Journal: International Journal of Biological Sciences

Article Title: MANF Promotes Unexplained Recurrent Miscarriages by Interacting with NPM1 and Downregulating Trophoblast Cell Migration and Invasion

doi: 10.7150/ijbs.85378

Figure Lengend Snippet: The levels of mesencephalic astrocyte-derived neurotrophic factor (MANF) in the serum and the expression levels of MANF, matrix metallopeptidase (MMP)2, and MMP9 in the villi of healthy pregnant women and pregnant women with unexplained recurrent miscarriage (URM), as well as cell proliferation and apoptosis. A. Comparison of MANF serum levels in 30 healthy pregnant women and 50 pregnant women with URM. B. Comparison of serum MANF levels in 20 healthy non-pregnant women of childbearing age and 19 women with URM, at the time of miscarriage and the non-pregnant period, more than 6 months after the abortion procedure. C. Immunohistochemistry of MANF, MMP2, and MMP9 in the villi of the two groups of participants, scale bar = 200 μm. D. Immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and TUNEL staining of villi tissue, scale bar = 200 μm, 50 μm. E. Western blot analysis to detect the levels of MANF, MMP2, MMP9, and PCNA in villi from both groups. F. Immunoblot band density quantification. G. mRNA levels of MANF, MMP2, MMP9, and PCNA in the villi from the two groups. All results are from three or more independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The packaged lentiviruses overexpressing MANF (oe-MANF), knocking down MANF (sh-MANF), and overexpressing NPM1 (oe-NPM1) were designed and synthesized by Gene-Chem (Shanghai, China).

Techniques: Derivative Assay, Expressing, Comparison, Immunohistochemistry, Immunohistochemical staining, Staining, TUNEL Assay, Western Blot

Journal: International Journal of Biological Sciences

Article Title: MANF Promotes Unexplained Recurrent Miscarriages by Interacting with NPM1 and Downregulating Trophoblast Cell Migration and Invasion

doi: 10.7150/ijbs.85378

Figure Lengend Snippet: The expression level of MANF, MMP2, and MMP9 in the decidua of patients with URM, as well as cell proliferation and apoptosis. A. Immunohistochemical staining of MANF, MMP2, and MMP9 in the two groups of decidua, scale bar = 200 μm. B. Immunohistochemistry of PCNA and TUNEL staining of decidua tissue, scale bar = 200 μm, 50 μm. C. Western blot analysis to detect the levels of MANF, MMP2, MMP9, and PCNA in decidual tissue from both groups. D. Quantification of immunoblot band densities. E. mRNA levels of MANF, MMP2, MMP9, and PCNA in decidual tissue from the two groups. All results are from three or more independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The packaged lentiviruses overexpressing MANF (oe-MANF), knocking down MANF (sh-MANF), and overexpressing NPM1 (oe-NPM1) were designed and synthesized by Gene-Chem (Shanghai, China).

Techniques: Expressing, Immunohistochemical staining, Staining, Immunohistochemistry, TUNEL Assay, Western Blot

Journal: International Journal of Biological Sciences

Article Title: MANF Promotes Unexplained Recurrent Miscarriages by Interacting with NPM1 and Downregulating Trophoblast Cell Migration and Invasion

doi: 10.7150/ijbs.85378

Figure Lengend Snippet: MANF expression levels differentially regulate MMP expression, as well as cell invasion and migration abilities. A. Levels of MMP2 and MMP9 in trophoblast cell lines with stable overexpression or knockdown of MANF, detected by western blotting. B. Proliferation of trophoblast cells at different levels of endogenous MANF assessed by EdU analysis, scale bar = 50 μm. C. Apoptosis of trophoblast cells at different levels of MANF using the TUNEL assay, scale bar = 50 μm. D. Wound healing assay showing the 24 h migration ability of trophoblasts expressing different MANF levels, scale bar = 100 μm. E. Transwell assay showing the 24-h invasion ability of trophoblast cells expressing different MANF levels, scale bar = 100 μm. All results are from three or more independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The packaged lentiviruses overexpressing MANF (oe-MANF), knocking down MANF (sh-MANF), and overexpressing NPM1 (oe-NPM1) were designed and synthesized by Gene-Chem (Shanghai, China).

