Journal: Bioactive Materials

Article Title: Graphene oxide/gallium nanoderivative as a multifunctional modulator of osteoblastogenesis and osteoclastogenesis for the synergistic therapy of implant-related bone infection

doi: 10.1016/j.bioactmat.2022.07.015

Figure Lengend Snippet: Investigation of the potential molecular mechanisms of the regulatory effects of GO/Ga nanocomposites on osteoblastogenesis and osteoclastogenesis in MC3T3-E1 and RAW 264.7 cells. (a) Expression features of BMP/SMAD signaling molecules during the osteoblast differentiation of MC3T3-E1 cells after coculture with different nanomaterials for 7 d. (b) Expression features of RANKL-stimulated MAPK signaling molecules during the osteoclast differentiation of RAW 264.7 cells after being pretreated with different nanomaterials for 4 h prior to RANKL (50 ng/mL) stimulation for 30 min. (c) Expression features of RANKL-stimulated NF-κB signaling molecules during the osteoclast differentiation of RAW 264.7 cells after being pretreated with different nanomaterials for 4 h prior to RANKL (50 ng/mL) stimulation for 30 min. The expression levels of NFATc1 and c-Jun were also confirmed after coculture for 3 d with the stimulation of RANKL (50 ng/mL). Overall, the relative expression levels of p-Smad 1/5, p-JNK, p-P38, p-ERK, p-IkBα, p-P65, NFATc1 and c-Jun were calculated and normalized by β-actin in terms of the gray band intensities as confirmed by ImageJ software. (d) Luciferase reporter gene assessment of BMP-2 and NFATc1 in MC3T3-E1 and RAW264.7 cells, respectively. Stably transfected cells were treated with different nanomaterials for 6 h with specific stimulation, and the luciferase activities were confirmed by a Pierce™ Gaussia Luminescence Assay kit. (e) Molecular docking of Ga3+ with JNK/P38 kinases as demonstrated in binding mode figures using PyMOL visualization software. (f) Schematic diagram of the regulatory mechanisms of GO/Ga nanocomposites involved in osteoblastogenesis and osteoclastogenesis (Created with BioRender.com). *p < 0.01 compared with CTRL and GaNPs, **p < 0.05 and ***p < 0.01 compared with CTRL, #p < 0.01 compared with GO, ##p < 0.05 compared with GO.

Article Snippet: To further confirm the crucial role of corresponding signaling axis involved in the osteogenic and osteoclast differentiation, a small interfering RNA (siRNA) mediated BMP-2 gene knockdown (Ribobio, Guangzhou, China), and specific activators of JNK (Anisomycin, 50 μM, S7409), P38 (Asiatic acid, 10 μM, S2266) and NF-κB (Betulinic acid, 15 μM, S3603) purchased from Selleck Chemicals (Houston, USA) with indicated concentration were used to perform reverse validation experiments aimed at verifying the stimulative effects of GO/Ga nanocomposites on BMP/Smad signaling pathways and the inhibitory effects on MAPK and NF-κB signaling pathways, as previously reported [ [53] , [54] , [55] , [56] ].

Techniques: Expressing, Software, Luciferase, Stable Transfection, Transfection, Luminescence Assay, Binding Assay

Journal: Advanced Science

Article Title: FOXA2 Suppression by TRIM36 Exerts Anti‐Tumor Role in Colorectal Cancer Via Inducing NRF2/GPX4‐Regulated Ferroptosis

doi: 10.1002/advs.202304521

Figure Lengend Snippet: FOXA2 expression is up‐regulated in CRC patients. A,B) FOXA2 expression profile in CRC tissues from TCGA cohort. C) Representative IHC images for FOXA2 in CRC tissues and normal tissues ( https://www.proteinatlas.org/ ). D) Kaplan‐Meier analysis for relapse‐free‐survival (RFS) of CRC patients with high ( n = 709) or low ( n = 633) FOXA2 expression based on the median expression of FOXA2 from the Kaplan‐Meier Plotter ( https://kmplot.com/analysis/index.php?p = service). E) Images of IHC staining for FOXA2 in CRC tissues and the paired adjacent normal tissues from our cohort. Scale bar = 120 µm. F) IHC scores of FOXA2 expression levels in paired normal and CRC samples were quantified. G) RT‐qPCR analysis for FOXA2 gene expression in human CRC tissues and the matched adjacent normal tissues (ANT) from our cohort ( n = 60). H) FOXA2 protein expression in eighteen paired CRC tissues was examined using western blot. I) RT‐qPCR ( n = 5) and J) western blot ( n = 4) assays for FOXA2 gene and protein expression levels in six CRC cell lines and non‐tumor cell line NCM460. Data are marked as the means ± SD. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: To establish the stable FOXA2‐knockdown, FOXA2‐overexpression, Nrf2‐overexpression, and TRIM36‐overexpression CRC cell lines, the human lentivirus‐sh‐FOXA2, FOXA2 overexpression (oe‐FOXA2) plasmids, Nrf2 and TRIM36 plasmids were purchased from GeneChem (Shanghai, China).

Techniques: Expressing, Immunohistochemistry, Quantitative RT-PCR, Gene Expression, Western Blot

Journal: Advanced Science

Article Title: FOXA2 Suppression by TRIM36 Exerts Anti‐Tumor Role in Colorectal Cancer Via Inducing NRF2/GPX4‐Regulated Ferroptosis

doi: 10.1002/advs.202304521

Figure Lengend Snippet: FOXA2 enhances the proliferation, migration and invasion of CRC cells in vitro or in vivo. CCK8 analysis for cell proliferation of HCT‐116 and SW480 cells with FOXA2 A) knockdown or B) over‐expression ( n = 4). C) EdU staining of HCT‐116 and SW480 cells transfected with sh‐FOXA2 or oe‐FOXA2 as shown ( n = 4). Scale bar = 50 µm. D) Transwell analysis for CRC cell migration and invasion after FOXA2 knockdown or over‐expression ( n = 5). Scale bar = 50 µm. E) RT‐qPCR analysis for EMT markers in HCT‐116 and SW480 cells after FOXA2 knockdown or over‐expression ( n = 3). F) Tumor samples from the indicated groups of mice expressing the control vector, sh‐FOXA2 or oe‐FOXA2 plasmids ( n = 5 or 4 in each). G) Tumor formation volume and H) tumor weights were measured. Data are marked as the means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To establish the stable FOXA2‐knockdown, FOXA2‐overexpression, Nrf2‐overexpression, and TRIM36‐overexpression CRC cell lines, the human lentivirus‐sh‐FOXA2, FOXA2 overexpression (oe‐FOXA2) plasmids, Nrf2 and TRIM36 plasmids were purchased from GeneChem (Shanghai, China).

Techniques: Migration, In Vitro, In Vivo, Knockdown, Over Expression, Staining, Transfection, Quantitative RT-PCR, Expressing, Control, Plasmid Preparation

Journal: Advanced Science

Article Title: FOXA2 Suppression by TRIM36 Exerts Anti‐Tumor Role in Colorectal Cancer Via Inducing NRF2/GPX4‐Regulated Ferroptosis

doi: 10.1002/advs.202304521

Figure Lengend Snippet: Positive correlation between FOXA2 and Nrf2/GPX4 signaling in CRC cell lines. A) Positive correlation between FOXA2 expression and GPX4, NFE2L2 (Nrf2), NQO1, G6PD, and SLC7A11 in CRC patients from TCGA database. B) RT‐qPCR analysis for genes including Nrf2, GCLC, NQO1, SOD1, GPX4, SLC7A11 and G6PD in HCT‐116 and SW480 cells with FOXA2 knockdown or over‐expression ( n = 3). C,D) IF staining for GPX4 expression in CRC cell lines transfected with sh‐FOXA2 or oe‐FOXA2 ( n = 4). Scale bar = 20 µm. E) Western blot analysis for GPX4, Nrf2, and NQO1 protein expression levels in FOXA2‐diminished or ‐over‐expressed HCT‐116 and SW480 cells ( n = 3). Data are marked as the means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To establish the stable FOXA2‐knockdown, FOXA2‐overexpression, Nrf2‐overexpression, and TRIM36‐overexpression CRC cell lines, the human lentivirus‐sh‐FOXA2, FOXA2 overexpression (oe‐FOXA2) plasmids, Nrf2 and TRIM36 plasmids were purchased from GeneChem (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Over Expression, Staining, Transfection, Western Blot

Journal: Advanced Science

Article Title: FOXA2 Suppression by TRIM36 Exerts Anti‐Tumor Role in Colorectal Cancer Via Inducing NRF2/GPX4‐Regulated Ferroptosis

doi: 10.1002/advs.202304521

Figure Lengend Snippet: FOXA2 suppression induces ferroptosis in CRC cell lines. A,B) ROS production was measured by DCF‐DA staining in CRC cells with FOXA2 knockdown or over‐expression. Scale bar = 50 µm. C,D) Lipid ROS generation in HCT‐116 and SW480 cells was measured by C11‐BODIPY581/591 staining post‐sh‐FOXA2 or oe‐FOXA2 plasmid transfection. Scale bar = 20 µm. E) MDA levels, (F) GSH contents and G) iron currents in the show groups of CRC cells were examined. H) HCT‐116 and SW480 cells with FOXA2 knockdown were treated with ferroptosis inhibitor (Fer‐1, 1 µM), necroptosis inhibitor (Nec‐1, 10 µM), or apoptosis inhibitor (Z‐VAD‐FMK, 10 µM) for another 24 h. Then, all cells were collected for cell viability examination using CCK‐8 analysis. Data are marked as the means ± SD ( n = 5 in each). ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns , no significant difference.

Article Snippet: To establish the stable FOXA2‐knockdown, FOXA2‐overexpression, Nrf2‐overexpression, and TRIM36‐overexpression CRC cell lines, the human lentivirus‐sh‐FOXA2, FOXA2 overexpression (oe‐FOXA2) plasmids, Nrf2 and TRIM36 plasmids were purchased from GeneChem (Shanghai, China).

Techniques: Staining, Knockdown, Over Expression, Plasmid Preparation, Transfection, CCK-8 Assay

Journal: Advanced Science

Article Title: FOXA2 Suppression by TRIM36 Exerts Anti‐Tumor Role in Colorectal Cancer Via Inducing NRF2/GPX4‐Regulated Ferroptosis

doi: 10.1002/advs.202304521

Figure Lengend Snippet: FOXA2 suppression sensitizes CRC cells to OXA treatment by facilitating ferroptosis. HCT‐116 and SW480 cells were transfected with sh‐FOXA2, followed by OXA treatments for another 24 h. Then, all cells were harvested for subsequent assays. A) CCK‐8 analysis for cell viability evaluation ( n = 4). B,C) DCF‐DA staining was used to examine ROS production ( n = 4). Scale bar = 50 µm. D,E) C11‐BODIPY581/591 staining was conducted for the measurements of lipid ROS production ( n = 4). Scale bar = 20 µm. F) MDA contents, G) GSH levels, and H) iron currents were assessed ( n = 4). I) Ferroptosis hallmarks including Nrf2, NQO1, SOD1, GPX4 and SLC7A11 were calculated by RT‐qPCR ( n = 3). Data are marked as the means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To establish the stable FOXA2‐knockdown, FOXA2‐overexpression, Nrf2‐overexpression, and TRIM36‐overexpression CRC cell lines, the human lentivirus‐sh‐FOXA2, FOXA2 overexpression (oe‐FOXA2) plasmids, Nrf2 and TRIM36 plasmids were purchased from GeneChem (Shanghai, China).

Techniques: Transfection, CCK-8 Assay, Staining, Quantitative RT-PCR

Journal: Advanced Science

Article Title: FOXA2 Suppression by TRIM36 Exerts Anti‐Tumor Role in Colorectal Cancer Via Inducing NRF2/GPX4‐Regulated Ferroptosis

doi: 10.1002/advs.202304521

Figure Lengend Snippet: FOXA2 knockdown induces ferroptosis in chemoresistant CRC cell lines. A) RT‐qPCR analysis for Nrf2, NQO1, SOD1, GPX4 and SLC7A11 gene expression levels in drug‐sensitive or ‐resistant HCT‐116 and SW480 cells ( n = 3). B) Western blot assay for GPX4, Nrf2, and NQO1 protein expression levels in HCT‐116 and SW480 cells with or without chemoresistance ( n = 4). C) Western blot assay for FOXA2, GPX4, Nrf2, and NQO1 protein expression levels in chemoresistant HCT‐116 and SW480 cells with or without FOXA2 knockdown ( n = 4). (D) GPX4 expression by IF staining in drug‐resistant CRC cells after transfection with sh‐FOXA2 ( n = 4). Scale bar = 20 µm. E) ROS and (F) lipid ROS production by DCF‐DA (Scale bar = 50 µm) and C11‐BODIPY581/591 (Scale bar = 20 µm) staining, respectively, in chemoresistant HCT‐116 and SW480 cells with FOXA2 knockdown ( n = 4). G) MDA levels, H) GSH contents, and I) iron currents in drug‐resistant HCT‐116 and SW480 cells after FOXA2 knockdown ( n = 4). Data are marked as the means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To establish the stable FOXA2‐knockdown, FOXA2‐overexpression, Nrf2‐overexpression, and TRIM36‐overexpression CRC cell lines, the human lentivirus‐sh‐FOXA2, FOXA2 overexpression (oe‐FOXA2) plasmids, Nrf2 and TRIM36 plasmids were purchased from GeneChem (Shanghai, China).

Techniques: Knockdown, Quantitative RT-PCR, Gene Expression, Western Blot, Expressing, Staining, Transfection

Journal: Advanced Science

Article Title: FOXA2 Suppression by TRIM36 Exerts Anti‐Tumor Role in Colorectal Cancer Via Inducing NRF2/GPX4‐Regulated Ferroptosis

doi: 10.1002/advs.202304521

Figure Lengend Snippet: FOXA2 knockdown CRC cells are sensitive to OXA‐induced ferroptosis via the Nrf2 activation. HCT‐116 cells with FOXA2 knockdown or over‐expression were incubated with OXA (10 µM) alone or combination with Nrf2 activator (ML334, 20 µM) or inhibitor (ML385, 5 µM) for an additional 24 h. SW480 cells co‐transfected with sh‐FOXA2 and Nrf2 plasmids, or oe‐FOXA2 and si‐Nrf2 were exposed to OXA (10 µM) treatment for another 24 h. Then, all HCT‐116 and SW480 cells were harvested for studies as follows. A‐H) DCF‐DA (Scale bar = 50 µm) and C11‐BODIPY581/591 (Scale bar = 20 µm) staining were performed to examine ROS and lipid ROS production in CRC cells treated as shown ( n = 4). I,J) MDA levels in HCT‐116 and SW480 cells were examined. K,L) Examination of iron currents in CRC cells. (M,N) Calculation for cellular MDA levels. O,P) Iron currents in CRC cells were assessed ( n = 5). Q‐T) Western blot analysis for Nrf2, NQO1, and GPX4 protein expression levels in CRC cells were conducted ( n = 3). Data are marked as the means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: To establish the stable FOXA2‐knockdown, FOXA2‐overexpression, Nrf2‐overexpression, and TRIM36‐overexpression CRC cell lines, the human lentivirus‐sh‐FOXA2, FOXA2 overexpression (oe‐FOXA2) plasmids, Nrf2 and TRIM36 plasmids were purchased from GeneChem (Shanghai, China).

Techniques: Knockdown, Activation Assay, Over Expression, Incubation, Transfection, Staining, Western Blot, Expressing

Journal: Advanced Science

Article Title: FOXA2 Suppression by TRIM36 Exerts Anti‐Tumor Role in Colorectal Cancer Via Inducing NRF2/GPX4‐Regulated Ferroptosis

doi: 10.1002/advs.202304521

Figure Lengend Snippet: RSL3 promotes the sensitivity of FOXA2‐overexpressed and chemoresistant CRC cells to OXA treatment. Drug‐sensitive CRC cells with FOXA2 over‐expression or chemoresistant CRC cells were subjected to OXA (10 µM), GPX4 inhibitor (RSL3, 100 nM) single or double incubation for another 24 h. Next, all cells were harvested for studies as following. A‐D) Western blot and IF staining analysis for GPX4 protein expression levels in cells treated as shown ( n = 3 or 4 in each). E,F) MDA levels and G,H) iron contents were examined ( n = 4). I,J) Lipid ROS production was measured using C11‐BODIPY581/591 staining ( n = 4). K,L) Cell viability of the shown CRC cells with or without chemoresistance was examined using CCK‐8 analysis ( n = 4). Data are marked as the means ± SD. Scale bar = 20 µm. + p < 0.05, ++ p < 0.01 versus the Ctrl group; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns , no significant difference.

Article Snippet: To establish the stable FOXA2‐knockdown, FOXA2‐overexpression, Nrf2‐overexpression, and TRIM36‐overexpression CRC cell lines, the human lentivirus‐sh‐FOXA2, FOXA2 overexpression (oe‐FOXA2) plasmids, Nrf2 and TRIM36 plasmids were purchased from GeneChem (Shanghai, China).

Techniques: Over Expression, Incubation, Western Blot, Staining, Expressing, CCK-8 Assay

Journal: Advanced Science

Article Title: FOXA2 Suppression by TRIM36 Exerts Anti‐Tumor Role in Colorectal Cancer Via Inducing NRF2/GPX4‐Regulated Ferroptosis

doi: 10.1002/advs.202304521

Figure Lengend Snippet: Identifying a potent suppressor of FOXA2. A) Working model of the protocols used to identify potent binding proteins and suppressors of FOXA2. B) SDS‐PAGE band showing the immunoprecipitated proteins in HCT‐116 and HEK293T cells with anti‐FOXA2 antibody vs the isotype‐controlled IgG. C) Immunoblotting assay to validate the presence of FOXA2 in the immunoprecipitated samples. D) There were a total of 31 overlapping FOXA2 binding proteins identified both in HCT‐116 and HEK293T cells, including the listed E3 ligases TRIM36, CHIP, TRIM25, RNF2, and MID1. E) Western blot indicating the FOXA2 expression in HCT‐116 and HEK293T cells transfected with the shown plasmids. F) TRIM36 expression profile in CRC tissues from TCGA cohort. G) Correlation between FOXA2 and TRIM36 from TCGA database. H) TRIM36 protein expression in eighteen paired CRC tissues was examined using western blot. I) Spearman's correlation between TRIM36 and FOXA2 protein expression in the eighteen CRC patients. J) Western blot analysis for TRIM36 and FOXA2 in CRC cells transfected with TRIM36 plasmids. (K) RT‐qPCR analysis for FOXA2 in CRC cells with or without TRIM36 over‐expression. L) FOXA2 protein expression levels in empty vector and TRIM36‐overexpressed HCT‐116 or SW480 cells were examined at the shown time after CHX (20 µg ml −1 ) incubation. M) Western blot indicating the influence of MG132 (10 µM) on FOXA2 expression levels in CRC cells with or without TRIM36 over‐expression. Data are marked as the means ± SD. ** p < 0.01, **** p < 0.0001.

Article Snippet: To establish the stable FOXA2‐knockdown, FOXA2‐overexpression, Nrf2‐overexpression, and TRIM36‐overexpression CRC cell lines, the human lentivirus‐sh‐FOXA2, FOXA2 overexpression (oe‐FOXA2) plasmids, Nrf2 and TRIM36 plasmids were purchased from GeneChem (Shanghai, China).

Techniques: Binding Assay, SDS Page, Immunoprecipitation, Western Blot, Expressing, Transfection, Quantitative RT-PCR, Over Expression, Plasmid Preparation, Incubation

