Journal: Cell Reports

Article Title: ALKBH5 modulates hematopoietic stem and progenitor cell energy metabolism through m 6 A modification-mediated RNA stability control

doi: 10.1016/j.celrep.2023.113163

Figure Lengend Snippet:

Article Snippet: GAPDH (14C10) Rabbit mAb , Cell Signaling Technology , Cat# 2118; RRID: AB_561053.

Techniques: Purification, Blocking Assay, Recombinant, Transfection, cDNA Synthesis, SYBR Green Assay, Protease Inhibitor, Membrane, Staining, Magnetic Beads, Methylation, Isolation, Mutagenesis, Colorimetric Assay, Malate Assay, Plasmid Preparation, Software

Journal: International Journal of Molecular Sciences

Article Title: Endoplasmic Reticulum Homeostasis Regulates TLR4 Expression and Signaling in Mast Cells

doi: 10.3390/ijms231911826

Figure Lengend Snippet: Activation of CBMCs promotes expression of IRE1α and PERK. CBMCs were sensitized by culturing them with human myeloma IgE (0.3 μg/mL). Three days later, cells were activated by rabbit anti-human IgE Ab (5 μg/mL, 4 h). ( A ) Equal amounts of whole cell extracts were analyzed by immunoblotting for phosphorylated and total IRE1α, PERK, phosphorylated and total JNK, and p97 as a loading control. A representative image of three independent experiments is shown. RT-qPCR analyses of ( B ) IRE1α, ( C ) sXBP1, and ( D ) ERdj4 following the indicated treatments, represented as the mean of three independent experiments normalized to β-actin ± SD. * p < 0.05. ( E ) IgE-sensitized CBMCs were pretreated with 3 µM JNK-IN-8 for 1 h, followed by activation with rabbit anti-human IgE Ab (5 μg/mL, 4 h). Equal amounts of whole cell extracts were analyzed by immunoblotting for phosphorylated and total IRE1α, PERK, and p97 as a loading control. ( F ) RT-qPCR analysis of sXBP1 following the indicated treatments, represented as the mean of relative mRNA levels normalized to β-actin ± SD, * p < 0.05. ( G ) CBMCs were sensitized with human myeloma IgE (0.3 μg/mL, 3 days), followed by activation with rabbit anti-human IgE Ab (5 μg/mL) for 1, 3, and 6 h. Equal amounts of whole cell extracts were analyzed by immunoblotting for PERK, total IRE1, ERO1, PDI, and p97 as a loading control. A representative image of three independent experiments is shown. ( H ) Fluorescence of ER-Tracker (green marker) in CBMCs following IgE-mediated activation for 4 h, analyzed by confocal microscopy (60× with immersion oil). The fluorescence signal from 50 cells was quantified. Data are expressed as mean intensity ±SD. * p < 0.05.

Article Snippet: The following primary antibodies were used: anti-phospho-IRE1α rabbit mAb [EPR5253] (phospho S724) (ab124945), purchased from Abcam (Cambridge, UK), as well as anti-IRE1α (14C10) rabbit mAb (ab3294), phospho-JNK (Thr183/Tyr185) mAb mouse antibody (ab9255), and SAPK/JNK antibody (total-JNK) pAb rabbit (ab9252), and β-actin pAb rabbit (4967), purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Expressing, Western Blot, Control, Quantitative RT-PCR, Fluorescence, Marker, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Endoplasmic Reticulum Homeostasis Regulates TLR4 Expression and Signaling in Mast Cells

doi: 10.3390/ijms231911826

Figure Lengend Snippet: Inhibition of IRE1 nuclease activity decreases the secretory response to IgE-mediated CBMC activation. Sensitized CBMCs were pretreated with DMSO or STF (30 or 60 μM) 1 h prior to activation with rabbit anti-human IgE Ab (5 μg/mL). Thirty minutes after activation, ( A ) supernatants and cell pellets underwent chromogenic assay for β-hex quantification. Data are shown as mean ± SD. * p < 0.05. ( B ) Supernatants were collected following 4 h of activation and analyzed for IL-6 by ELISA. Each bar represents the average of three independent measurements ± SD. * p < 0.05. ( C ) RT-qPCR analysis of IL-6, and ( D ) TNF following 4 h of activation. The average of mRNA levels normalized to β-actin ±SD of three independent experiments is shown. * p < 0.05.