Techniques: Expressing, Migration, Over Expression, Knockdown, Western Blot, TUNEL Assay, Wound Healing Assay, Transwell Assay

Journal: International Journal of Biological Sciences

Article Title: MANF Promotes Unexplained Recurrent Miscarriages by Interacting with NPM1 and Downregulating Trophoblast Cell Migration and Invasion

doi: 10.7150/ijbs.85378

Figure Lengend Snippet: The interaction between MANF and NPM1 in the trophoblasts of URM patients leads to increased ubiquitination and degradation of NPM1. A. SDS-PAGE silver staining of co-IP samples using the MANF antibody in the villi tissue of patients in the URM group. MANF and target bands are labeled. B. Secondary mass spectra of NPM1-specific peptides identified by LC-MS/MS. C. Co-localization of MANF and NPM1 in the nucleus of trophoblast cells in the villi of patients with URM, detected by immunofluorescence double-labeling, scale bar = 100 μm. D. Co-IP confirming the interaction between MANF and NPM1 in the villi of patients with URM. E. A glutathione S-transferase (GST) pull-down assay revealed that MANF and NPM1 interact in vitro . F. Western blotting and statistical analysis of NPM1 levels in trophoblast cells with MANF overexpression or knockdown. G. Effect of addition of 100 μM CHX to HTR-8/SVneo cells overexpressing MANF and corresponding vector controls. Western blotting is used to detect changes in the half-life of the NPM1 protein and perform statistical analysis. H. Western blotting was used to detect the effect of adding or not adding MANF in HTR-8/SVneo cells on the ubiquitination level of NPM1. I. NPM1 mRNA levels in MANF overexpression or knockdown trophoblast cells. J. MANF mRNA levels in NPM1 overexpression or knockdown trophoblast cells. All results are from three or more independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The packaged lentiviruses overexpressing MANF (oe-MANF), knocking down MANF (sh-MANF), and overexpressing NPM1 (oe-NPM1) were designed and synthesized by Gene-Chem (Shanghai, China).

Techniques: Ubiquitin Proteomics, SDS Page, Silver Staining, Co-Immunoprecipitation Assay, Labeling, Liquid Chromatography with Mass Spectroscopy, Immunofluorescence, Pull Down Assay, In Vitro, Western Blot, Over Expression, Knockdown, Plasmid Preparation

Journal: International Journal of Biological Sciences

Article Title: MANF Promotes Unexplained Recurrent Miscarriages by Interacting with NPM1 and Downregulating Trophoblast Cell Migration and Invasion

doi: 10.7150/ijbs.85378

Figure Lengend Snippet: MANF negatively regulates NPM1 expression in trophoblast cells, affecting cell proliferation, invasion, and migration ability. A. Western blotting of MANF, MMP2, and MMP9 expression in trophoblast cells with NPM1 overexpression and knockdown. B. Western blot band density quantification. C. CCK-8assay showing the cell proliferation activity of trophoblast cells overexpressing and knocked down for NPM1. D. Western blotting of the expression of MMP2 and MMP9 after simultaneous knockdown of NPM1 and MANF in trophoblast cells. E. Immunoblot band density quantification. F. Results and statistical analysis of wound healing experiments in trophoblast cells with NPM1 overexpression and knockdown. G. Results and statistical analysis of 24-h transwell experiments in trophoblast cells with NPM1 overexpression and knockdown. H. Results and statistical analysis of wound healing in trophoblast cells with stable MANF knockdown and simultaneous inhibition of NPM1 for 24 h. I. Determination and statistical analysis of cell invasion capacity of trophoblast cells with stable MANF knockdown and simultaneous inhibition of NPM1 for 24 h. All results are from three or more independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The packaged lentiviruses overexpressing MANF (oe-MANF), knocking down MANF (sh-MANF), and overexpressing NPM1 (oe-NPM1) were designed and synthesized by Gene-Chem (Shanghai, China).

Techniques: Expressing, Migration, Western Blot, Over Expression, Knockdown, Activity Assay, Inhibition

Journal: International Journal of Biological Sciences

Article Title: MANF Promotes Unexplained Recurrent Miscarriages by Interacting with NPM1 and Downregulating Trophoblast Cell Migration and Invasion

doi: 10.7150/ijbs.85378

Figure Lengend Snippet: Establishment of the mouse URM model and the effect of MANF protein level changes on mouse pregnancy. A. Representative photos of the gestational day (GD)14.5 uterus, fetus, and placenta of the control (n = 10) and URM mice (n = 15), and statistics of absorbed fetuses versus surviving fetal mice. B. Comparative images of GD14.5 uterus, fetus, and placenta of the URM group (n = 15), AAV9-shMANF-treated group (n = 15), and AAV9-shMANF+NSC348884-treated group (n = 15), and statistics of absorbed fetuses versus surviving fetal mice. The AAV9-shMANF+DMSO condition (n = 6) was used as the NSC348884-treated vehicle control. C. MANF protein concentrations in the peripheral blood of CBA/J female mice after two weeks in the blank treatment group (n = 6), AAV9-shMANF (n = 6), and AAV9-vector (n = 6). D. MANF protein concentrations in peripheral blood in the URM group, AAV9-shMANF-treated group, and AAV9-shMANF+NSC348884-treated group at GD14.5, with the AAV9-shMANF+DMSO control. E. Representative images and statistical analysis of immunohistochemical staining of MANF protein in three groups of mouse decidua tissue, scale bar = 100 μm, 50 μm. F. Representative images and statistical analysis of immunohistochemical staining of MMP2, MMP9, and PCNA in mouse decidua tissue, scale bar = 100 μm. G. Levels of MANF, NPM1 oligomers, p53, MMP2, MMP9, and PCNA in decidua tissue detected by western blotting. All results are from three or more independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The packaged lentiviruses overexpressing MANF (oe-MANF), knocking down MANF (sh-MANF), and overexpressing NPM1 (oe-NPM1) were designed and synthesized by Gene-Chem (Shanghai, China).