Journal: Advanced Science

Article Title: FOXA2 Suppression by TRIM36 Exerts Anti‐Tumor Role in Colorectal Cancer Via Inducing NRF2/GPX4‐Regulated Ferroptosis

doi: 10.1002/advs.202304521

Figure Lengend Snippet: TRIM36 interacts with FOXA2 and induces its K48‐linked polyubiquitination. A) Co‐IP analysis of HEK293T cells transfected with Flag‐tagged FOXA2 and HA‐tagged TRIM36. Anti‐Flag and anti‐HA antibodies were used for western blot assay. B) GST precipitation indicating the direct interaction of TRIM36 with FOXA2 using purified GST‐FOXA2 and His‐tagged TRIM36 (left) or purified GST‐TRIM36 and His‐FOXA2 (right) by western blot analysis. GST was defined as a control. C) IF images of HEK293T cells co‐transfected with 24 h of Flag‐tagged FOXA2 (red) and HA‐tagged TRIM36 (green). Scale bar = 15 µm. D,E) Schematic indicating full‐length and truncated TRIM36 (top) and FOXA2 (top) with representative Co‐IP assays (bottom) for the mapping analysis of the domains responsible for the TRIM36/FOXA2 interaction in HEK293T cells. F) Lysates of HEK293T cells transfected with plasmids expressing HA‐FOXA2, Myc‐Ub and increasing amounts of Flag‐TIRM36 were immunoprecipitated with anti‐HA beads and immunoblotted using an anti‐Myc antibody. G) Western blots showing FOXA2 ubiquitination in HEK293T cells transfected with the indicated plasmids in different combinations. ∆CC, deletion of the CC domain. H) Western blot analysis showing K48 ubiquitination of FOXA2 in HEK293T cells co‐transfected with Flag‐TRIM36 and Myc‐K48‐Ub or Myc‐K63‐Ub. I) Western blot analysis showing K48 ubiquitination of FOXA2 in HEK293T cells co‐transfected with Flag‐TRIM36, Flag‐TRIM36 (ΔCC), and Myc‐K48‐Ub. (J) FOXA2 protein expression levels and its ubiquitination levels in 24 h of OXA‐treated HCT‐116 and SW480 cells.

Article Snippet: To establish the stable FOXA2‐knockdown, FOXA2‐overexpression, Nrf2‐overexpression, and TRIM36‐overexpression CRC cell lines, the human lentivirus‐sh‐FOXA2, FOXA2 overexpression (oe‐FOXA2) plasmids, Nrf2 and TRIM36 plasmids were purchased from GeneChem (Shanghai, China).

Techniques: Co-Immunoprecipitation Assay, Transfection, Western Blot, Purification, Control, Expressing, Immunoprecipitation, Ubiquitin Proteomics

Journal: Advanced Science

Article Title: FOXA2 Suppression by TRIM36 Exerts Anti‐Tumor Role in Colorectal Cancer Via Inducing NRF2/GPX4‐Regulated Ferroptosis

doi: 10.1002/advs.202304521

Figure Lengend Snippet: Conditional knockout of FOXA2 in IECs ameliorates colitis‐associated tumorigenesis in vivo. A) Scheme protocols of recombination in Villin‐Cre ; FOXA2 f/f mice. B,C) RT‐qPCR and western blot assays for FOXA2 gene and protein expression levels in colon crypts from the mice treated with or without AOM/DSS ( n = 6). D) H&E and IHC staining of FOXA2 in colon from FOXA2 f/f and FOXA2 cKO mice (n = 8). Scale bar = 120 µm. E) Inflammation scores were quantified. (F) FOXA2 expression by IHC staining was calculated. G) Body weights of mice were recorded ( n = 10–15 in each group). H) Overall survival rates for each group of mice ( n = 10–15 in each group). I) Images for colons from all groups of mice. J) Colon length was measured ( n = 15). (K) Tumor number on colon was examined ( n = 15). L) IHC staining for KI‐67 in colon tissues was performed ( n = 5). Scale bar = 50 µm. Data are marked as the means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns , no significant difference.

Article Snippet: To establish the stable FOXA2‐knockdown, FOXA2‐overexpression, Nrf2‐overexpression, and TRIM36‐overexpression CRC cell lines, the human lentivirus‐sh‐FOXA2, FOXA2 overexpression (oe‐FOXA2) plasmids, Nrf2 and TRIM36 plasmids were purchased from GeneChem (Shanghai, China).

Techniques: Knock-Out, In Vivo, Quantitative RT-PCR, Western Blot, Expressing, Immunohistochemistry