Journal: Arthritis and rheumatism

Article Title: A Wnt/β-catenin negative feedback loop inhibits interleukin-1-induced matrix metalloproteinase expression in human articular chondrocytes.

doi: 10.1002/art.34425

Figure Lengend Snippet: Figure 3. Cross-talk of Wnt/-catenin and interleukin-1 (IL-1) pathways. A, Messenger RNA expression of Wnt-7B, MMP-1, and MMP-13, and the Wnt antagonists Dkk-1, Frizzled-related protein (FRZB), and Wnt inhibitory factor 1 (WIF-1) was evaluated at the indicated time points after stimulation with IL-1 by quantitative polymerase chain reaction (qPCR). Data represent the mean fold change in cells from 3 donors relative to untreated cells, which was set at 1. Each experiment was performed in triplicate. At 4 hours, P 0.05 for MMP-1, MMP-13, Dkk-1, and WIF-1 versus untreated cells; at 72 hours, P 0.05 for Wnt-7B and MMP-13 versus untreated cells; for comparisons at all other time points, P 0.01. At 4 hours and 72 hours, FRZB expression was not significantly changed. B, Human chondrocytes were stimulated with 20 ng/ml IL-1 for 48 hours, and nuclear and cytoplasmic -catenin protein expression was detected by immunoblotting. C, Human Wnt-3 and Wnt-7B were overexpressed using lentiviral transduction in human chondrocytes, and MMP mRNA expression was measured by qPCR. Lentiviral green fluorescent protein (GFP)–transduced cells (Lenti-GFP) were used as control. D, Wnt-7B was knocked down by lentivirus-mediated expression of short hairpin RNA (shRNA) in human chondrocytes using scrambled shRNA (shScr) as control. MMP-1 and MMP-13 mRNA expression was measured 24 hours after stimulation with 10 ng/ml IL-1 and compared with control. Values in C and D are the mean SD of triplicate cultures (n 1 donor). See Figure 1 for other definitions.

Article Snippet: Human Wnt-3 and Wnt-7B cDNAs (OriGene) were cloned into a pBOB lentiviral vector (plasmid 12335; Addgene) (22).

Techniques: RNA Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transduction, Control, shRNA

Journal: Frontiers in Oncology

Article Title: PMAIP1 regulates the progression of follicular thyroid carcinoma through the Wnt3/FOSL1 pathway

doi: 10.3389/fonc.2025.1502391

Figure Lengend Snippet: Primer sequences used in this study.

Article Snippet: Knockdown plasmid of PMAIP1 (sh-PMAIP1: 5’-GTGCTACTCAACTCAGGAGAT -3’), sh-negative control (sh-NC), over-expression plasmid of PMAIP1 (NM_001382618.1), Vector, knockdown plasmid of Wnt3 (sh-Wnt3: 5’-GCGCTTCTGCCGCAATTACAT-3’), siRNA of FOSL1 (siFOSL1:5’-CCUCAGCUCAUCGCAAGAGUA-3’) and siNC were ordered form Guangzhou Fulengen Co, Ltd. 5μg plasmids (or 5μmol siRNA) were transfected into FTC133 and FTC238 by using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions.

Techniques:

Journal: Frontiers in Oncology

Article Title: PMAIP1 regulates the progression of follicular thyroid carcinoma through the Wnt3/FOSL1 pathway

doi: 10.3389/fonc.2025.1502391

Figure Lengend Snippet: PMAIP1 was upregulated in FTC. (A) RNA-sequencing expression profiles and corresponding clinical data for FTC (n=106) were obtained from TCGA dataset. Normal control datasets (n=653) were sourced from the GTEx data portal. PMAIP1 mRNA expression was higher in FTC tissues relative to normal tissues at all stages, *** P < 0.001. (B) In stages I (n=58), II (n=23), III (n=18), and IV (n=6), the expression level of PMAIP1 was all significantly elevated in FTC tissues compared to normal tissues (n=653), *** P < 0.001. (C) 8 FTC tissues and 8 paired adjacent normal tissues were detected by qRT-PCR, showing that PMAIP1 expression was upregulated in FTC tissues compared with paired adjacent normal tissues, ** P < 0.01. (D) Cell lines were used to explore the PMAIP1 level. Compared with NTHY-ORI3-1, PMAIP1 expression was upregulated in both FTC133 and FTC238, *** P < 0.001.

Article Snippet: Knockdown plasmid of PMAIP1 (sh-PMAIP1: 5’-GTGCTACTCAACTCAGGAGAT -3’), sh-negative control (sh-NC), over-expression plasmid of PMAIP1 (NM_001382618.1), Vector, knockdown plasmid of Wnt3 (sh-Wnt3: 5’-GCGCTTCTGCCGCAATTACAT-3’), siRNA of FOSL1 (siFOSL1:5’-CCUCAGCUCAUCGCAAGAGUA-3’) and siNC were ordered form Guangzhou Fulengen Co, Ltd. 5μg plasmids (or 5μmol siRNA) were transfected into FTC133 and FTC238 by using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions.

Techniques: RNA Sequencing, Expressing, Control, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: PMAIP1 regulates the progression of follicular thyroid carcinoma through the Wnt3/FOSL1 pathway

doi: 10.3389/fonc.2025.1502391

Figure Lengend Snippet: Knockdown of PMAIP1 significantly inhibited the proliferation and metastasis of FTC in vitro . (A) Stable knockdown of PMAIP1 by shRNA in FTC133 and FTC238 was confirmed by WB, * P < 0.05, ** P < 0.01. (B) CCK-8 assay showed that the knockdown of PMAIP1 in FTC133 and FTC238 cells resulted in a significant decrease in cell proliferation compared with the NC group, *** P < 0.001. (C) Colony formation assay showed the knockdown of PMAIP1 markedly decreased the colony formation number of FTC133 and FTC238 cells compared with the NC group, ** P < 0.01. (D) The wound healing assay suggested the knockdown of PMAIP1 substantially impaired the wound-healing ability of FTC133 and FTC238 cells, *** P < 0.001. (E) Migration and invasion assay showed the knockdown of PMAIP1 significantly reduced the number of invasive and migrating FTC133 cells, ** P < 0.01, *** P < 0.001. (F) Migration and invasion assay showed the knockdown of PMAIP1 significantly decreased the number of invasive and migrating FTC238 cells, *** P < 0.001, * P < 0.05.

Article Snippet: Knockdown plasmid of PMAIP1 (sh-PMAIP1: 5’-GTGCTACTCAACTCAGGAGAT -3’), sh-negative control (sh-NC), over-expression plasmid of PMAIP1 (NM_001382618.1), Vector, knockdown plasmid of Wnt3 (sh-Wnt3: 5’-GCGCTTCTGCCGCAATTACAT-3’), siRNA of FOSL1 (siFOSL1:5’-CCUCAGCUCAUCGCAAGAGUA-3’) and siNC were ordered form Guangzhou Fulengen Co, Ltd. 5μg plasmids (or 5μmol siRNA) were transfected into FTC133 and FTC238 by using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions.

Techniques: Knockdown, In Vitro, shRNA, CCK-8 Assay, Colony Assay, Wound Healing Assay, Migration, Invasion Assay

Journal: Frontiers in Oncology

Article Title: PMAIP1 regulates the progression of follicular thyroid carcinoma through the Wnt3/FOSL1 pathway

doi: 10.3389/fonc.2025.1502391

Figure Lengend Snippet: Knockdown of PMAIP1 significantly inhibited the proliferation and metastasis of FTC in vivo . (A) Experimental flowchart of subcutaneous tumor transplantation in NTG mice. (B) Compared with the NC group, the weight and volume of tumors of FTC133 were significantly reduced (*** P < 0.001) and the body weight of mice was increased (*** P < 0.001) by knockdown of PMAIP1. (C) Compared with the NC group, the weight and volume of tumors of FTC238 were significantly reduced (*** P < 0.001) and the body weight of mice was increased (** P < 0.01) by knockdown of PMAIP1. (D) Knockdown of PMAIP1 decreased the Ki67 positive cells percentage, MMP2 and MMP9 positive area percentage in FTC133 subcutaneous xenografts determined by IHC staining, *** P < 0.001. (E) Knockdown of PMAIP1 decreased the Ki67 positive cells percentage, MMP2 and MMP9 positive area percentage in FTC238 subcutaneous xenografts determined by IHC staining, *** P < 0.001.

Article Snippet: Knockdown plasmid of PMAIP1 (sh-PMAIP1: 5’-GTGCTACTCAACTCAGGAGAT -3’), sh-negative control (sh-NC), over-expression plasmid of PMAIP1 (NM_001382618.1), Vector, knockdown plasmid of Wnt3 (sh-Wnt3: 5’-GCGCTTCTGCCGCAATTACAT-3’), siRNA of FOSL1 (siFOSL1:5’-CCUCAGCUCAUCGCAAGAGUA-3’) and siNC were ordered form Guangzhou Fulengen Co, Ltd. 5μg plasmids (or 5μmol siRNA) were transfected into FTC133 and FTC238 by using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions.

Techniques: Knockdown, In Vivo, Transplantation Assay, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: PMAIP1 regulates the progression of follicular thyroid carcinoma through the Wnt3/FOSL1 pathway

doi: 10.3389/fonc.2025.1502391

Figure Lengend Snippet: PMAIP1 activated the Wnt signaling pathway. (A) Venn diagram of downregulated genes in FTC133 and FTC238 cells and 82 common genes were detected. (B) The top 10 enrichment KEGG pathways of 82 intersection genes were shown. (C) As the Wnt signaling pathway showed, expression of Wnt, fra-1, and PLC were decreased after the knockdown of PMAIP1. (D) Spearman’s correlation analysis of TCGA dataset suggested that the expression level of PMAIP1 was directly proportional to Wnt3 in FTC and the expression levels of PMAIP1 are directly proportional to FOSL1 in FTC. (E) In FTC133 and FTC238 cell lines, the expression levels of Wnt3 and FOSL1 were both decreased after PMAIP1 was knocked down, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Knockdown plasmid of PMAIP1 (sh-PMAIP1: 5’-GTGCTACTCAACTCAGGAGAT -3’), sh-negative control (sh-NC), over-expression plasmid of PMAIP1 (NM_001382618.1), Vector, knockdown plasmid of Wnt3 (sh-Wnt3: 5’-GCGCTTCTGCCGCAATTACAT-3’), siRNA of FOSL1 (siFOSL1:5’-CCUCAGCUCAUCGCAAGAGUA-3’) and siNC were ordered form Guangzhou Fulengen Co, Ltd. 5μg plasmids (or 5μmol siRNA) were transfected into FTC133 and FTC238 by using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Knockdown

Journal: Frontiers in Oncology

Article Title: PMAIP1 regulates the progression of follicular thyroid carcinoma through the Wnt3/FOSL1 pathway

doi: 10.3389/fonc.2025.1502391

Figure Lengend Snippet: PMAIP1 modulated the proliferation and metastasis of FTC by regulating the Wnt signaling pathway. (A) Overexpression of PMAIP1 in FTC133 and FTC238 was confirmed by qRT-PCR. The overexpression of PMAIP1 led to elevated levels of Wnt3 and FOSL1. Conversely, the expression levels of Wnt3 and FOSL1 were significantly diminished in the Vector+shWnt3 group compared to the Vector+shNC group; in the meantime, a comparative analysis revealed that the expression levels of Wnt3 and FOSL1 were significantly reduced in the PMAIP1+shWnt3 group relative to the PMAIP1+shNC group, ** P < 0.01, *** P < 0.001. (B) CCK-8 assay was used to determine the proliferation ability of cells. The proliferation ability of FTC133 and FTC238 in the PMAIP1+shNC group were promoted compared with the Vector+shNC group, *** P < 0.001. Compared with the Vector+shNC group, the knockdown of Wnt3 significantly decreased the proliferation ability of FTC133 and FTC238 in the Vector+shWnt3 group, *** P < 0.001. Compared with the PMAIP1+shNC group, the knockdown of Wnt3 significantly decreased the proliferation ability of FTC133 and FTC238 in the PMAIP1+shWnt3 group, *** P < 0.001. (C) The colony formation numbers of FTC133 and FTC238 in the PMAIP1+shNC group were promoted compared with the Vector+shNC group, ** P < 0.01, ***P < 0.001. Compared with the Vector+shNC group, knockdown of Wnt3 significantly decreased the colony formation numbers of FTC133 and FTC238 in the Vector+shWnt3 group, * P < 0.05, ** P < 0.01. Compared with the PMAIP1+shNC group, knockdown of Wnt3 significantly decreased the colony formation numbers of FTC133 and FTC238 in the PMAIP1+shWnt3 group, ** P < 0.01. (D) The wound-healing rates of FTC133 and FTC238 in the PMAIP1+shNC group were promoted compared with the Vector+shNC group, ** P < 0.01. Compared with the Vector+shNC group, the knockdown of Wnt3 significantly decreased the wound-healing rate of FTC133 and FTC238 in the Vector+shWnt3 group, ** P < 0.01. Compared with the PMAIP1+shNC group, the knockdown of Wnt3 significantly decreased the wound-healing rate of FTC133 and FTC238 in the PMAIP1+shWnt3 group, ** P < 0.01. (E, F) Migration and invasion cell numbers of FTC133 and FTC238 in the PMAIP1+shNC group were increased compared with the Vector+shNC group, ** P < 0.01, *** P < 0.001. Compared with the Vector+shNC group, migration and invasion cells of FTC133 and FTC238 significantly decreased in the Vector+shWnt3 group, ** P < 0.01, *** P < 0.001. Compared with the PMAIP1+shNC group, migration and invasion cells of FTC133 and FTC238 significantly decreased in the PMAIP1+shWnt3 group, ** P < 0.01, *** P < 0.001.

Article Snippet: Knockdown plasmid of PMAIP1 (sh-PMAIP1: 5’-GTGCTACTCAACTCAGGAGAT -3’), sh-negative control (sh-NC), over-expression plasmid of PMAIP1 (NM_001382618.1), Vector, knockdown plasmid of Wnt3 (sh-Wnt3: 5’-GCGCTTCTGCCGCAATTACAT-3’), siRNA of FOSL1 (siFOSL1:5’-CCUCAGCUCAUCGCAAGAGUA-3’) and siNC were ordered form Guangzhou Fulengen Co, Ltd. 5μg plasmids (or 5μmol siRNA) were transfected into FTC133 and FTC238 by using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions.

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Plasmid Preparation, CCK-8 Assay, Knockdown, Migration

Journal: Frontiers in Oncology

Article Title: PMAIP1 regulates the progression of follicular thyroid carcinoma through the Wnt3/FOSL1 pathway