Article Snippet: The following primary antibodies were used: anti-phospho-IRE1α rabbit mAb [EPR5253] (phospho S724) (ab124945), purchased from Abcam (Cambridge, UK), as well as anti-IRE1α (14C10) rabbit mAb (ab3294), phospho-JNK (Thr183/Tyr185) mAb mouse antibody (ab9255), and SAPK/JNK antibody (total-JNK) pAb rabbit (ab9252), and β-actin pAb rabbit (4967), purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Activity Assay, Activation Assay, Chromogenic Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Endoplasmic Reticulum Homeostasis Regulates TLR4 Expression and Signaling in Mast Cells

doi: 10.3390/ijms231911826

Figure Lengend Snippet: PMC activation through FcεRI promotes expression of IRE1α without UPR activation. ( A ) WT PMCs were sensitized with mouse IgE-anti-DNP (0.5 µg/mL, 18 h) and then activated with DNP-BSA (50 ng/mL, 4 h). Equal amounts of whole cell extracts were analyzed by immunoblotting for total IRE1α, phosphorylated and total JNK, and p97 as a loading control. A typical immunoblot is shown. ( B ) WT PMCs were treated for 4 h as mentioned in ( A ) or treated with Tg (1 µg/mL) as a positive control. Equal whole cellular protein extracts were analyzed by immunoblotting for phosphorylated and total IRE1α, and p97 as loading control. Shown is a representative image of three independent experiments. ( C ) RT-qPCR analyses of IRE1α, ( D ) sXBP1, and ( E ) ERdj4 following 4 h of activation represented as the average of the mRNA levels normalized to GAPDH ± SD. ( F ) PMCs were sensitized with rat myeloma IgE (1 µg/mL, 18 h) followed by activation with goat anti-rat IgG F(ab’) 2 Ab (1 µg/mL, 4 h). Identical quantity of total cellular protein extracts was analyzed by immunoblotting against total IRE1α and p97 as a loading control. ( G ) IgE sensitized WT PMCs with IgE-anti-DNP (0.5 µg/mL, 18 h) were pretreated with 3 µM JNK-IN-8 for 1 h followed by activation with DNP-BSA (50 ng/mL, 4 h). An identical quantity of total cellular protein extracts was analyzed by immunoblotting against total IRE1α, PERK, and p97 as a loading control. ( H ) WT PMCs were sensitized with mouse IgE–anti-DNP (0.5 µg/mL, 18 h), followed by activation with DNP-BSA (50 ng/mL) for 1, 3, 6, or 24 h. An identical quantity of protein was analyzed by immunoblotting against PERK, total IRE1α, and p97 as a loading control. ( I ) WT PMCs were sensitized with mouse IgE–anti-DNP (0.5 µg/mL, 18 h), followed by activation with DNP-BSA (50 ng/mL) for 1, 3, and 6 h. Equal amounts of whole cell extracts were analyzed by immunoblotting for PERK, total IRE1α, ERO1, PDI, and p97 as a loading control. ( J ) Fluorescence of ER-Tracker in PMCs following IgE-mediated activation for 4 h, analyzed by confocal microscopy at magnification of 60× with immersion oil. Mean fluorescence signal from 50 cells was quantified using ImageJ 1.53t software (U. S. National Institutes of Health, Bethesda, MD, USA). The average intensity and standard deviation are shown. * p < 0.05.

