 24 h and processed for western blot analysis as described in Section . Numbers at the bottom of the autoradiograms indicate treatment‐dependent changes in the level of the proteins and equal loading control (Ponceau). The number of determinations for each experimental condition were as follows: C: n = 2, D: n = 2.

Article Snippet: Salubrinal (#HY‐15486), CB‐5083 (#HY‐12861), and HA15 (#HY‐100437) were from MedChem Express (NJ).

Techniques: Control, Western Blot

Journal: iScience

Article Title: Depletion of HIV reservoir by activation of ISR signaling in resting CD4 + T cells

doi: 10.1016/j.isci.2022.105743

Figure Lengend Snippet: Activation of ISR disrupts latent HIV and reduces HIV+ cells in the primary CD4 + T cell model of latency with minimal impact in bystander CD4 + T cells The primary CD4 + T cell model of HIV latency was treated with or without HA15. Cells were collected 12 h, 1 day, 2 days and 4 days after treatment. Percentage of GFP+ cells was analyzed by flow cytometry (A). HIV RNA was analyzed by qPCR (B). Protein expression of ATF4 was measured 24 h after treatment where actin served as loading controls (insert in A). Proposed ISR/ATF4 downstream signaling was shown in (C). Total cell number was counted with Trypan blue staining (D), while flow cytometry was performed to analyze cell apoptosis (Annexin V) (E). Similar as in A-B, the primary CD4 + T cell model of latency was treated with 20 or 40 μM HA15, and cells were collected 4 days after treatment. Flow cytometry was performed to analyze HIV expression (GFP), cell apoptosis (cleaved PARP1) and viability (F and G). Then, HIV negative primary CD4 + T cells were treated with 20 μM HA15 (H and I). Cells were collected 1 day, 2 days and 4 days after treatment. ATF4 protein expression are analyzed 2 days after 20 μM HA15 treatment (G, insert) whereas total cell number was counted with Trypan blue staining (G). Flow cytometry was performed to analyze cell apoptosis (Annexin V) and viability (I). ∗, p<0.05; ∗, p<0.01; ∗∗∗, p<0.001; ∗∗∗∗, p<0.00001, analyzed with one-way ANOVA or two-tailed t test compared with the control treatment (n = 3).

Article Snippet: HA15 , Selleckchem , Cat#S8299.

Techniques: Activation Assay, Flow Cytometry, Expressing, Staining, Two Tailed Test, Control

Journal: iScience

Article Title: Depletion of HIV reservoir by activation of ISR signaling in resting CD4 + T cells

doi: 10.1016/j.isci.2022.105743

Figure Lengend Snippet: ISR/ATF4 signaling activation in T cells with or without latent HIV infection HIV negative primary CD4 + T cells (H18) or primary CD4 + T cell models (E47) of latency were treated with 20 or 40 μM HA15 for 1–2 days. Cells were collected. Then, the whole cell protein lysates were prepared and subjected to Western blot to measure the protein expression in ISR/ATF4 pathways. ∗, band of CHOP protein.

Article Snippet: HA15 , Selleckchem , Cat#S8299.

Techniques: Activation Assay, Infection, Western Blot, Expressing

Journal: iScience

Article Title: Depletion of HIV reservoir by activation of ISR signaling in resting CD4 + T cells

doi: 10.1016/j.isci.2022.105743

Figure Lengend Snippet: Activation of ISR signaling reduces HIV DNA/RNA without impact on global cell number of rCD4 + T cells isolated from PLWH on ART The rCD4 + T cells isolated from ART-suppressed PLWH (n = 4) were treated with 20 μM HA15. Cells were collected 1 day, 2 days and 3 days after the treatment (n = 3). Total cell number was counted with Trypan blue staining. (A–C) Trypan blue staining and flow cytometry were performed to analyze cell viability and apoptosis (Annexin V). (D) Total RNA was extracted from the intestinal tissues in the SIV acutely infected rhesus macaques (n = 5). ATF4 gene expression and SIV RNA were quantitated by qRT-PCR. Then, the correlation of gene expression in the same animals was analyzed by linear regression. (E and F) Patient primary CD4 + T cells were treated with DMSO or 20 μM HA15 for 4 days. Then, the expression of ATF4 and CHOP was measured by RT-qPCR. ∗, p < 0.05, analyzed with two-tailed t test compared with the control treatment (n = 3). (G) Resting CD4 + T cells (n = 3) were treated with or without 20 μM HA15 for 4 days. Cells were collected, then HIV env DNA was extracted and subjected for droplet digital PCR (ddPCR) (n = 3).

Article Snippet: HA15 , Selleckchem , Cat#S8299.

Techniques: Activation Assay, Isolation, Staining, Flow Cytometry, Infection, Gene Expression, Quantitative RT-PCR, Expressing, Two Tailed Test, Control, Digital PCR

Journal: iScience

Article Title: Depletion of HIV reservoir by activation of ISR signaling in resting CD4 + T cells

doi: 10.1016/j.isci.2022.105743

Figure Lengend Snippet: Activation of ISR signaling depletes replication competent HIV in the rCD4 + T cells isolated from PLWH on ART (A) Resting CD4 + T cells were treated with or without 20 μM HA15 for 4 days in the presence of ART plus 20 U/mL IL-2 (n = 5). Then, the treatment was washed out, followed by PHA (2 μg/mL) reactivation for 24 h. PHA was then washed out and cells were co-cultured with CD8-depleted human PBMCs to measure viral outgrowth. Cells were collected 21 days after co-culture with CD8-depleted human PBMCs. (B–D) Cell and supernatants were collected. Cell-associated HIV gag RNA, HIV gag DNA and cell-free HIV gag RNA were determined by RT-ddPCR and/or ddPCR.

Article Snippet: HA15 , Selleckchem , Cat#S8299.

Techniques: Activation Assay, Isolation, Cell Culture, Co-Culture Assay

Journal: iScience

Article Title: Depletion of HIV reservoir by activation of ISR signaling in resting CD4 + T cells

doi: 10.1016/j.isci.2022.105743

Figure Lengend Snippet: Targeting eIF2α/GADD34/CReP by Ritonavir enhances the reduction of GFP+ cells elicited by HA15 in the primary CD4 + T cell model of latency Model of ISR/ATF4 signaling pathway to activate HIV and induce cell death which is negatively regulated by ATF4-induced gene GADD34 and HIV protease. Breaking these negative feedback loops by the inhibition of GADD34 by Sephin 1 or HIV protease by Ritonavir (Rit) may enhance the cell death in HIV+ CD4 + T cells. (A) Primary CD4 + T cell model of latency was treated with 20 μM HA15, 20 μM Sephin-1, 10 μM Rit alone or in combination. (B–D) Anti-CD3/CD28 treatment was used as a positive control. Cells were collected 4 days after treatment, and the percentage of GFP positive cells and cellular viability were analyzed by flow cytometry. ∗, p<0.05; ∗∗, p<0.01; ∗∗∗∗, p<0.0001 (n = 3). Data were analyzed by One-way ANOVA.

Article Snippet: HA15 , Selleckchem , Cat#S8299.

Techniques: Inhibition, Positive Control, Flow Cytometry

Journal: iScience

Article Title: Depletion of HIV reservoir by activation of ISR signaling in resting CD4 + T cells

doi: 10.1016/j.isci.2022.105743

Figure Lengend Snippet: ISR/ATF4 activation by HA15 treatment enhances cleaved PARP in HIV RNA+ cells in the primary CD4 + T cell model of latency Primary T cell model were treated with or without HA15 (20 μM) for 2 days. Cells were collected and prepared for HIV RNA FISH-Flow. (A) Flow gating strategy and representative flow charts of HIV RNA and cleaved PARP staining from the treated or untreated cells. (B) Positive control (probe for Human RFL13A) and negative control (probe for Bacteria DAPB) during the flow cytometry. (C–E) Percentages of cleaved PARP1+ cells and cleaved PARP1+ cells in RNA+ or HIV RNA-cells with or without ISR/ATF4 activation. ∗∗∗∗, p<0.0001 analyzed by two-tailed T-test.

Article Snippet: HA15 , Selleckchem , Cat#S8299.

Techniques: Activation Assay, Staining, Positive Control, Negative Control, Bacteria, Flow Cytometry, Two Tailed Test

Journal: iScience

Article Title: Depletion of HIV reservoir by activation of ISR signaling in resting CD4 + T cells

doi: 10.1016/j.isci.2022.105743

Figure Lengend Snippet: Single cell RNA-seq analysis identifies major modulated pathways that related to HIV+ cell reduction after ISR activation by HA15 in primary CD4 + T cell model of latency (A) The transcription of HIV Tat and Rev across all cells plotted in two-dimensional UMAP space. SCT-normalized expression data are plotted, higher expression is indicated in purple. (B) Cells were treated with or without HA15 for 2 days, then the cells were collected for scRNA-seq analysis. Enriched gene ontology terms related to cell death or apoptosis, translation, stress or stimulus response, innate immunity, or similar pathways were plotted depending on treatment and HIV status. FDR <0.05 was considered significant.

Article Snippet: HA15 , Selleckchem , Cat#S8299.

Techniques: RNA Sequencing, Activation Assay, Expressing

Journal: iScience

Article Title: Depletion of HIV reservoir by activation of ISR signaling in resting CD4 + T cells

doi: 10.1016/j.isci.2022.105743

Figure Lengend Snippet: ISR/ATF4 activation by HA15 activates anti-viral IFIT1/cell death signaling in primary CD4 + T cell model of latency The primary CD4 + T cell model of latency was treated with or without 20 μM HA15 for 48 h. Cells were collected 12 h, 1 day, and 2 days after the treatment. (A) Total RNA was isolated, and RT-qPCR was performed to analyze the expression of IFN-I/IFIT signaling genes. p<0.05; ∗∗∗, p<0.001; analyzed with One-way ANOVA (n = 3). (B–D) Primary CD4 + T cell model are treated with DMSO or HA15 (20 μM) for 4 days. Cells are collected and prepared for HIV RNA FISH-Flow to determine the percentage of IFIT1+ cells The percentage of IFIT+/HIV RNA+ cells and cell death (% of cleaved PARP1+) was determined by HIV RNA FISH-Flow ∗, p<0.05; ∗∗, p<0.01; ∗∗∗, p<0.001; ∗∗∗∗, p<0.0001; analyzed with two-tailed t -test (n = 3–6).

Article Snippet: HA15 , Selleckchem , Cat#S8299.

Techniques: Activation Assay, Isolation, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: iScience

Article Title: Depletion of HIV reservoir by activation of ISR signaling in resting CD4 + T cells

doi: 10.1016/j.isci.2022.105743

Figure Lengend Snippet:

Article Snippet: HA15 , Selleckchem , Cat#S8299.

Techniques: Recombinant, Staining, Reverse Transcription, Cell Isolation, Software

Journal: Cell reports methods

Article Title: A size-exclusion-based approach for purifying extracellular vesicles from human plasma

doi: 10.1016/j.crmeth.2021.100055

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: EXP®2 Stem Trap , Optimize Technologies , Part# 04003HNC18.

Techniques: Purification, Recombinant, Sequencing, Modification, Mass Spectrometry, Saline, Isolation, Software, Pore Size