Techniques: Control, Plasmid Preparation, Immunohistochemical staining, Staining, Western Blot

Journal: International Journal of Biological Sciences

Article Title: MANF Promotes Unexplained Recurrent Miscarriages by Interacting with NPM1 and Downregulating Trophoblast Cell Migration and Invasion

doi: 10.7150/ijbs.85378

Figure Lengend Snippet: Schematic diagram of MANF and NPM1 interaction in the trophoblast during URM pathogenesis. In the trophoblasts of patients with URM, overexpressed MANF protein appears to undergo nuclear translocation and interacts with NPM1, resulting in increased ubiquitination of NPM1, decreased expression of NPM1, increased activation of the p53 signaling pathway, limited proliferation of trophoblastic cells, decreased invasion and migration functions, and induced miscarriage.

Article Snippet: The packaged lentiviruses overexpressing MANF (oe-MANF), knocking down MANF (sh-MANF), and overexpressing NPM1 (oe-NPM1) were designed and synthesized by Gene-Chem (Shanghai, China).

Techniques: Translocation Assay, Ubiquitin Proteomics, Expressing, Activation Assay, Migration

Journal: Signal Transduction and Targeted Therapy

Article Title: Phosphorylated NFS1 weakens oxaliplatin-based chemosensitivity of colorectal cancer by preventing PANoptosis

doi: 10.1038/s41392-022-00889-0

Figure Lengend Snippet: In vivo CRISPR screening reveals that NFS1 deficiency enhances the sensitivity of CRC cells to oxaliplatin (Oxa). a Diagram showing the strategy of the CRISPR-based screen in vivo ( n = 6). b Volcano plot illustrating the depleted or enriched genes in the oxaliplatin-treatment group compared with the control group based on the depletion or enrichment of sgRNAs. Each dot represents a gene whose knockout can enhance (blue) or reduce (red) the sensitivity of cells to oxaliplatin treatment. c Illustration of the top ten candidates depleted in the oxaliplatin-treatment group. The analyzed CRISPR screening data are provided in Supplementary Table . d Schematic illustration of Fe–S cluster biogenesis and the main enzymes involved in this process. e MTS analysis of the proliferation of HCT116 cells in which NFS1 or FDX2 is silenced. f Quantification of colony formation analysis reflecting the proliferation of control and NFS1 -knockdown HCT116 and DLD1 cells. g , h Cell viability of HCT116 and DLD1 cells treated with different concentrations of oxaliplatin for 48 h after NFS1 knockdown. i , j LDH analysis indicating the cytotoxicity of different concentrations of oxaliplatin for 48 h in HCT116 and DLD1 cells with NFS1 knockdown. k Live/dead viability/cytotoxicity assay showing the dead (red) and live (green) cells among control and NFS1 -knockdown HCT116 cells treated with 40 µM oxaliplatin for 24 h. Scale bar = 100 μm. l Quantification of the relative number of dead cells in ( k ). m , n Cell viability ( m ) and cytotoxicity ( n ) assessments of control and NFS1 -knockdown HCT116 cells treated or not treated with 40 µM oxaliplatin for 24 h in combination with the apoptosis inhibitor Z-VAD-FMK (VAD, 25 µM), the necroptosis inhibitor necrostatin (Nec, 20 µM), the ferroptosis inhibitor ferrostatin-1 (Fer, 10 µM), the pyroptosis inhibitors Ac-DMPD/DMLD-CMK (DMPD/DMLD, 20 µM) and disulfiram (dis, 1 µM) or the autophagy inhibitor 3-methyladenine (3-me, 10 µM). The data in ( e – j ) and ( l – n ) are representative of three independent experiments and presented as the mean ± SD. The P values in ( e – h ) were calculated by two-way ANOVA with Dunnett’s multiple comparisons test, those in ( l – n ) were calculated by one-way ANOVA with Tukey’s multiple comparisons test, and those in ( i , j ) were calculated by two-tailed unpaired Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Lentiviruses packaging NFS1 short hairpin RNAs (shRNAs) were synthesized by OBiO Technology (Shanghai, China).

Techniques: In Vivo, CRISPR, Control, Knock-Out, Knockdown, Cytotoxicity Assay, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Phosphorylated NFS1 weakens oxaliplatin-based chemosensitivity of colorectal cancer by preventing PANoptosis

doi: 10.1038/s41392-022-00889-0

Figure Lengend Snippet: NFS1 deficiency synergizes with oxaliplatin treatment to induce PANoptosis. a Representative images showing YP1 + cells (green) that may undergo apoptosis or necroptosis and PI + cells (red) that may undergo apoptosis, necroptosis, pyroptosis, or ferroptosis in control and NFS1 -knockdown HCT116 cells treated with 40 µM oxaliplatin for 24 h. The bottom panel shows representative bright fields, and the red arrowheads indicate the large bubbles emerging from the plasma membrane. Scale bar = 100 μm. b Quantification of the YP1 + and PI + cells from ( a ). c , d Flow cytometry ( c ) and quantification analysis ( d ) with Annexin V/PI staining evaluating the percentages of live cells (Annexin V − /PI − ), early apoptotic cells (Annexin V + /PI − ) and late apoptotic cells (Annexin V + /PI + ) among the control and NFS1 -knockdown DLD1 cells treated with PBS or oxaliplatin (80 μM, 24 h). e The lipid ROS levels in control and NFS1 -knockdown HCT116 cells treated with oxaliplatin (40 μM, 24 h) were assessed by the BODIPY™ 581/591 C11 probe assay. f – i Western blotting analysis of caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7, caspase-8, cleaved caspase-8, caspase-9, cleaved caspase-9, phosphorylated MLKL, total MLKL, phosphorylated RIP1, total RIP1, cleaved RIP1, GSDME, cleaved GSDME, GSDMD, cleaved GSDMD, TFRC, FTH1 expression in control and NFS1 -knockdown HCT116 ( f , g ) and DLD1 ( h , i ) cells treated with oxaliplatin (40 μM for HCT116 and 80 μM for DLD1, 24 h), GSDME and GSDMD are not expressed in DLD1 cells. Vinculin was included as a loading control. The data in ( b , d , e ) are representative of three independent experiments and presented as the mean ± SD. The P values in ( b , d ) were calculated by two-way ANOVA and those in ( e ) were calculated by one-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Lentiviruses packaging NFS1 short hairpin RNAs (shRNAs) were synthesized by OBiO Technology (Shanghai, China).

Techniques: Control, Knockdown, Clinical Proteomics, Membrane, Flow Cytometry, Staining, Western Blot, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Phosphorylated NFS1 weakens oxaliplatin-based chemosensitivity of colorectal cancer by preventing PANoptosis

doi: 10.1038/s41392-022-00889-0

Figure Lengend Snippet: NFS1 deficiency enhances the antitumor effect of oxaliplatin in vivo. a , b Statistical analysis of CDX tumor volumes ( a ) and weights ( b ) in nude mice after implantation of NFS1 -knockdown or control DLD1 cells, followed by i.p. injection of oxaliplatin (7.5 mg/kg) or PBS ( n = 5, Bliss synergy P value is shown). c , d Quantification of the proliferation index (Ki67 staining) ( c ) and apoptotic index (TUNEL staining) ( d ) of DLD1-based xenograft tumors. e Illustration of the methodology used to establish CRC PDX models. f Photographs of the excised tumors from PDX #1 (left) and PDX #2 (right) models after intratumoral injection of in vivo-optimized NFS1 inhibitor (si NFS1 ) or the control, followed by i.p. injection of oxaliplatin (7.5 mg/kg) or PBS (PDX #1, n = 5; PDX #2, n = 4) and comparison of the tumor sizes. g , h Statistical analysis of the tumor volumes ( g , Bliss synergy P values are shown) and weights ( h ) in nude mice from the PDX #1 (left) and PDX #2 (right) models. i Representative H&E and IHC staining images of NFS1, Ki67, and TUNEL in PDX #1-based paraffin-embedded subcutaneous tumor sections. Scale bar = 50 μm. j , k Quantification of the proliferation index (Ki67 staining) ( j ) and apoptotic index (TUNEL staining) ( k ) of the PDX #1 (left) and PDX #2 (right) models. The data in ( a – d , g , h , j , k ) (PDX #1) are representative of five independent experiments and those in ( g , h , j , k ) (PDX #2) are representative of four independent experiments. All the data are presented as mean ± SD. The P values in ( a , g ) were calculated by two-way ANOVA, and those in ( b – d , h , j , k ) were calculated by one-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Lentiviruses packaging NFS1 short hairpin RNAs (shRNAs) were synthesized by OBiO Technology (Shanghai, China).