Article Snippet: The following primary antibodies were used: anti-phospho-IRE1α rabbit mAb [EPR5253] (phospho S724) (ab124945), purchased from Abcam (Cambridge, UK), as well as anti-IRE1α (14C10) rabbit mAb (ab3294), phospho-JNK (Thr183/Tyr185) mAb mouse antibody (ab9255), and SAPK/JNK antibody (total-JNK) pAb rabbit (ab9252), and β-actin pAb rabbit (4967), purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Expressing, Western Blot, Control, Positive Control, Quantitative RT-PCR, Fluorescence, Confocal Microscopy, Software, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Endoplasmic Reticulum Homeostasis Regulates TLR4 Expression and Signaling in Mast Cells

doi: 10.3390/ijms231911826

Figure Lengend Snippet: The absence of IRE1α does not affect PMC secretory response to IgE-mediated activation. ( A ) PMCs expanded from the peritoneal lavage of the WT, XBP1 KO, and DKO strain mice were sorted for YFP expression and visualized by TEM. Typical cells from 2 independent separations are shown. Red arrows show the perinuclear ER, which is not discerned in the DKO PMCs. Bars: 500 nm (upper panel) and 2 µm (lower panel). ( B ) PMCs were sensitized with mouse IgE-anti-DNP (0.5 µg/mL, 18 h), followed by activation with DNP-BSA (50 ng/mL, 1 h). Following activation, supernatants and cell pellets underwent a chromogenic assay for β-hex quantification. Data are shown as the mean ± SD of three independent experiments. Supernatants were analyzed for secreted cytokines as a response to IgE-mediated activation of 4 h in YFP-sorted XBP1 KO PMCs and DKO PMCs, as compared to WT PMCs. The amounts of ( C ) IL-6 and ( D ) TNF were determined by ELISA. Each bar represents the average ± SD of three independent experiments. * p < 0.05.

Article Snippet: The following primary antibodies were used: anti-phospho-IRE1α rabbit mAb [EPR5253] (phospho S724) (ab124945), purchased from Abcam (Cambridge, UK), as well as anti-IRE1α (14C10) rabbit mAb (ab3294), phospho-JNK (Thr183/Tyr185) mAb mouse antibody (ab9255), and SAPK/JNK antibody (total-JNK) pAb rabbit (ab9252), and β-actin pAb rabbit (4967), purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Expressing, Chromogenic Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Endoplasmic Reticulum Homeostasis Regulates TLR4 Expression and Signaling in Mast Cells

doi: 10.3390/ijms231911826

Figure Lengend Snippet: TLR4 surface expression and function is supported by the IRE1/XBP1 pathway. TNF and IL-6 levels in the supernatants in response to LPS stimulation (1 μg/mL, 4 h) were measured by ELISA for an equal number of YFP-sorted XBP1 KO and DKO PMCs, as compared to WT PMCs. The average of three independent experiments ± SD is shown for ( A ) IL-6 and ( B ) TNF. * p < 0.05. ( C ) Flow cytometry analysis of TLR4 cell surface expression of XBP1 KO (green histogram) and DKO PMCs (red histogram), as compared to WT PMCs (blue histogram). ( D ) Quantification of TLR4 expression levels in WT, XBP1 KO, and DKO PMCs. Data are presented as the average ± SD of the mean fluorescence intensity minus the background (ΔMFI) of three independent analyses. * p < 0.05. ( E ) Flow cytometry analysis of TLR2 expression in WT, XBP1 KO, and DKO PMCs (blue, green, and red histograms, respectively). ( F ) RT-qPCR analysis of sXBP1 in PMCs treated with DMSO or 60 μM STF, 1 h prior to stimulation with LPS (1 μg/mL) for 3, 6, and 12 h. Bars are represented as the mean of the relative mRNA levels normalized to GAPDH ± SD of three independent experiments. Cytokines levels in the supernatants for ( G ) IL-6 and ( H ) TNF were determined by ELISA. Each bar represents the mean ± SD of three independent experiments. * p < 0.05.