doi: 10.3389/fonc.2025.1502391

Figure Lengend Snippet: PMAIP1 regulated the proliferation and metastasis of FTC by FOSL1. (A) Knockdown of FOSL1 in FTC133 and FTC238 was confirmed by qRT-PCR. The expression level of FOSL1 were significantly diminished in the Vector+siFOSL1 group compared to the Vector+siNC group; the expression level of FOSL1 were significantly reduced in the PMAIP1+ siFOSL1 group relative to the PMAIP1+siNC group, ** P < 0.01, *** P < 0.001. (B) Compared with the Vector+siNC group, the knockdown of FOSL1 significantly decreased the proliferation ability of FTC133 and FTC238 in the Vector+siFOSL1 group, *** P < 0.001. Compared with the PMAIP1+siNC group, the knockdown of FOSL1 significantly decreased the proliferation ability of FTC133 and FTC238 in the PMAIP1+siFOSL1 group, *** P < 0.001. (C) Compared with the Vector+siNC group, the knockdown of FOSL1 significantly decreased the colony formation numbers of FTC133 and FTC238 in the Vector+siFOSL1 group, * P < 0.05, ** P < 0.01. Compared with the PMAIP1+siNC group, knockdown of FOSL1 significantly decreased the colony formation numbers of FTC133 and FTC238 in the PMAIP1+siFOSL1 group, ** P < 0.01. (D) Compared with the Vector+siNC group, knockdown of FOSL1 significantly decreased the wound-healing rate of FTC133 and FTC238 in the Vector+siFOSL1 group, *** P < 0.001. Compared with the PMAIP1+siNC group, knockdown of FOSL1 significantly decreased the wound-healing rate of FTC133 and FTC238 in the PMAIP1+siFOSL1 group, *** P < 0.001. (E, F) Compared with the Vector+ siNC group, migration and invasion cells of FTC133 and FTC238 significantly decreased in the Vector+siFOSL1 group, * P < 0.05, ** P < 0.01, *** P < 0.001. Compared with the PMAIP1+siNC group, migration and invasion cells of FTC133 and FTC238 significantly decreased in the PMAIP1+ siFOSL1 group, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Knockdown plasmid of PMAIP1 (sh-PMAIP1: 5’-GTGCTACTCAACTCAGGAGAT -3’), sh-negative control (sh-NC), over-expression plasmid of PMAIP1 (NM_001382618.1), Vector, knockdown plasmid of Wnt3 (sh-Wnt3: 5’-GCGCTTCTGCCGCAATTACAT-3’), siRNA of FOSL1 (siFOSL1:5’-CCUCAGCUCAUCGCAAGAGUA-3’) and siNC were ordered form Guangzhou Fulengen Co, Ltd. 5μg plasmids (or 5μmol siRNA) were transfected into FTC133 and FTC238 by using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions.

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Plasmid Preparation, Migration

Journal: eLife

Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

doi: 10.7554/eLife.77376

Figure Lengend Snippet: ( A ) Confocal images of normal gastric epithelial cell line (HFE-145) and gastric cancer (GC) cell lines (MKN-28, MKN-7, and AGS) expressing LifeAct-GFP to visualise actin-based structures. Yellow arrows indicate examples of filopodia. ( B ) Quantification of filopodia length in GC cell lines MKN-28, MKN-7, and AGS (n=7, 8, 25; n=number of cells). Significance is calculated by Student’s t-test. ( C ) Immunofluorescent images of HFE, MKN-28, MKN-7, and AGS, stained with antibodies against Wnt3 (green) and actin (Phalloidin-iFluor594, red). Scale bar 10 µm. High-magnification images indicate an example of a Wnt3-bearing cytonemes. Scale bar 2.5 µm. ( D ) Quantification of Wnt3-positive filopodia in gastric epithelial (HFE-145) and cancer (AGS) cells as a percentage of total filopodia (number of cells analysed = 6, 8, 6). Significance is calculated by Student’s t-test. ( E ) Immunohistochemistry (IHC) images of AGS cells overexpressing Wnt3 and stained with an antibody against Wnt3 (green) and actin (iFluor594, red). Scale bar 10µm. High-magnification images highlight cytonemes. Scale bar 2.5 µm. ( F ) IHC images of AGS cells treated with the Porcupine inhibitor IWP2 (100 µM, 48 hr) and stained with an antibody against Wnt3 (green) and actin (iFluor594, red). ( G ) Live confocal cell imaging of AGS cells expressing Wnt3-mCherry and LifeAct-GFP. Cytoneme-localised Wnt3-mCherry highlighted by yellow arrows. ( H–J ) IHC images of AGS cells stained with antibodies against ( H ) Myosin-X (MyoX) and ( I ) Evi/Wntless (red) and Wnt3 (red) and ( J ) Evi/Wntless (green Scale bars 10 µm). Phalloidin labels actin (FITC-Phalloidin, green; Phalloidin-iFluor350, blue). Figure 1—source data 1. Western blot images of HFE, AGS, MKN7, and MKN28 cell lysates stained for Flot2. Beta-actin was used as the loading control.

Article Snippet: The following plasmids were used in transfections and/or microinjections: pCS2 + membrane-mCherry , pCAG-mGFP membrane-bound GFP (Addgene 14757), pEGFP-N1 Flot2-GFP , pEGFP-N1 ΔN-Flot2-GFP , 7×TRE SuperTOPFlash-NLS-mCherry , JNK KTR-mCherry , pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7-eGFP (from Rüdiger Rudolf), pEGFP-N1 LAMP1-mTurq2 (Addgene #98828), IRSp534K-mCherry/GFP , pCS2 + Wnt8a-mCherry , pCS2 + Ror2-mCherry (cloned in with ClaI and XbaI), pEGFP-N3 mRor2-∆CRD-GFP (XhoI-XhoI mRor2 insert taken from pcDNA-mRor2, subcloned into SalI site of pEGFP-N3 vector), pCS2 + Ror2-eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3-mCherry (cloned from Addgene plasmid pcDNA3.2-Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI).

Techniques: Expressing, Staining, Immunohistochemistry, Imaging, Western Blot, Control

Journal: eLife

Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

doi: 10.7554/eLife.77376

Figure Lengend Snippet: ( A ) Quantification of Flot2 expression levels (mRNA and protein) in HFE-145, AGS, MKN-28, and MKN-7 cells. Significance is calculated by Student’s t-test. ( B ) Quantification of filopodia length in HFE and AGS cells expressing indicated constructs. Significance is calculated by Student’s t-test with Bonferroni correction for multiple comparisons. ( C ) Antibody staining of AGS cells expressing ΔN-Flot2-GFP and stained for endogenous WT Flot2 (red), showing the effect of the dominant-negative mutant on Flot2 localisation. Scale bar 20 µm. ( D ) Analysis of efficiency of siRNA-mediated knock-down of Flot2 in AGS cells by Western blot. ( E ) Confocal image of an AGS cell with depleted Flot2 (siRNA-mediated) and expressing LifeAct-GFP to visualise actin. Yellow arrow highlights an example of lamellipodia frequently seen in Flot2-depleted cells. Scale bars 10 µm. Figure 3—figure supplement 1—source data 1. Flotillin-2 promotes filopodia formation. Figure 3—figure supplement 1—source data 2. Flotillin-2 promotes filopodia elongation.

Article Snippet: The following plasmids were used in transfections and/or microinjections: pCS2 + membrane-mCherry , pCAG-mGFP membrane-bound GFP (Addgene 14757), pEGFP-N1 Flot2-GFP , pEGFP-N1 ΔN-Flot2-GFP , 7×TRE SuperTOPFlash-NLS-mCherry , JNK KTR-mCherry , pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7-eGFP (from Rüdiger Rudolf), pEGFP-N1 LAMP1-mTurq2 (Addgene #98828), IRSp534K-mCherry/GFP , pCS2 + Wnt8a-mCherry , pCS2 + Ror2-mCherry (cloned in with ClaI and XbaI), pEGFP-N3 mRor2-∆CRD-GFP (XhoI-XhoI mRor2 insert taken from pcDNA-mRor2, subcloned into SalI site of pEGFP-N3 vector), pCS2 + Ror2-eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3-mCherry (cloned from Addgene plasmid pcDNA3.2-Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI).

Techniques: Expressing, Construct, Staining, Dominant Negative Mutation, Knockdown, Western Blot

Journal: eLife

Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

doi: 10.7554/elife.77376

Figure Lengend Snippet: Figure 5. Flotillin-2 is required for Ror2 membrane localisation, Ror2/PCP signalling and Ror2-mediated cytoneme formation. (A) Immunohistochemistry (IHC) analysis of AGS cells stained for Ror2 (red) and Flot2 (green). Flot2 and Ror2 show co-localisation with a Pearson’s correlation coefficient (PCC) of 0.65 (n=10), highlighted at the membrane by arrows. Scale bars represent 10 µm, and in high-magnification images, 2.5 µm (right). (B–D), Confocal live-cell imaging of AGS cells expressing Ror2-BFP with Flot2-GFP (B), Flot2 siRNA (c) or ∆N-Flot2-GFP (D) and memCherry. Arrows highlight subcellular

Article Snippet: The following plasmids were used in transfections and/or microinjections: pCS2 + membrane- mCherry (Mattes et al., 2018), pCAG- mGFP membrane- bound GFP (Addgene 14757), pEGFP- N1 Flot2- GFP (Neumann- Giesen et al., 2004), pEGFP- N1 ∆N- Flot2- GFP (Neumann- Giesen et al., 2004), 7×TRE SuperTOPFlash- NLS- mCherry (Moro et al., 2012), JNK KTR- mCherry (Regot et al., 2014), pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7- eGFP (from Rüdiger Rudolf), pEGFP- N1 LAMP1- mTurq2 (Addgene #98828), IRSp534K- mCherry/GFP (Stanganello et al., 2015), pCS2 + Wnt8a- mCherry (Stanganello et al., 2015), pCS2 + Ror2- mCherry (cloned in with ClaI and XbaI), pEGFP- N3 mRor2-∆CRD- GFP (XhoI- XhoI mRor2 insert taken from pcDNA- mRor2, subcloned into SalI site of pEGFP- N3 vector), pCS2 + Ror2- eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3- mCherry (cloned from Addgene plasmid pcDNA3.2- Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI).

Techniques: Membrane, Immunohistochemistry, Staining, Live Cell Imaging, Expressing

Journal: eLife

Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

doi: 10.7554/elife.77376

Figure Lengend Snippet: Figure 1. Gastric epithelial normal and cancer cell lines utilise cytonemes to transport Wnt3 intercellularly. (A) Confocal images of normal gastric epithelial cell line (HFE-145) and gastric cancer (GC) cell lines (MKN-28, MKN-7, and AGS) expressing LifeAct-GFP to visualise actin-based structures. Yellow arrows indicate examples of filopodia. (B) Quantification of filopodia length in GC cell lines MKN-28, MKN-7, and AGS (n=7, 8, 25; n=number of cells). Significance is calculated by Student’s t-test. (C) Immunofluorescent images of HFE, MKN-28, MKN-7, and AGS, stained with antibodies against Wnt3 (green) and actin (Phalloidin-iFluor594, red). Scale bar 10 µm. High-magnification images indicate an example of a Wnt3-bearing cytonemes. Scale bar 2.5 µm. (D) Quantification of Wnt3-positive filopodia in gastric epithelial (HFE-145) and cancer (AGS) cells as a percentage of total filopodia (number of cells analysed = 6, 8, 6). Significance is calculated by Student’s t-test. (E) Immunohistochemistry (IHC) images of AGS cells overexpressing Wnt3 and stained with an antibody against Wnt3 (green) and actin (iFluor594, red). Scale bar 10µm. High-magnification images highlight cytonemes. Scale bar 2.5 µm. (F) IHC images of AGS cells treated with the Porcupine inhibitor IWP2 (100 µM, 48 hr) and stained with an antibody against Wnt3 (green) and actin (iFluor594, red). (G) Live confocal cell imaging of AGS cells expressing Wnt3-mCherry and LifeAct-GFP. Cytoneme-localised Wnt3-mCherry highlighted by yellow arrows. (H–J) IHC images of AGS cells stained with antibodies against (H) Myosin-X (MyoX) and (I) Evi/Wntless (red) and Wnt3 (red) and (J) Evi/Wntless (green Scale bars 10 µm). Phalloidin labels actin (FITC-Phalloidin, green; Phalloidin-iFluor350, blue).

Article Snippet: The following plasmids were used in transfections and/or microinjections: pCS2 + membrane- mCherry (Mattes et al., 2018), pCAG- mGFP membrane- bound GFP (Addgene 14757), pEGFP- N1 Flot2- GFP (Neumann- Giesen et al., 2004), pEGFP- N1 ∆N- Flot2- GFP (Neumann- Giesen et al., 2004), 7×TRE SuperTOPFlash- NLS- mCherry (Moro et al., 2012), JNK KTR- mCherry (Regot et al., 2014), pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7- eGFP (from Rüdiger Rudolf), pEGFP- N1 LAMP1- mTurq2 (Addgene #98828), IRSp534K- mCherry/GFP (Stanganello et al., 2015), pCS2 + Wnt8a- mCherry (Stanganello et al., 2015), pCS2 + Ror2- mCherry (cloned in with ClaI and XbaI), pEGFP- N3 mRor2-∆CRD- GFP (XhoI- XhoI mRor2 insert taken from pcDNA- mRor2, subcloned into SalI site of pEGFP- N3 vector), pCS2 + Ror2- eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3- mCherry (cloned from Addgene plasmid pcDNA3.2- Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI).

