Journal: Cell reports

Article Title: IRE1α promotes phagosomal calcium flux to enhance macrophage fungicidal activity.

doi: 10.1016/j.celrep.2025.115694

Figure Lengend Snippet: Figure 1. Candida albicans infection results in activation of macrophage IRE1α (A) iBMDM cell lines (WT or IRE1ΔR) were infected with C. albicans (Ca), treated with LPS, thapsi- gargin, or mock for 4 h. Xbp1 mRNA splicing was measured by semi-quantitative RT-PCR amplifi- cation of the Xbp1 transcript, followed by treat- ment with PstI, which recognizes a cleavage site within the 26-bp intron that is removed by IRE1α, resulting in cleavage of the unspliced isoform, specifically. (B) Immunoblot analysis of lysates from WT or IRE1ΔR iBMDM to confirm IRE1α truncation in IRE1ΔR cells resulting from removal of floxed exons 20 and 21. (C) Expression of spliced Xbp1 over a time course following Ca infection. (D) Expression of spliced Xbp1 at 4 h post-infec- tion (hpi) with the indicated isolates. (E) Expression of spliced Xbp1 at 4 h following Ca infection or LPS treatment of WT or CARD9 KO iBMDM. (F) Expression of spliced Xbp1 at 4 h following Ca infection and depleted zymosan (d-zymosan) or LPS treatment of WT or IRE1ΔR iBMDM. (G) Expression of spliced Xbp1 at 4 h following Ca infection, LPS treatment, or d-zymosan treatment of WT or TLR2/4/9 KO iBMDM. (H) Expression of spliced Xbp1 at 4 h following Ca infection or LPS treatment of two pairs of clonal iBMDM (WT or TRAF6 KO). Closed symbols are data from WT-1 and KO-1; open symbols are data from WT-2 and KO-2. Data are representative of 3–4 individual experiments. Graphs show the mean ± SEM of biological repli- cates (C–H). For all experiments, Ca MOI = 1, LPS treatment = 100 ng/mL, d-zymosan = 100 μg/mL, thapsigargin = 5 μM. Statistical analysis details are in STAR Methods.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal anti-IRE1α Cell Signaling Cat#3924; RRID:AB_823545 Rabbit polyclonal anti-XBP1 Abcam Cat#ab37152; RRID:AB_778939 Rabbit polyclonal anti-CARD9 Cell Signaling Cat#12283; RRID:AB_2797869 Mouse monoclonal anti-ACTIN ThermoFisher Cat#MA5-11869; RRID:AB_11004139 Rabbit monoclonal anti-TRAF6 Abcam Cat#ab40675; RRID:AB_778573 Rabbit polyclonal anti-K48-linkage specific polyubiquitin Cell Signaling Cat#4289; RRID:AB_10557239 Rabbit monoclonal anti-ATF6 Cell Signaling Cat#65880; RRID:AB_2799696 Rabbit polyclonal anti-phosphoeIF2α Ser51 Cell Signaling Cat#9721; RRID:AB_330951 Mouse monoclonal anti- eIF2α Cell Signaling Cat#2103; RRID:AB_836874 Rabbit monoclonal anti-ATF4 Cell Signaling Cat#11815; RRID:AB_2616025 Rabbit polyclonal anti-Candida albicans-FITC LSBio Cat#LS-C103355 Rat monoclonal anti-LAMP1 DSHB Cat#1D4B; RRID:AB_2134500 Mouse monoclonal anti-EEA1 ThermoFisher Cat#MA5-31575 Rabbit monoclonal anti-KDEL Abcam Cat#ab176333; RRID:AB_2819147 Rat monoclonal anti-CD11b, BV421-conjugated Biolegend Cat#414-0112-82 Bacterial and virus strains MRSA-mCherry Methicillin-resistant Staphyloccocus aureus USA300 LAC harboring pSarA-mCherry plasmid Boles et al. 200873 N/A Chemicals, peptides, and recombinant proteins Thioflavin T Cayman Chemical Cat#32553; CAS Number 2390-54-7 TRIzol reagent Thermo Fisher Cat#15596018 Turbo DNase I Thermo Fisher Cat#AM2238 Paraformaldehyde Electron Microscopy Services Cat#15700 Calcofluor white Sigma-Aldrich Cat#18909 Bovine serum albumin Thermo Fisher Cat#BP9706-100 Sulforhodamine B Sigma-Aldrich Cat#230162; CAS Number 3520-42-1 Critical commercial assays Lookout Mycoplasma PCR Detection Kit Sigma-Aldrich Cat#MP0035 iScript cDNA synthesis kit BioRad Cat# 1708891 PowerUP SYBR Green qPCR master mix Thermo Fisher Cat# A25742 Fluo-4 Calcium Imaging Kit Invitrogen Cat#F10489 LysoSensor Yellow/Blue DND-160 Thermo Fisher Cat#L7545 LysoTracker Deep Red Invitrogen Cat#L12492 Duolink In Situ PLA Kit Sigma-Aldrich Cat#DUO92008 Cat#DUO92002 Cat#DUO92004 Fluorospheres Thermo Fisher Cat#F8801 (Continued on next page) Cell Reports 44, 115694, May 27, 2025 15

Techniques: Infection, Activation Assay, Quantitative RT-PCR, Western Blot, Expressing

Journal: Cell reports

Article Title: IRE1α promotes phagosomal calcium flux to enhance macrophage fungicidal activity.

doi: 10.1016/j.celrep.2025.115694

Figure Lengend Snippet: Figure 4. IRE1α promotes phagosome maturation during C. albicans infection (A) Representative images showing LAMP1 (yellow) recruitment to phagosomes containing iRFP-expressing C. albicans (magenta) in IRE1 WT or IRE1ΔR iBMDM at indicated times post-infection. (B) Quantification of LAMP1 recruitment to phagosomes containing C. albicans in IRE1 WT or IRE1ΔR iBMDM, as measured by LAMP1 mean fluorescence in- tensity associated with C. albicans-expressed iRFP. (C) Quantification of C. albicans killing in IRE1 WT and IRE1ΔR macrophages treated with BafA or DMSO control.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal anti-IRE1α Cell Signaling Cat#3924; RRID:AB_823545 Rabbit polyclonal anti-XBP1 Abcam Cat#ab37152; RRID:AB_778939 Rabbit polyclonal anti-CARD9 Cell Signaling Cat#12283; RRID:AB_2797869 Mouse monoclonal anti-ACTIN ThermoFisher Cat#MA5-11869; RRID:AB_11004139 Rabbit monoclonal anti-TRAF6 Abcam Cat#ab40675; RRID:AB_778573 Rabbit polyclonal anti-K48-linkage specific polyubiquitin Cell Signaling Cat#4289; RRID:AB_10557239 Rabbit monoclonal anti-ATF6 Cell Signaling Cat#65880; RRID:AB_2799696 Rabbit polyclonal anti-phosphoeIF2α Ser51 Cell Signaling Cat#9721; RRID:AB_330951 Mouse monoclonal anti- eIF2α Cell Signaling Cat#2103; RRID:AB_836874 Rabbit monoclonal anti-ATF4 Cell Signaling Cat#11815; RRID:AB_2616025 Rabbit polyclonal anti-Candida albicans-FITC LSBio Cat#LS-C103355 Rat monoclonal anti-LAMP1 DSHB Cat#1D4B; RRID:AB_2134500 Mouse monoclonal anti-EEA1 ThermoFisher Cat#MA5-31575 Rabbit monoclonal anti-KDEL Abcam Cat#ab176333; RRID:AB_2819147 Rat monoclonal anti-CD11b, BV421-conjugated Biolegend Cat#414-0112-82 Bacterial and virus strains MRSA-mCherry Methicillin-resistant Staphyloccocus aureus USA300 LAC harboring pSarA-mCherry plasmid Boles et al. 200873 N/A Chemicals, peptides, and recombinant proteins Thioflavin T Cayman Chemical Cat#32553; CAS Number 2390-54-7 TRIzol reagent Thermo Fisher Cat#15596018 Turbo DNase I Thermo Fisher Cat#AM2238 Paraformaldehyde Electron Microscopy Services Cat#15700 Calcofluor white Sigma-Aldrich Cat#18909 Bovine serum albumin Thermo Fisher Cat#BP9706-100 Sulforhodamine B Sigma-Aldrich Cat#230162; CAS Number 3520-42-1 Critical commercial assays Lookout Mycoplasma PCR Detection Kit Sigma-Aldrich Cat#MP0035 iScript cDNA synthesis kit BioRad Cat# 1708891 PowerUP SYBR Green qPCR master mix Thermo Fisher Cat# A25742 Fluo-4 Calcium Imaging Kit Invitrogen Cat#F10489 LysoSensor Yellow/Blue DND-160 Thermo Fisher Cat#L7545 LysoTracker Deep Red Invitrogen Cat#L12492 Duolink In Situ PLA Kit Sigma-Aldrich Cat#DUO92008 Cat#DUO92002 Cat#DUO92004 Fluorospheres Thermo Fisher Cat#F8801 (Continued on next page) Cell Reports 44, 115694, May 27, 2025 15

Techniques: Infection, Expressing, Fluorescence, Control

Journal: Cell reports

Article Title: IRE1α promotes phagosomal calcium flux to enhance macrophage fungicidal activity.

doi: 10.1016/j.celrep.2025.115694

Figure Lengend Snippet: Figure 5. IRE1α promotes phagosomal calcium flux and contact sites between the ER and endosomal network (A) Representative micrographs of WT or IRE1ΔR iBMDM following phagocytosis of C. albicans (20 min post-infection; MOI 2) showing early cellular calcium flux, and influx of calcium specifically in the phagosome following phagocytosis of C. albicans. Scale bar, 10 μm. (B) Quantification of calcium-high phagosomes, defined by a 1.25-fold increase of the mean fluorescence intensity of the cell (20 min post-infection; MOI 2). (C) Violin plot of the ratio of phagosomal to cytosolic mean fluorescence intensity of Fluo4 (20 min post-infection; MOI 2). (D) Quantification of LAMP1 recruitment to phagosomes containing C. albicans in IRE1 WT or IRE1ΔR iBMDM with or without treatment of BAPTA-AM (calcium chelator), as measured by LAMP1 mean fluorescence intensity associated with C. albicans-expressed iRFP. (E) Representative confocal micrographs of PLA puncta representing sites of proximity between the ER and early endosomes/phagosomes at resting state (mock) or during C. albicans infection. Scale bar, 10 μm. (F) Quantification of PLA puncta in IRE1 WT or IRE1ΔR iBMDM at resting state (mock). Values are the mean ± SEM from 3 to 4 biological replicates, as indicated by data points. Statistical analysis details are in STAR Methods.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal anti-IRE1α Cell Signaling Cat#3924; RRID:AB_823545 Rabbit polyclonal anti-XBP1 Abcam Cat#ab37152; RRID:AB_778939 Rabbit polyclonal anti-CARD9 Cell Signaling Cat#12283; RRID:AB_2797869 Mouse monoclonal anti-ACTIN ThermoFisher Cat#MA5-11869; RRID:AB_11004139 Rabbit monoclonal anti-TRAF6 Abcam Cat#ab40675; RRID:AB_778573 Rabbit polyclonal anti-K48-linkage specific polyubiquitin Cell Signaling Cat#4289; RRID:AB_10557239 Rabbit monoclonal anti-ATF6 Cell Signaling Cat#65880; RRID:AB_2799696 Rabbit polyclonal anti-phosphoeIF2α Ser51 Cell Signaling Cat#9721; RRID:AB_330951 Mouse monoclonal anti- eIF2α Cell Signaling Cat#2103; RRID:AB_836874 Rabbit monoclonal anti-ATF4 Cell Signaling Cat#11815; RRID:AB_2616025 Rabbit polyclonal anti-Candida albicans-FITC LSBio Cat#LS-C103355 Rat monoclonal anti-LAMP1 DSHB Cat#1D4B; RRID:AB_2134500 Mouse monoclonal anti-EEA1 ThermoFisher Cat#MA5-31575 Rabbit monoclonal anti-KDEL Abcam Cat#ab176333; RRID:AB_2819147 Rat monoclonal anti-CD11b, BV421-conjugated Biolegend Cat#414-0112-82 Bacterial and virus strains MRSA-mCherry Methicillin-resistant Staphyloccocus aureus USA300 LAC harboring pSarA-mCherry plasmid Boles et al. 200873 N/A Chemicals, peptides, and recombinant proteins Thioflavin T Cayman Chemical Cat#32553; CAS Number 2390-54-7 TRIzol reagent Thermo Fisher Cat#15596018 Turbo DNase I Thermo Fisher Cat#AM2238 Paraformaldehyde Electron Microscopy Services Cat#15700 Calcofluor white Sigma-Aldrich Cat#18909 Bovine serum albumin Thermo Fisher Cat#BP9706-100 Sulforhodamine B Sigma-Aldrich Cat#230162; CAS Number 3520-42-1 Critical commercial assays Lookout Mycoplasma PCR Detection Kit Sigma-Aldrich Cat#MP0035 iScript cDNA synthesis kit BioRad Cat# 1708891 PowerUP SYBR Green qPCR master mix Thermo Fisher Cat# A25742 Fluo-4 Calcium Imaging Kit Invitrogen Cat#F10489 LysoSensor Yellow/Blue DND-160 Thermo Fisher Cat#L7545 LysoTracker Deep Red Invitrogen Cat#L12492 Duolink In Situ PLA Kit Sigma-Aldrich Cat#DUO92008 Cat#DUO92002 Cat#DUO92004 Fluorospheres Thermo Fisher Cat#F8801 (Continued on next page) Cell Reports 44, 115694, May 27, 2025 15

Techniques: Infection, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: HIF1α-dependent hypoxia response in myeloid cells requires IRE1α

doi: 10.1186/s12974-023-02793-y

Figure Lengend Snippet: HIF1α and IRE1α interact during myeloid cell response to hypoxia. A Schematic representation of the OIR mouse model. B Heat map of gene set variation analysis (GSVA) enrichment scores of RNA-seq data from OIR and normoxic retinas at P14 and C P17. Pathways associated with hypoxia response are enriched at P14 when the retina is still avascular, and pathways involved in hypoxia, inflammatory responses and angiogenesis are significantly upregulated at P17 when there is maximal preretinal neovascularization; n = 2–3 mice per condition. For P14, P < 0.05 and > 0.2 logFC and for P17, p adj < 0.05 and > 0.2 logFC. D Immunoblot showing HIF1α stabilization in mononuclear phagocytes (CD45 low , Gr1 − , CD11b + , F4/80 + ) cell-sorted from normoxic and OIR retinas at P14. E STRING database representation of the protein interaction network of HIF1α immunoprecipitated from J774 macrophages under hypoxia (2% O 2 for 8 h) and subjected to tandem mass spectrometry (MS/MS). Proteins including the unfolded protein response (UPR) such as GRP78 are highlighted in blue, and the interaction score ranked from 0 to 1 is noted below. F Co-immunoprecipitation of HIF1α in J774 macrophages under normoxia (21% O 2 ) and hypoxia (2% O 2 ) for 1 h followed by immunoblotting (IB) for UPR sensors IRE1α, PERK and ATF6 ( n = 3 independent experiments)

Article Snippet: Anti-total IRE1α , Cell Signaling , Cat# 14C10.

Techniques: RNA Sequencing, Western Blot, Immunoprecipitation, Mass Spectrometry, Tandem Mass Spectroscopy

Journal: Journal of Neuroinflammation

Article Title: HIF1α-dependent hypoxia response in myeloid cells requires IRE1α

doi: 10.1186/s12974-023-02793-y

Figure Lengend Snippet: IRE1α kinase activity is required for HIF1α stabilization in macrophage response to hypoxia. A Immunoblot timecourse from J774 macrophage cell lysates under hypoxia probed for HIF1α stabilization, IRE1α phosphorylation and expression. ( n = 3 independent experiments). B Co-immunoprecipitation of HIF1α and IRE1α in hypoxic (2% O 2 for 1 h) J774 macrophages preincubated for 1 h with IRE1α endoribonuclease inhibitor 4µ8c (100µM) or IRE1α kinase inhibitor KIRA6 (1µM) ( n = 3 independent experiments). Red box highlights Co-IP results upon KIRA6 treatment. C Immunoblots for HIF1α stabilization in cytosolic and nuclear fractions of hypoxic (2% O 2 for 1 h) J774 cells pretreated with IRE1α kinase inhibitor KIRA6 (1µM) for 1 h. LDH was used to assess the purity of the cytosolic fraction ( n = 3 independent experiments). D RT - qPCR analysis of Hif1a mRNA expression in hypoxic (2% O 2 for 8 h) J774 cells preincubated for 1 h with IRE1α kinase inhibitor KIRA6 (1µM). n = 3–8 per condition, unpaired two-tailed t-test. E RT-qPCR analysis of Hif1a and F Ern1 mRNA expression in LysM- Hif1a −/− or LysM- Ern1 −/− peritoneal macrophages and their control LysM-cre /HIF1a + / + /Ern1 + / + mice under normoxic (21% O 2 ) or hypoxic (2% O 2 for 8 h) conditions. n = 3–12 per condition. Data expressed as mean ± S.E.M. Statistical analysis ( D , F , G ): one-way ANOVA with Bonferroni post hoc analysis; * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Anti-total IRE1α , Cell Signaling , Cat# 14C10.

Techniques: Activity Assay, Western Blot, Phospho-proteomics, Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Two Tailed Test, Control

Journal: Journal of Neuroinflammation

Article Title: HIF1α-dependent hypoxia response in myeloid cells requires IRE1α

doi: 10.1186/s12974-023-02793-y

Figure Lengend Snippet: IRE1α/XBP1 and HIF1α crosstalk regulates the myeloid inflammatory response secondary to a hypoxic stimulus. A Co-immunoprecipitation of XBP1 and immunoblot for HIF1α in cytosolic and B nuclear fractions of hypoxic (2% O 2 for 1 h) J774 cells ( n = 2 independent experiments). C , D Schematic representation and RT-qPCR analysis of Il6, Il1b , and Vegfa mRNA expression in hypoxic (2% O 2 for 8 h) J774 cells preincubated for 1 h with IRE1α endoribonuclease inhibitor 4µ8c (100µM) or IRE1α kinase inhibitor KIRA6 (1µM) ( n = 3–8 per condition). E HIF1α or mock (IgG) ChIP-qPCR at Il6, Il1b , and Vegfa loci in hypoxic (2% O 2 for 8 h) J774 macrophages preincubated for 1 h with IRE1α endoribonuclease inhibitor 4µ8c (100µM) or IRE1α kinase inhibitor KIRA6 (1µM) ( n = 3 independent experiments). Percent of input represents the signals obtained from the HIF1α ChIP over signals from respective input samples. Data expressed as mean ± S.E.M. Statistical analysis ( D , E ): one-way ANOVA with Bonferroni post hoc analysis; * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Anti-total IRE1α , Cell Signaling , Cat# 14C10.

Techniques: Immunoprecipitation, Western Blot, Quantitative RT-PCR, Expressing, ChIP-qPCR

Journal: Journal of Neuroinflammation

Article Title: HIF1α-dependent hypoxia response in myeloid cells requires IRE1α

doi: 10.1186/s12974-023-02793-y

Figure Lengend Snippet: Myeloid-resident HIF1α and IRE1α influence sterile inflammation. A RT-qPCR analysis of Vegfa , Tnf , Il1b , and Il6 mRNA expression in retinas from LysM- Hif1a −/− and LysM- Ern1 −/− mice and their control LysM-cre /Hif1a + / + /Ern1 + / + mice conditions at P14 and B P17 under normoxia and OIR conditions. n = 5–8 retinas per condition. Results are shown as a fold change relative to respective normoxia control for each time point ± S.E.M. C–H LysM- Hif1a −/− , LysM- Ern1 −/− , LysM-cre /Hif1a fl/f /Ern1 fl/fl mice and their control LysM-cre /Hif1a + / + /Ern1 + / + mice were subjected to OIR, and retinas were collected at P14 and P17, flat-mounted, and stained with isolectin B4. C , D Representative photomicrographs of isolectin B4-stained LysM- Hif1a −/− , LysM- Ern1 −/− , LysM-cre /Hif1a fl/f /Ern1 fl/fl and LysM-cre /Hif1a + / + /Ern1 + / + mice at P14 with highlighted avascular hypoxic regions, and their quantification. E , F Representative photomicrographs of isolectin B4-stained LysM- Hif1a −/− , LysM- Ern1 −/− , LysM-cre /Hif1a fl/f /Ern1 fl/fl and LysM-cre /Hif1a + / + /Ern1 + / + mice at P17 with highlighted pathological neovascularization, and their quantification. G , H Representative photomicrographs of isolectin B4-stained LysM- Hif1a −/− , LysM- Ern1 −/− , LysM-cre /Hif1a fl/f /Ern1 fl/fl and LysM-cre /Hif1a + / + /Ern1 + / + mice at P17 with highlighted avascular hypoxic regions, and their quantifications. n = 5–13 retinas per group ( C – H ). Scale bars: 1 µm (for the whole flat mount of retina) and µm (for one petal of retina flat mount). Data expressed as mean ± S.E.M. Statistical analysis ( A , B , D , F , H ): one-way ANOVA with Bonferroni post hoc analysis; * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Anti-total IRE1α , Cell Signaling , Cat# 14C10.

Techniques: Sterility, Quantitative RT-PCR, Expressing, Control, Staining

Journal: Journal of Neuroinflammation

Article Title: HIF1α-dependent hypoxia response in myeloid cells requires IRE1α

doi: 10.1186/s12974-023-02793-y

Figure Lengend Snippet:

Article Snippet: Anti-total IRE1α , Cell Signaling , Cat# 14C10.

Techniques: Purification, Control, Plasmid Preparation, Amplification, Western Blot, Magnetic Beads, Software

Journal: Frontiers in Pharmacology

Article Title: A Nudibranch Marine Extract Selectively Chemosensitizes Colorectal Cancer Cells by Inducing ROS-Mediated Endoplasmic Reticulum Stress

doi: 10.3389/fphar.2021.625946

Figure Lengend Snippet: Relationship among ER stress, cell cycle modulation, and caspase-dependent apoptotic cell death in colon cancer cells treated with Dolabella auricularia extract (NB). (A) Effect of increasing concentrations of NB extract on the cell cycle of HCT-116 and CCD-18Co cells after 24 h. HCT-116 cells were pretreated with a pan-caspase inhibitor, zVAD (20 μM), for 2 h prior to the addition of 10 μg/ml NB extract for 24 h to study the effect of NB extract on apoptosis. (B) Measurement of apoptotic cells (% Annexin V positive cells), (C) caspases 3/7 and 8, and (D) the G2/M phase. Data are presented as the mean ± SD of three independent experiments. p -values were calculated by comparison with the untreated cell line (*) and NB condition ( # ) using ANOVA. * or # p -value < 0.05, ** or ## p -value < 0.01, *** or ### p -value < 0.001, and ****or #### p -value < 0.0001. The effects of NB extract on the modulation of ER stress–related proteins and apoptosis were analyzed by western blot analysis. The effect of NB extract on ER stress–related proteins (E) and its relationship to caspase activation (F) . Caspases 8 and 9 in HCT-116 were silenced with siRNA, and cells were treated with 10 μg/ml NB extract for 24 h. ER stress–related proteins (PARP, phospho-IRE1α, total-IRE1α, phospho-JNK, total-JNK, ATF4, CHOP, and caspases 8 and 9) were analyzed by western blot analysis. β-Actin was used as a loading control in the western blot assay. Densitometry analyses of E and F are shown in and , respectively.

Article Snippet: The antibodies PARP Rabbit mAb Antibody #9532, ATF4 mAb Rabbit (D4B8) #1815, ATF-6 (D4Z8V) mAb Rabbit #65880, total-IRE1α (14C10) mAb Rabbit #3294, Phospho-JNK (Thr183/Tyr185) mAb Mouse antibody #9255, SAPK/JNK Antibody (total-JNK) pAb Rabbit #9252, CHOP (L63F7) mAb Mouse antibody #2895, BiP or GRP78 (C50B12) mAb Rabbit #3177, XBP-1s (D2C1F) mAb Rabbit #12782, Phospho-eIF2α (Ser51) (D9G8) mAb Rabbit #3398, total-eIF2α (D7D3) mAb Rabbit #5324, Caspase-3 Antibody #9662, and Caspase-9 #9502 were obtained from Cell Signaling Technology Inc., Beverly, MA, USA.

Techniques: Comparison, Western Blot, Activation Assay, Control

Journal: Frontiers in Pharmacology

Article Title: A Nudibranch Marine Extract Selectively Chemosensitizes Colorectal Cancer Cells by Inducing ROS-Mediated Endoplasmic Reticulum Stress

doi: 10.3389/fphar.2021.625946

Figure Lengend Snippet: Relationships between ROS and ER stress induced by NB extract in the colon cancer HCT-116 cell model. Cells were pretreated with the ROS scavenger N-acetyl-cysteine (NAC, 5 mM) and ER stress inhibitor 4-phenylbutric acid (4-PBA, 5 mM) for 2 h prior to 10 μg/ml NB extract treatment. NB extract modulation of ER stress–related proteins (PARP, phospho-IRE1α, total-IRE1α, phospho-JNK, total-JNK, ATF4, and CHOP) was analyzed by western blot analysis (A) . β-Actin was used as a loading control. The experiment was repeated twice. Apoptosis (B) , G2/M phase (C), and phosphorylation of H2A.γ (as DNA damage indicator) (E) were measured using a Muse ® cell analyzer according to the manufacturer’s instructions. ROS levels were measured using the 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCF-DA) fluorescence label (D) . Data are presented as the mean ± SD of three independent experiments. p -values were calculated by comparison with the control (untreated cells plus less than 0.2% dimethyl sulfoxide, C) ( * ) and NB condition ( # ) using ANOVA. * or # p -value < 0.05, ** or ## p -value < 0.01, *** or ### p -value < 0.001, and **** or #### p -value < 0.0001.

Article Snippet: The antibodies PARP Rabbit mAb Antibody #9532, ATF4 mAb Rabbit (D4B8) #1815, ATF-6 (D4Z8V) mAb Rabbit #65880, total-IRE1α (14C10) mAb Rabbit #3294, Phospho-JNK (Thr183/Tyr185) mAb Mouse antibody #9255, SAPK/JNK Antibody (total-JNK) pAb Rabbit #9252, CHOP (L63F7) mAb Mouse antibody #2895, BiP or GRP78 (C50B12) mAb Rabbit #3177, XBP-1s (D2C1F) mAb Rabbit #12782, Phospho-eIF2α (Ser51) (D9G8) mAb Rabbit #3398, total-eIF2α (D7D3) mAb Rabbit #5324, Caspase-3 Antibody #9662, and Caspase-9 #9502 were obtained from Cell Signaling Technology Inc., Beverly, MA, USA.

Techniques: Western Blot, Control, Phospho-proteomics, Fluorescence, Comparison