Article Snippet: The following primary antibodies were used: anti-phospho-IRE1α rabbit mAb [EPR5253] (phospho S724) (ab124945), purchased from Abcam (Cambridge, UK), as well as anti-IRE1α (14C10) rabbit mAb (ab3294), phospho-JNK (Thr183/Tyr185) mAb mouse antibody (ab9255), and SAPK/JNK antibody (total-JNK) pAb rabbit (ab9252), and β-actin pAb rabbit (4967), purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Endoplasmic Reticulum Homeostasis Regulates TLR4 Expression and Signaling in Mast Cells

doi: 10.3390/ijms231911826

Figure Lengend Snippet: IRE1 activation supports cooperativity between TLR4 and FcεRI in CBMCs. ( A ) IgE-sensitized CBMCs were pretreated with DMSO or STF (30 or 60 μM) for 1 h followed by activation with rabbit anti-human IgE Ab (5 μg/mL, 4 h). Equal amounts of whole cell protein extracts were analyzed by immunoblotting for TLR4 and p97 as a loading control. RT-qPCR analysis of sensitized CBMCs after activation with rabbit anti-human IgE Ab (5 μg/mL), stimulation with 100 ng/mL LPS, or both treatments for 4 h, for ( B ) sXBP1, ( C ) TNF, and ( D ) IL-6. Bars represent the mean of relative mRNA levels normalized to β-actin ± SD of three independent experiments. * p < 0.05. ( E ) IgE-sensitized CBMCs were pretreated with DMSO or STF (60 μM) for 1 h followed by activation with rabbit anti-human IgE Ab (5 μg/mL), 100 ng/mL LPS, or both for 4 h. IL-6 levels in the supernatants were quantified by ELISA. Bars represent the mean ± SD of three independent experiments. * p < 0.05. PMCs were sensitized overnight with mouse IgE-anti-DNP (0.5 µg/mL, 18 h). Later, cells were stimulated with 100 ng/mL LPS, activated with DNP-BSA (25 or 50 ng/mL), or stimulated with both for 4 h. Levels of IL-6 ( F ) and TNF ( G ) in supernatants were determined by ELISA. Each bar represents the mean ± SD of three independent experiments. * p < 0.05.

Article Snippet: The following primary antibodies were used: anti-phospho-IRE1α rabbit mAb [EPR5253] (phospho S724) (ab124945), purchased from Abcam (Cambridge, UK), as well as anti-IRE1α (14C10) rabbit mAb (ab3294), phospho-JNK (Thr183/Tyr185) mAb mouse antibody (ab9255), and SAPK/JNK antibody (total-JNK) pAb rabbit (ab9252), and β-actin pAb rabbit (4967), purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Western Blot, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Endoplasmic Reticulum Homeostasis Regulates TLR4 Expression and Signaling in Mast Cells

doi: 10.3390/ijms231911826

Figure Lengend Snippet: List of human primer sequences for RT-qPCR.

Article Snippet: The following primary antibodies were used: anti-phospho-IRE1α rabbit mAb [EPR5253] (phospho S724) (ab124945), purchased from Abcam (Cambridge, UK), as well as anti-IRE1α (14C10) rabbit mAb (ab3294), phospho-JNK (Thr183/Tyr185) mAb mouse antibody (ab9255), and SAPK/JNK antibody (total-JNK) pAb rabbit (ab9252), and β-actin pAb rabbit (4967), purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Endoplasmic Reticulum Homeostasis Regulates TLR4 Expression and Signaling in Mast Cells

doi: 10.3390/ijms231911826

Figure Lengend Snippet: List of murine primer sequences for RT-qPCR.

Article Snippet: The following primary antibodies were used: anti-phospho-IRE1α rabbit mAb [EPR5253] (phospho S724) (ab124945), purchased from Abcam (Cambridge, UK), as well as anti-IRE1α (14C10) rabbit mAb (ab3294), phospho-JNK (Thr183/Tyr185) mAb mouse antibody (ab9255), and SAPK/JNK antibody (total-JNK) pAb rabbit (ab9252), and β-actin pAb rabbit (4967), purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: