DOI: 10.5061/dryad.d2547d8c6
title: Data from: The role of BiP and the IRE1α-XBP1 axis in rhabdomyosarcoma pathology

description: Background: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma
 in children, and is associated with a poor prognosis in patients
 presenting with recurrent or metastatic disease. The unfolded protein
 response (UPR) plays pivotal roles in tumor development and resistance to
 therapy, including RMS. Methods: In this study, we used
 immunohistochemistry and a tissue microarray (TMA) on human RMS and normal
 skeletal muscle to evaluate the expression of key UPR proteins (GRP78/BiP,
 IRE1α and cytosolic/nuclear XBP1 (spliced XBP1-sXBP1)) in the four main
 RMS subtypes: alveolar (ARMS), embryonal (ERMS), pleomorphic (PRMS) and
 sclerosing/spindle cell (SRMS) RMS. We also investigated the correlation
 of these proteins with the risk of RMS and several clinicopathological
 indices, such as lymph node involvement, distant metastasis, tumor stage
 and tumor scores. Results: Our results revealed that the expression of
 BiP, sXBP1, and IRE1α, but not cytosolic XBP1, are significantly
 associated with RMS (BiP and sXBP1 p-value = 0.0001, IRE1 p-value = 0.001)
 in all of the studied types of RMS tumors (n = 192) compared to normal
 skeletal muscle tissues (n = 16). In addition, significant correlations of
 BiP with the lymph node score (p = 0.05), and of IRE1α (p value = 0.004),
 cytosolic XBP1 (p = 0.001) and sXBP1 (p value = 0.001) with the stage
 score were observed. At the subtype level, BiP and sXBP1 expression were
 significantly associated with all subtypes of RMS, whereas IRE1α was
 associated with ARMS, PRMS and ERMS, and cytosolic XBP1 expression was
 associated with ARMS and SRMS. Importantly, the expression levels of IRE1α
 and sXBP1 were more pronounced in ARMS than in any of the other subtypes.
 The results also showed correlations of BiP with the lymph node score in
 ARMS (p value = 0.05), and of sXBP1 with the tumor score in PRMS (p value
 = 0.002). Conclusions: In summary, this study demonstrates that the
 overall UPR is upregulated and, more specifically, that the IRE1/sXBP1
 axis is active in RMS. The subtype and stage-specific dependency on the
 UPR machinery in RMS may open new avenues for the development of novel
 targeted therapeutic strategies and the identification of specific tumor
 markers in this rare but deadly childhood and young-adult disease.
description: <strong>2.1. Tissue Microarray
 (TMA)</strong> The tissue microarray (TMA)
 analysis was conducted on rhabdomyosarcoma (RMS) and normal (control)
 striated muscle samples. A rhabdomyosarcoma tissue array kit containing
 104 cases and 208 cores was utilized, purchased from US Biomax, Inc.
 (Catalog no. SO2082a; RKV, MD, USA). This array provides detailed
 information on tumor characteristics, including TNM (tumor, node,
 metastasis) staging, clinical stages, and pathology grades, ensuring
 comprehensive clinical and pathological data for each sample.
 The TMA includes cores from four subtypes of RMS:
 <strong>15 cases of spindle
 cell/sclerosing RMS (SRMS)</strong> 
 <strong>27 cases of embryonal RMS
 (ERMS)</strong> 
 <strong>30 cases of pleomorphic RMS
 (PRMS)</strong> 
 <strong>24 cases of alveolar RMS
 (ARMS)</strong> Additionally, it includes
 <strong>8 normal skeletal muscle tissue cores</strong> as
 controls, with all samples processed in duplicate. Each of the 208 tissue
 cores is embedded on a single slide, with two cores derived from each case
 or control. Detailed histopathological characteristics for all samples are
 presented in Supplementary Table S1. 
 <strong>2.2. Immunohistochemistry
 (IHC)</strong> TMA slides (5-μm thick) were
 subjected to immunohistochemistry (IHC) analysis to evaluate protein
 expression profiles. Slides were first deparaffinized by incubation at
 60°C for 30 minutes and subsequently rehydrated using xylene and a graded
 alcohol series. Heat-mediated antigen retrieval was performed using a 0.01
 M sodium citrate buffer (pH 6.0), following previously described
 protocols. To minimize non-specific background
 staining, slides were washed with phosphate-buffered saline (PBS) and
 blocked with a blocking solution (composition: 1.5 mL maleic acid buffer,
 0.5 mL FBS, 0.5 mL stock blocking solution, 50 μL 10% Tween-20, and 2.5 mL
 PBS) at room temperature for 30 minutes. Endogenous peroxidase activity
 was quenched by incubation in freshly prepared 3% hydrogen peroxide
 (H₂O₂). Slides were then incubated sequentially with Avidin blocking
 solution (15 minutes; Vector SP-2001) and Biotin blocking solution (15
 minutes; Vector WP-2001). The following primary
 antibodies were used for IHC analysis: 
 <strong>Mouse monoclonal antibody (mAb) against IRE1
 (1:100; Abcam, cat. no. ab96481)</strong> 
 <strong>Rabbit monoclonal antibody against BiP
 (1:200; Cell Signaling, cat. no. 3177)</strong>
 <strong>Rabbit polyclonal antibody
 against XBP1 (1:200; Abcam, cat. no. ab37152)</strong>
 The <strong>ab37152 antibody
 against XBP1</strong> is validated for the detection of both
 cytosolic (unspliced) and nuclear (spliced) isoforms of XBP1, allowing for
 a comprehensive analysis of its localization and functional
 activity. Slides were incubated with these primary
 antibodies overnight at 4°C. Following thorough PBS washes, slides were
 incubated with biotinylated secondary antibodies specific to each primary
 antibody for 1 hour at room temperature. After additional PBS washes,
 slides were treated with horseradish peroxidase-labeled streptavidin
 (1:200 dilution) for 30 minutes at room temperature.
 Signal detection was performed using 3,3′-diaminobenzidine (DAB)
 substrate for 2 minutes at room temperature. Slides were then
 counterstained with Mayer’s Hematoxylin (10 drops in 1.25 mL PBS; Vector
 H-3404) for 1–4 minutes. Negative controls, processed without primary
 antibodies, were included to ensure specificity of staining.
 <strong>2.3. Tissue Microarray
 Scoring</strong> IHC results were independently
 evaluated by three pathologists in a blinded manner. Scoring was based on
 the intensity of staining, categorized as follows: 
 <strong>None (N)</strong>
 <strong>Weak
 (W)</strong> 
 <strong>Moderate (M)</strong> 
 <strong>Strong (S)</strong>
 For XBP1, both <strong>cytosolic
 and nuclear staining</strong> were assessed, ensuring a detailed
 analysis of its functional isoforms and their differential
 localization. <strong>2.4. Statistical
 Analysis</strong> Statistical analysis was
 performed using SPSS software (version 16.0; SPSS, Inc., Chicago, IL,
 USA). Data are expressed as numbers (n) and percentages (%). Group
 comparisons were conducted using a chi-square test or Fisher’s exact test,
 as appropriate. All p-values are presented as two-tailed, with a
 significance threshold of <strong>p &lt;
 0.05</strong>.
description: # The Role of BiP and the IRE1α-XBP1 Axis in Rhabdomyosarcoma Pathology
 [https://doi.org/10.5061/dryad.d2547d8c6](https://doi.org/10.5061/dryad.d2547d8c6) It is Tissue microarray data ## Code/software Any software for image (adobe photoshop, image j) and excel/csv.  ## Description of the data and file structure **Detailed Description of the BIP File:** ### **1. File Name: TMA Patients Data - Cancers Paper - BIP.csv** This dataset presents patient-level data related to tissue microarrays (TMA) used to study the **cytosolic expression of BIP (Binding Immunoglobulin Protein)**, an essential molecular chaperone in the endoplasmic reticulum involved in protein folding and stress responses. The cytosolic score corresponds to the **quantified expression of BIP in the cytosol**, which is important in understanding cancer development and progression, particularly in rhabdomyosarcoma and other cancers. --- **2. Structure of the Dataset** * **Columns Overview:** * Position: Position identifier in the tissue microarray. * Organ/Anatomic Site: The anatomical site or organ where the sample was taken. * Pathology Diagnosis: The histopathological diagnosis of the cancer subtype (e.g., spindle cell, pleomorphic, alveolus rhabdomyosarcoma). * TNM: Tumor-Node-Metastasis staging system, indicating the extent of cancer spread. * Grade: Tumor grade, reflecting the degree of differentiation and aggressiveness. * Stage: Clinical stage of cancer (e.g., IIA, IIB). * Type: Indicates whether the tissue sample is **malignant** or **normal (control)** tissue. * Tissue ID: Unique identifier for each tissue specimen. * **Cytosolic Score: Represents the level of BIP expression in the cytosol of the tissue sample, with values ranging from 0 (no expression) to 3 (high expression). Missing data is marked as none.** --- **3. Detailed Variable Descriptions** **Sample Characteristics:** **Anatomical Sites and Histological Diagnoses:** * Common cancer sites include the **testis, prostate, thigh, striated muscle, pleura, bladder, uterus, abdominal cavity, skin**, among others. * The dataset focuses on rhabdomyosarcoma subtypes, including: * **Spindle cell rhabdomyosarcoma** * **Embryonal rhabdomyosarcoma** * **Pleomorphic rhabdomyosarcoma** * **Alveolus rhabdomyosarcoma** These subtypes are critical to understanding disease progression, as each presents distinct molecular and pathological features. **Pathological and Clinical Staging:** * The **TNM classification** describes tumor size and spread: * T: Tumor size and extent. * N: Involvement of lymph nodes. * M: Presence of metastases. * Example: T2N0M0 indicates a larger localized tumor without lymph node involvement or distant spread. * **Tumor Grade and Stage:**\ The grade (G2, G3, or G4) reflects the aggressiveness of the cancer. Higher grades (e.g., G4) indicate poorly differentiated, more aggressive tumors.\ Stages (IIA, IIB, IIIB, IV) correspond to clinical progression. **Malignant vs. Normal Tissue:** * **Type column:** Distinguishes between **malignant tissue** samples and **normal (control) tissue samples**, primarily involving skeletal or striated muscle as the control group.\ These control tissues are used to compare BIP expression in malignant samples. **BIP Cytosolic Expression (Cytosolic Score):** * The **Cytosolic Score** quantifies BIP expression in the cytosol, providing critical data on the molecular response within cancer cells. * **Range of Scores:** * 0: No detectable BIP expression in the cytosol. * 1: Low BIP expression. * 2: Moderate BIP expression. * 3: High BIP expression. * none: Data not available or not applicable. * The presence and intensity of BIP cytosolic expression are vital for understanding its role in cancer progression and stress response. --- **4. Observations and Patterns** * **Multiple specimens per samples:**\ Some samples have more than one specimens, reflecting either different sampling sites or multiple sections analyzed.\ Example: Rows with the same patient No. and organ but different Tissue IDs correspond to different experimental tissue replicates. * **Expression trends across sites:**\ Sites such as the testis, thigh, and striated muscle are common due to the frequent occurrence of rhabdomyosarcoma in these regions. * **Normal vs. Malignant:**\ Malignant tissues generally exhibit higher BIP cytosolic scores, indicating elevated stress response compared to normal tissues, where the score often remains at 0 or 1. --- **5. Insights and Applications** * **BIP as a Biomarker:**\ The variation in BIP expression across samples can be linked to cancer aggressiveness, metastasis, and response to treatment. Elevated cytosolic BIP levels may indicate increased protein folding demand or stress due to cancer cell proliferation. * **Normal vs. Malignant Comparisons:**\ By comparing cytosolic BIP levels in normal control tissues and malignant samples, researchers can identify significant molecular differences and targetable pathways. * **Correlation with Clinical Features:**\ The dataset can be used to correlate BIP expression with variables such as **age, tumor grade, stage, and TNM classification**, providing insights into its prognostic or predictive potential. **Detailed Description of the IRE1 File:** --- **1. File Name:** ### TMA Patients (samples) Data - Cancers Paper - IRE1.csv This dataset provides clinical and pathological information associated with **IRE1 cytosolic expression** in various cancer tissue microarrays (TMAs). IRE1 (Inositol-Requiring Enzyme 1) is a key sensor in the unfolded protein response (UPR) pathway, activated under endoplasmic reticulum (ER) stress. The **cytosolic score** in this context quantifies the **cytoplasmic localization and activity of IRE1**, which is relevant in understanding its role in oncogenesis and cellular stress management. --- **2. Structure of the Dataset** The dataset consists of columns capturing sample characteristics, cancer types, and the IRE1 cytosolic score. * **Columns Overview:** * Position: TMA slide-specific position of the sample. * Organ/Anatomic Site: Location or organ where the tissue sample was taken (e.g., testis, prostate, thigh). * Pathology Diagnosis: Describes the type and subtype of cancer diagnosed (e.g., spindle cell, pleomorphic, or alveolar rhabdomyosarcoma). * TNM: Tumor-Node-Metastasis classification, describing the size and spread of cancer. * Grade: Reflects the aggressiveness of the cancer (e.g., well-differentiated (low grade) or poorly differentiated (high grade)). * Stage: Clinical stage of cancer progression (e.g., IIA, IIIB, IVA). * Type: Whether the sample represents malignant or normal tissue. * Tissue ID: Identifying code for the tissue sample. * **Cytosolic Score**: A value indicating the level of IRE1 expression in the cytoplasm, where: * 0: No detectable cytosolic IRE1 expression. * 1: Low IRE1 expression. * 2: Moderate IRE1 expression. * 3: High IRE1 expression. * \- or None: Data unavailable. --- **3. Detailed Description of the Variables** **Anatomic Sites and Pathological Diagnoses:** * Tissue samples originate from a wide range of anatomic sites, including the testis, thigh, prostate, bladder, uterus, striated muscle, and more. * Cancer subtypes commonly observed in the dataset are: * **Spindle cell rhabdomyosarcoma** * **Embryonal rhabdomyosarcoma** * **Pleomorphic rhabdomyosarcoma** * **Alveolar rhabdomyosarcoma** Each of these subtypes exhibits distinct pathological features, with implications for tumor aggressiveness, treatment response, and patient outcomes. **TNM, Grade, and Stage Classifications:** * **TNM Classification:** Describes the extent of cancer spread: * T: Size and extent of the primary tumor. * N: Involvement of nearby lymph nodes. * M: Metastasis to distant organs. * Example: T2N0M0 denotes a larger tumor with no lymph node or metastatic involvement. * **Grade:** The dataset classifies tumors by grades such as G2, G3, and G4, reflecting the level of differentiation, with higher grades indicating more aggressive and less differentiated tumors. * **Stage:** Clinical staging, such as IIA, IIIB, or IV, indicates how far the cancer has progressed and correlates with prognosis and treatment strategies. **Malignant vs. Normal Samples:** * Samples labeled as **malignant** represent various cancer subtypes, while **normal (control)** samples are derived from healthy tissues, primarily striated or skeletal muscle. * Normal tissues serve as critical controls for comparing IRE1 expression patterns in cancerous tissues. **IRE1 Cytosolic Score:** * The cytosolic score quantifies the presence of IRE1 within the cytoplasm of cells: * **High IRE1 expression (score 2 or 3)** could indicate increased cellular stress and activation of the unfolded protein response, which is often associated with tumor progression and survival mechanisms. * **Low or absent expression (scores 0 or 1)** may indicate reduced UPR activity or variations in stress response across cancer types. **4. Observations and Patterns** * **Multiple Specimens for Each Sample:**\ The same sample may have multiple specimen taken from different sections of the tumor or different experimental replicates. These are distinguished by unique tissue IDs. * **Variable IRE1 Expression Across Cancer Types:** * Higher IRE1 expression scores (2 or 3) are often seen in samples from aggressive tumors, reflecting the role of ER stress and UPR in cancer cell survival and adaptation. * Normal samples consistently show lower scores (0 or 1), highlighting the upregulation of IRE1 in pathological conditions. * **Organs with High Representation:**\ Frequent sampling sites include the testis, thigh, striated muscle, and retroperitoneum, due to the common occurrence of rhabdomyosarcoma in these locations. **5. Insights and Applications** * **IRE1 as a Biomarker:**\ Cytosolic expression of IRE1 can serve as a potential biomarker for ER stress and UPR activation in cancer, aiding in prognosis and treatment response prediction. * **Comparison with BIP Expression:**\ Since BIP and IRE1 are both involved in ER stress, cross-referencing this dataset with BIP expression levels can reveal valuable insights into the coordination of UPR signaling and its impact on cancer cell survival. * **Therapeutic Targeting:**\ Elevated IRE1 expression may indicate cancer cells’ reliance on UPR for survival, suggesting that therapeutic strategies targeting this pathway could be effective in certain subtypes. ### **Detailed Description of the XBP1 File:** **1. File Name:** **TMA Patients (samples) Data - Cancers Paper - Cytosolic and Nuclear XBP1** This dataset provides clinical, pathological, and molecular data related to the cytosolic (inactive) and nuclear (active) expression of **XBP1** in various cancer tissue microarrays (TMAs). XBP1 is a key transcription factor in the **unfolded protein response (UPR)** pathway. The dataset captures its role in **ER stress signaling** by evaluating both **cytosolic XBP1 (unspliced, inactive)** and **nuclear XBP1 (spliced, active)** expression. The cytosolic score reflects **baseline ER stress**, while the nuclear score quantifies **active UPR and transcriptional regulation** related to tumor progression. --- **2. Structure of the Dataset:** The dataset is organized with columns detailing patient information, cancer characteristics, and cytosolic/nuclear XBP1 scores. * **Columns Overview:** * **Position:** TMA-specific location of the sample. * **Organ/Anatomic Site:** Tissue location where the sample was taken (e.g., testis, thigh, prostate). * **Pathology Diagnosis:** Type and subtype of the diagnosed cancer (e.g., spindle cell or pleomorphic rhabdomyosarcoma). * **TNM:** Tumor-Node-Metastasis classification indicating cancer severity and spread. * **Grade:** Histological grading of the tumor (e.g., G2, G3, G4). * **Stage:** Clinical cancer stage (e.g., IIA, IIIB, IV). * **Type:** Classification as either **malignant** or **normal** tissue. * **Tissue ID:** Identifying code for tissue samples. * **Cytosolic Score:** Measures the expression of **inactive XBP1** in the cytoplasm. * **0:** No expression. * **1:** Low expression. * **2:** Moderate expression. * **3:** High expression. * **Nuclear Score:** Reflects the level of **active sXBP1** in the nucleus. * **0:** No expression. * **1:** Low expression. * **2:** Moderate expression. * **3:** High expression. --- **3. Detailed Description of the Variables** **Anatomic Sites and Pathological Diagnoses:** * Samples are derived from various organs, such as the **testis, thigh, prostate, bladder, uterus, striated muscle, and pleura**. * The dataset primarily focuses on **rhabdomyosarcoma subtypes**, including: * **Spindle cell rhabdomyosarcoma** * **Embryonal rhabdomyosarcoma** * **Pleomorphic rhabdomyosarcoma** * **Alveolar rhabdomyosarcoma** These cancer subtypes have distinct prognostic and therapeutic implications due to differences in **tumor aggressiveness and treatment responses**. **TNM, Grade, and Stage Classifications:** * **TNM Classification:** Describes the extent of cancer spread: * **T:** Primary tumor size and extent. * **N:** Lymph node involvement. * **M:** Presence of metastases. * **Example:** T2N0M0 indicates a large tumor with no lymph node or distant metastasis involvement. * **Grade:** Differentiation level of the tumor cells, ranging from **G2 to G4**. Higher grades indicate **less differentiation and more aggressive cancer**. * **Stage:** Describes cancer progression with stages such as **IIA, IIIB, and IV**, used for prognosis and treatment planning. **Malignant vs. Normal Samples:** * **Malignant samples** are derived from various tumor tissues, while **normal samples** come from healthy tissue, primarily **skeletal or striated muscle**, and serve as controls. --- **4. XBP1 Cytosolic and Nuclear Scores** * **Cytosolic Score:** Reflects the expression of inactive XBP1 in the cytoplasm. It is indicative of **baseline ER stress** and can suggest whether the cell is in a **pre-stressed or unstressed state**. * **Low Cytosolic XBP1 (0 or 1):** Indicates low baseline ER stress or limited UPR activation. * **High Cytosolic XBP1 (2 or 3):** Reflects increased ER stress or incomplete UPR. * **Nuclear Score:** Measures the level of active sXBP1 in the nucleus, which drives the expression of UPR-related genes, such as **BIP, CHOP**, and **ERAD components**. * **Low Nuclear XBP1 (0 or 1):** Indicates mild or non-activated UPR signaling. * **High Nuclear XBP1 (2 or 3):** Suggests strong ER stress response and transcriptional activation, often associated with **tumor adaptation or progression**. --- **5. Observations and Patterns** * **Multiple Specimens per Patient:**\ Many samples have multiple specimens analyzed, either from different parts of the tumor or technical replicates, distinguished by **Tissue IDs**. * **Patterns of XBP1 Expression:** * **High nuclear XBP1 (2 or 3)** is often seen in aggressive tumors or late-stage cancers, reflecting a **highly activated UPR**. * **Normal samples** generally have **low cytosolic (0 or 1) and nuclear scores**, showing minimal stress activation under normal conditions. * **Organs with High Representation:**\ Frequent sample sites include the **testis, thigh, retroperitoneum, striated muscle, and bladder**, reflecting the common occurrence of rhabdomyosarcoma in these tissues. --- **6. Biological Significance** * **Cytosolic XBP1:**\ High cytosolic expression without corresponding nuclear localization may indicate **incomplete UPR activation** or conditions where ER stress is being managed without severe consequences. * **Nuclear XBP1:**\ Nuclear localization of sXBP1 indicates **active UPR**, which enables cancer cells to adapt to stress by promoting **autophagy, protein folding, and degradation of misfolded proteins**. This mechanism can **enhance cancer cell survival** under harsh microenvironmental conditions, such as **hypoxia or nutrient deprivation**. **7. Insights and Applications** * **XBP1 as a Biomarker:**\ The differential expression of **cytosolic vs. nuclear XBP1** could help stratify patients based on ER stress and UPR activation, serving as a prognostic or therapeutic biomarker. * **Comparison with IRE1 and BIP:** * **IRE1:** Acts upstream by splicing XBP1 mRNA, so high IRE1 expression correlates with high nuclear XBP1 levels. * **BIP:** Works as a molecular chaperone to regulate UPR activation. Cross-referencing BIP and XBP1 expression provides insights into UPR balance and adaptive vs. apoptotic responses. * **Therapeutic Implications:** * Tumors with **high nuclear XBP1** rely on UPR for survival, making them candidates for therapies targeting ER stress pathways. * Inhibition of **IRE1-XBP1 signaling** could be explored to overcome chemoresistance and **sensitize tumors to ER stress-inducing treatments**. **Description of the TMA Image Files** **1. File Name:** ### BIP Protein TMA This file contains high-resolution tissue microarray (TMA) images captured at **40x magnification** under **phase-contrast microscopy**, showing **BIP (Binding Immunoglobulin Protein) expression**. BIP, a key chaperone protein in the **unfolded protein response (UPR)** pathway, plays a central role in **mitigating ER stress** by assisting in protein folding and controlling UPR activation. each **row folder**, the **sample localization** is identified using the **row name and column number**. For example, **"A1"** means the sample is located in **row A, column 1**. * **Staining Overview:**\ The TMA slides are immunostained specifically for BIP, allowing the visualization of its expression levels across different cancer types and tissue sites. * **Phase-Contrast Imaging:**\ The use of phase-contrast imaging provides enhanced contrast of cellular structures without requiring dyes, highlighting the localization of BIP within **cytoplasmic or perinuclear regions**. * **Contents:**\ The file includes TMAs covering diverse tissues, ranging from normal controls (skeletal/striated muscle) to malignant samples from sites such as the **testis, thigh, retroperitoneum, and uterus**. Each sample is labeled with its **Tissue ID** and its corresponding **BIP expression**. **2. File Name:** ### IRE1 Alpha Protein TMA This TMA file contains images of cancer and normal tissue samples stained for **IRE1α (Inositol-Requiring Enzyme 1 alpha)** and captured at **40x magnification** under **phase-contrast microscopy**. IRE1α is a critical ER stress sensor that activates the **UPR pathway** through its kinase and RNase functions, particularly by **splicing XBP1 mRNA** and triggering downstream adaptive responses. each **row folder**, the **sample localization** is identified using the **row name and column number**. For example, **"A1"** means the sample is located in **row A, column 1**. * **Staining Overview:**\ The images showcase the distribution and intensity of IRE1α expression in the **cytoplasm** of various cancer types, highlighting its role in detecting and responding to ER stress. * **High IRE1α expression** is often observed in aggressive tumors, where UPR activation supports survival under adverse conditions. * **Phase-Contrast Imaging:**\ The phase-contrast setting enables the clear distinction of cellular boundaries and subcellular localization of IRE1α without additional dyes. * **Cytoplasmic staining** is prominent in cases with high stress or active UPR signaling. * **Contents:**\ This file includes TMAs derived from multiple anatomic sites, with a focus on rhabdomyosarcoma subtypes. The **IRE1α staining intensity** serves as a proxy for ER stress and is particularly relevant in tumors with elevated adaptive responses. --- **3. File Name:** ### XBP1 TMA This dataset includes **TMA images** captured at **40x magnification** to detect **XBP1 expression**, including both **inactive (cytosolic XBP1) and active (nuclear sXBP1)** forms. XBP1 is a critical transcription factor that orchestrates the transcription of genes involved in **protein folding, degradation of misfolded proteins**, and **autophagy** during the UPR. each **row folder**, the **sample localization** is identified using the **row name and column number**. For example, **"A1"** means the sample is located in **row A, column 1**. * **Staining Overview:**\ The images are immunostained to show the **cytosolic localization of unspliced XBP1** (inactive) and **nuclear localization of spliced XBP1 (sXBP1)**, which reflects active transcriptional activity. * **Nuclear XBP1 staining** correlates with high UPR activity and potential tumor adaptation mechanisms. * **Phase-Contrast Imaging:**\ The phase-contrast images provide clear visualization of **cellular compartments**, distinguishing cytoplasmic XBP1 from nuclear sXBP1 localization, essential for interpreting UPR activation. * **Contents:**\ The file includes a comprehensive set of **TMA samples** from different types of Rhabdomyosarcoma along with normal tissues, making it valuable for **comparative analysis of UPR activation**. Each image is linked to patient data for further clinical correlation.