Techniques: In Vivo, Knockdown, Control, Injection, Staining, TUNEL Assay, Comparison, Immunohistochemistry

Journal: Signal Transduction and Targeted Therapy

Article Title: Phosphorylated NFS1 weakens oxaliplatin-based chemosensitivity of colorectal cancer by preventing PANoptosis

doi: 10.1038/s41392-022-00889-0

Figure Lengend Snippet: Oxidative stress is critical for NFS1 deficiency-induced PANoptosis under oxaliplatin treatment. a Gene Ontology (GO) enrichment analysis of genes that exhibited twofold upregulation under oxaliplatin treatment (40 μM, 24 h) in the NFS1-knockdown group compared with the control group. b ROS analysis of control and NFS1 -knockdown HCT116 and DLD1 cells under PBS or oxaliplatin treatment (40 μM/80 μM, 24 h). c Cell viability of HCT116 cells treated with 40 μM oxaliplatin combined with 5 mM NAC or 5 mM GSH for 24 h after NFS1 knockdown. d Lipid ROS analysis of HCT116 cells treated with 40 μM oxaliplatin combined with 5 mM NAC for 24 h after NFS1 knockdown. e Representative images showing YP1 + cells (green) which may undergo apoptosis or necroptosis and PI + cells (red) which may undergo apoptosis, necroptosis, pyroptosis, or ferroptosis among control and NFS1 -knockdown HCT116 cells treated with 40 μM oxaliplatin combined with 5 mM NAC (24 h) after NFS1 knockdown. The bottom panel shows representative bright fields and the red arrowheads indicate the large bubbles emerging from the plasma membrane. Scale bar = 100 μm. f ROS analysis of control and NFS1 -knockdown HCT116 cells under cisplatin (40 μM, 24 h) or H 2 O 2 (100 μM, 24 h) treatment. g , h Cell viability of HCT116 cells treated with 100 μM H 2 O 2 ( g ) and 40 μM cisplatin ( h ) combined with 5 mM NAC for 24 h after NFS1 knockdown. i Cell cytotoxicity assessments of control and NFS1 -knockdown HCT116 cells under cisplatin (40 μM, 24 h) or H 2 O 2 (100 μM, 24 h) treatment. j Western blotting analysis of caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7, caspase-8, cleaved caspase-8, caspase-9, cleaved caspase-9, phosphorylated MLKL, total MLKL, GSDME, cleaved GSDME and TFRC expression in control and NFS1 -knockdown HCT116 cells after treatment with 40 μM oxaliplatin combined with 5 mM NAC (24 h). k – m Photograph showing the gross comparison ( k ), tumor volumes ( l ), and weights ( m ) of control and NFS1 -knockdown HCT116 CDX tumors in nude mice subjected to i.p. injection of oxaliplatin (7.5 mg/kg) and NAC (1 mg/ml) in the drinking water ( n = 5). Vinculin was included as a loading control. The data in ( b – d , f – i ) are representative of three independent experiments and those in ( l , m ) are representative of five independent experiments, and all are presented as the mean ± SD. The P values in ( b , l ) were calculated by two-way ANOVA, and those in ( c , d , f – i , m ) were calculated by one-way ANOVA with Tukey’s multiple comparisons test. ** P < 0.01, *** P < 0.001

Article Snippet: Lentiviruses packaging NFS1 short hairpin RNAs (shRNAs) were synthesized by OBiO Technology (Shanghai, China).

Techniques: Knockdown, Control, Clinical Proteomics, Membrane, Western Blot, Expressing, Comparison, Injection

Journal: Signal Transduction and Targeted Therapy

Article Title: Phosphorylated NFS1 weakens oxaliplatin-based chemosensitivity of colorectal cancer by preventing PANoptosis

doi: 10.1038/s41392-022-00889-0

Figure Lengend Snippet: NFS1 prevents PANoptosis under oxaliplatin treatment depending on its phosphorylation level at S293. a Western blotting detection of NFS1 expression in HCT116 and 293T cells treated with different concentrations of oxaliplatin for 48 h. b IP analysis demonstrating the post-translational modifications level of NFS1 in 293T cells treated with oxaliplatin (40 μM, 24 h), including phosphorylation (P-Thr/Ser), acetylation (Pan-Ace), and ubiquitination (Pan-ubi). c IP analysis showing the level of phosphorylated threonine (P-Thr) and serine (P-Ser) residues of NFS1 in 293T cells treated with oxaliplatin (40 μM, 24 h). d , e IP analysis demonstrating the serine phosphorylation level of NFS1 in cells overexpressing WT NFS1 or the S293A or S293D mutant of NFS1 in the absence ( d ) or presence of oxaliplatin (40 μM, 24 h) ( e ). f ACO1 activity analysis of NFS1 in control and NFS1 -knockdown HCT116 cells when overexpressed rNFS1 WT and S293A or S293D mutant with oxaliplatin treatment (40 μM, 24 h). g – j ROS level ( g ), cell viability ( h ), YP1 + and PI + cells ( i ), and lipid ROS levels ( j ) in HCT116 cells overexpressing rNFS1 WT and S293A or S293D mutant with or without oxaliplatin treatment (40 μM, 24 h). The bottom panel in ( i ) shows representative bright fields, and the red arrowheads indicate the large bubbles emerging from the plasma membrane, Scale bar = 100 μm. k Western blotting analysis of caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7, caspase-8, cleaved caspase-8, caspase-9, cleaved caspase-9, phosphorylated MLKL, total MLKL, GSDME, cleaved GSDME, and TFRC expression in HCT116 cells overexpressing rNFS1 WT or the S293A or S293D mutant in the absence or presence of oxaliplatin (40 μM, 24 h). l , m Statistical analysis of the tumor volumes ( l ) and weights ( m ) in nude mice after implantation of NFS1 -knockdown HCT116 cells overexpressing rNFS1 WT or the S293A or S293D mutant, followed by an i.p. injection of oxaliplatin (7.5 mg/kg) ( n = 5). Vinculin or flag was included as a loading control. The data in ( f – h , j ) are representative of three independent experiments, and those in ( l , m ) are representative of five independent experiments, and all are presented as the mean ± SD. The P values in ( f – h , j , m ) were calculated by one-way ANOVA, and those in ( l ) were calculated by two-way ANOVA with Tukey’s multiple comparisons test. ** P < 0.01, *** P < 0.001

Article Snippet: Lentiviruses packaging NFS1 short hairpin RNAs (shRNAs) were synthesized by OBiO Technology (Shanghai, China).

Techniques: Phospho-proteomics, Western Blot, Expressing, Ubiquitin Proteomics, Mutagenesis, Activity Assay, Control, Knockdown, Clinical Proteomics, Membrane, Injection

Journal: Signal Transduction and Targeted Therapy

Article Title: Phosphorylated NFS1 weakens oxaliplatin-based chemosensitivity of colorectal cancer by preventing PANoptosis

doi: 10.1038/s41392-022-00889-0

Figure Lengend Snippet: NFS1 is transcriptionally regulated by MYC. a Hallmark enriched pathway analysis showing pathways associated with the expression of NFS1. b , c Gene set enrichment analysis suggesting that NFS1 is positively correlated with MYC pathways. d Q-PCR analysis showing that NFS1 expression is positively correlated with MYC expression in CRC tumor tissues from SYSUCC ( n = 115, Pearson’s correlation analysis). e , f Q-PCR analysis ( e ) and western blotting analysis ( f ) of NFS1 and MYC expression in HCT116 and 293T cells with control or silenced expression of MYC. g , h Dual-luciferase promoter activity analysis showing the transcriptional activity of NFS1 in HCT116 and 293T cells with MYC downregulation ( g ) or upregulation ( h ). i Schematic illustration of the NFS1 promoter containing two main MYC-binding sites (−531 to −495 and −485 to −454). The strategy for mutating the promoter is also shown. j , k Agarose gel electrophoresis ( j ) and Q-PCR ( k ) assay after ChIP analysis showing the occupancy of MYC on the NFS1 promoter (region −478 to −338) in HCT116 and 293T cells. l Luciferase promoter activity analysis of NFS1 transcriptional activity in 293T cells overexpressing NFS1 WT or truncation mutant ( i ). Vinculin was included as a loading control. The data in ( e , g , h , k , l ) are representative of three independent experiments and presented as the mean ± SD. The P values in ( e ) were calculated by two-way ANOVA with Dunnett’s multiple comparisons test, those in ( g , h , k , l ) were calculated by two-tailed unpaired Student’s t test, and this in ( d ) was calculated by Pearson’s correlation analysis and chi-square test. ** P < 0.01, *** P < 0.001

Article Snippet: Lentiviruses packaging NFS1 short hairpin RNAs (shRNAs) were synthesized by OBiO Technology (Shanghai, China).

Techniques: Expressing, Western Blot, Control, Luciferase, Activity Assay, Binding Assay, Agarose Gel Electrophoresis, Mutagenesis, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Phosphorylated NFS1 weakens oxaliplatin-based chemosensitivity of colorectal cancer by preventing PANoptosis

doi: 10.1038/s41392-022-00889-0

Figure Lengend Snippet: NFS1 is highly expressed in CRC and indicates poor prognosis. a – c Q-PCR detection of NFS1 mRNA expression in 115 pairs of primary CRC tumor tissues (T) and adjacent normal tissues (N) ( a ); 26 pairs of lymph-node metastases (LNM) ( b ); and 48 pairs of CRC tissues with recurrence (Re) and without recurrence (T) ( c ) . d Western blotting analysis of NFS1 protein expression in eight pairs of CRC tumor tissues (T) and adjacent normal tissues (N). e , f Representative IHC staining images of NFS1. Scale bar = 100 μm ( e ); and IHC staining scores of NFS1 expression in paired primary CRC tumor tissues (T) and adjacent normal tissues (N) from patients at different cancer phases (phase I, n = 22; phase II, n = 109; phase III, n = 176; phase IV, n = 64) ( f ). All samples were obtained from SYSUCC. g Overall survival (left) and disease-free survival (right) assays of patients with CRC based on NFS1 protein level from ( e , f ) . h Representative IHC staining images showing NFS1, Ki67, and MYC expression in CRC tumor tissues. Scale bar = 100 μm. i , j Correlations between NFS1 expression and Ki67 ( i ) and MYC ( j ) expression ( n = 340). k Representative images showing NFS1 expression in patients with FOLFOX or XELOX chemotherapy. l Correlation between NFS1 expression with the response of patients with CRC to FOLFOX or XELOX chemotherapy ( n = 61, PD progressive disease, SD stable disease, PR partial response, CR complete response). m Proposed working model based on this study. The model shows that phosphorylated NFS1 weakens platinum-based chemosensitivity by reducing the level of ROS to prevent PANoptosis. Vinculin was included as a loading control. The data in ( a – c , f ) are presented in the form of box and whisker plots (minimum–maximum) with the horizontal line in each box representing the median, and those in ( i , j , l ) are presented as the percentage of total samples. The P values in ( a , c , f ) were calculated by two-tailed paired Student’s t test, this in ( b ) was calculated by one-way ANOVA with Tukey’s multiple comparisons test, this in ( g ) was calculated by Kaplan–Meier analysis with the log-rank test, two-sided, and those in ( i , j , l ) were calculated by Pearson’s correlation analysis and chi-square test. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Lentiviruses packaging NFS1 short hairpin RNAs (shRNAs) were synthesized by OBiO Technology (Shanghai, China).

Techniques: Expressing, Western Blot, Immunohistochemistry, Control, Whisker Assay, Two Tailed Test

Journal: Frontiers in Physiology

Article Title: Long Non-coding RNA MIAT Knockdown Prevents the Formation of Intracranial Aneurysm by Downregulating ENC1 via MYC

doi: 10.3389/fphys.2020.572605

Figure Lengend Snippet: The shRNA sequences.

Article Snippet: In this experiment, the construction, identification and sequencing of lentiviral vectors for MIAT and ENC1 genes, plasmid extraction and packaging of lentiviruses were all completed by Shanghai SunBio Biomedical Technology Co., Ltd. (Shanghai, China).

Techniques: shRNA

Journal: Frontiers in Physiology

Article Title: Long Non-coding RNA MIAT Knockdown Prevents the Formation of Intracranial Aneurysm by Downregulating ENC1 via MYC

doi: 10.3389/fphys.2020.572605

Figure Lengend Snippet: Primer sequence for RT-qPCR.

Article Snippet: In this experiment, the construction, identification and sequencing of lentiviral vectors for MIAT and ENC1 genes, plasmid extraction and packaging of lentiviruses were all completed by Shanghai SunBio Biomedical Technology Co., Ltd. (Shanghai, China).

Techniques: Sequencing

Journal: Frontiers in Physiology

Article Title: Long Non-coding RNA MIAT Knockdown Prevents the Formation of Intracranial Aneurysm by Downregulating ENC1 via MYC

doi: 10.3389/fphys.2020.572605

Figure Lengend Snippet: MIAT upregulates ENC1 expression through MYC. (A) The subcellular localization of MIAT. CN RCI < 0 indicates that lncRNA is expressed in the nucleus, and CN RCI > 0 indicates that lncRNA is expressed in the cytoplasm. (B) After the nucleus and cytoplasm of HBEC-5i cells were separated, MIAT expression was detected by RT-qPCR. GAPDH was a marker of cytoplasm and U6 was a marker of nucleus. (C) The downstream TFs of MIAT analyzed by PPI analysis. The redder the color, the higher the core degree was Vice versa, the bluer the color, the lower the core degree was. (D) RNA pull down to detect the binding of MYC with MIAT. (E) RIP to detect the binding of MYC with MIAT. (F) The Venn map of the differentially expressed genes in the microarray GSE75436 and the downstream genes of TF MYC, and the intersection was ENC1. (G) The enrichment of MYC binding to the promoter region of ENC1 by ChIP. (H) MIAT expression in endothelial cells after overexpressing or silencing MIAT detected by RT-qPCR. (I) MYC mRNA expression in endothelial cells after overexpressing or silencing MIAT detected by RT-qPCR. (J) ENC1 expression in endothelial cells after overexpressing or silencing MIAT detected by RT-qPCR. (K) Protein expression of MYC and ENC1 in endothelial cells after overexpressing or silencing MIAT determined by Western blot analysis. (L) The regulatory effect on ENC1 by MIAT and MYC detected by dual luciferase reporter gene assay. * p < 0.05, ns p > 0.05. The measurement data were expressed as mean ± standard derivation, comparisons among multiple groups were analyzed by one-way ANOVA and followed by Tukey’s post hoc test, and the cell experiment was conducted three times independently.

Article Snippet: In this experiment, the construction, identification and sequencing of lentiviral vectors for MIAT and ENC1 genes, plasmid extraction and packaging of lentiviruses were all completed by Shanghai SunBio Biomedical Technology Co., Ltd. (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Marker, Binding Assay, Microarray, Western Blot, Luciferase, Reporter Gene Assay

Journal: Frontiers in Physiology

Article Title: Long Non-coding RNA MIAT Knockdown Prevents the Formation of Intracranial Aneurysm by Downregulating ENC1 via MYC

doi: 10.3389/fphys.2020.572605

Figure Lengend Snippet: ENC1 expression is augmented in IA patients, and ENC1 silencing negates MIAT-induced endothelial cell apoptosis. (A) ENC1 expression in ruptured IA, unruptured IA and control samples determined by RT-qPCR. (B) Analysis of the correlation between ENC1 expression and MIAT expression. (C) The correlation between the expression of ENC1 and disease-free survival of patients with IA. (D) The correlation between the expression of ENC1 and overall survival of patients with IA. (E) The protein expression of ENC1 in vascular endothelial cells in vitro in response to oe-MIAT and sh-ENC1 alone or in combination measured by Western blot analysis. (F) Flow cytometry analysis for apoptosis of endothelial cells in response to oe-MIAT and sh-ENC1 alone or in combination. (G) The expression of apoptosis-related factors (cleaved Caspase-3, cleaved PARP1, Bax, and Bcl-2) in response to oe-MIAT and sh-ENC1 alone or in combination measured by Western blot analysis. * p < 0.05. The measurement data were expressed as mean ± standard derivation. Comparisons between two groups were analyzed by unpaired t test, while the correlation between the expression of ENC1 in patients with IA and their disease-free survival and overall survival was analyzed by Kaplan–Meier method (long-rank test). Comparisons among multiple groups were analyzed by one-way ANOVA followed by Tukey’s post hoc test, and the cell experiment was conducted three times independently.

Article Snippet: In this experiment, the construction, identification and sequencing of lentiviral vectors for MIAT and ENC1 genes, plasmid extraction and packaging of lentiviruses were all completed by Shanghai SunBio Biomedical Technology Co., Ltd. (Shanghai, China).

Techniques: Expressing, Control, Quantitative RT-PCR, In Vitro, Western Blot, Flow Cytometry

Journal: Frontiers in Physiology

Article Title: Long Non-coding RNA MIAT Knockdown Prevents the Formation of Intracranial Aneurysm by Downregulating ENC1 via MYC

doi: 10.3389/fphys.2020.572605

Figure Lengend Snippet: Silencing ENC1 reduces endothelial cell apoptosis in vivo to protect rats from IA. Rats were induced with IA model and treated with sh-ENC1 or sh-NC, with the sham-operated rats as control. (A) The expression of ENC1 in rats determined by RT-qPCR. (B) HE staining (×200) for the ACA/OA branch sections of rats. (C) The representative images of TUNEL staining (×200) for apoptosis of rats (arrows indicate the arterial wall). (D) Quantitative analysis of cell apoptosis in rats evaluated by TUNEL staining. (E) The expression of apoptosis-related factors (cleaved Caspase-3, cleaved PARP1, Bax, and Bcl-2) as determined by Western blot analysis and quantified by Image J software. * p < 0.05. The measurement data were expressed as mean ± standard derivation. Comparisons between two groups were analyzed by unpaired t test, and comparisons among multiple groups were analyzed by one-way ANOVA and followed by Tukey’s post hoc test. The cell experiment was conducted three times independently. The sham group: n = 12, the IA + sh-NC group: n = 12, and the IA + sh-ENC1 group: n = 6.

Article Snippet: In this experiment, the construction, identification and sequencing of lentiviral vectors for MIAT and ENC1 genes, plasmid extraction and packaging of lentiviruses were all completed by Shanghai SunBio Biomedical Technology Co., Ltd. (Shanghai, China).

Techniques: In Vivo, Control, Expressing, Quantitative RT-PCR, Staining, TUNEL Assay, Western Blot, Software

Journal: Frontiers in Physiology

Article Title: Long Non-coding RNA MIAT Knockdown Prevents the Formation of Intracranial Aneurysm by Downregulating ENC1 via MYC

doi: 10.3389/fphys.2020.572605

Figure Lengend Snippet: The mechanism diagram illustrating the effects of the MIAT/MYC/ENC1 axis on endothelial cell apoptosis in IA. MIAT enhanced the expression of ENC1 through MYC, thereby promoting vascular endothelial cell apoptosis and further inducing the pathogenesis of IA.

Article Snippet: In this experiment, the construction, identification and sequencing of lentiviral vectors for MIAT and ENC1 genes, plasmid extraction and packaging of lentiviruses were all completed by Shanghai SunBio Biomedical Technology Co., Ltd. (Shanghai, China).

Techniques: Expressing