Techniques: Expressing, Staining, Immunohistochemistry, Imaging

Journal: eLife

Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

doi: 10.7554/elife.77376

Figure Lengend Snippet: Figure 2. Wnt3 cytonemes regulate paracrine Wnt/β-catenin signalling and proliferation. (A) Experimental protocol for measuring paracrine Wnt signalling activation. HFE cells expressing the SuperTOPFlash (STF) reporter, 7×TCF-NLS-mCherry, were cocultivated with AGS cells expressing indicated constructs. Fluorescence of STF mCherry reporter was measured after 48 hr and compared to untransfected control cells. (B) Representative

Article Snippet: The following plasmids were used in transfections and/or microinjections: pCS2 + membrane- mCherry (Mattes et al., 2018), pCAG- mGFP membrane- bound GFP (Addgene 14757), pEGFP- N1 Flot2- GFP (Neumann- Giesen et al., 2004), pEGFP- N1 ∆N- Flot2- GFP (Neumann- Giesen et al., 2004), 7×TRE SuperTOPFlash- NLS- mCherry (Moro et al., 2012), JNK KTR- mCherry (Regot et al., 2014), pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7- eGFP (from Rüdiger Rudolf), pEGFP- N1 LAMP1- mTurq2 (Addgene #98828), IRSp534K- mCherry/GFP (Stanganello et al., 2015), pCS2 + Wnt8a- mCherry (Stanganello et al., 2015), pCS2 + Ror2- mCherry (cloned in with ClaI and XbaI), pEGFP- N3 mRor2-∆CRD- GFP (XhoI- XhoI mRor2 insert taken from pcDNA- mRor2, subcloned into SalI site of pEGFP- N3 vector), pCS2 + Ror2- eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3- mCherry (cloned from Addgene plasmid pcDNA3.2- Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI).

Techniques: Activation Assay, Expressing, Construct, Fluorescence, Control

Journal: eLife

Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

doi: 10.7554/elife.77376

Figure Lengend Snippet: Figure 3. Flotillin-2 is over-expressed and promotes filopodia formation and elongation in gastric cancer cells. (A) Flot2 protein levels in HFE-145 and AGS cells as quantified by Western blot after normalising to beta-actin levels (n=3) and by RT-qPCR after normalising to Glyceraldehyde-3- Phosphate Dehydrogenase (GAPDH) as a housekeeping gene (n=4). Relative protein and mRNA levels are compared to HFE-145. Error bars represent SEM. Significance is calculated by Student’s t-test. (B) Representative images of HFE and AGS cells expressing membrane-mCherry and indicated Flotillin-2 (Flot2) constructs or siRNA after 48 hr. Scale bars 10 µm. (C–D) Filopodia quantifications of HFE and AGS cells transfected with indicated Flot2 plasmids or siRNA. Significance calculated by Student’s t-test with Bonferroni correction for multiple comparisons. Average cumulative filopodia length (C), average filopodia number per cell (D). (n per condition [HFE]=22, 19, 25, 23, 24). (n per condition [AGS]=25, 21, 25, 25, 25; n=number of cells measured). (E) Distribution of filopodia, categorised by length as a percentage of total filopodia per HFE or AGS cell 48 hr post-transfection with indicated Flot2 plasmids or siRNA. A Pearson’s χ2 test was performed to test for significance between control (ctrl) group (expected) and experimental groups (observed) with 5 degrees of freedom (df) and a p-value <0.05. The specific χ2 values are as follows, HFE: ctrl siRNA 0.86, Flot2 0.001, dnFlot2 <0.001, Flot2 siRNA <0.001, and for AGS: ctrl siRNA 0.65, Flot2 0.007, dnFlot2 <0.001, and Flot2 siRNA <0.001. Asterisks mark significant differences.

Article Snippet: The following plasmids were used in transfections and/or microinjections: pCS2 + membrane- mCherry (Mattes et al., 2018), pCAG- mGFP membrane- bound GFP (Addgene 14757), pEGFP- N1 Flot2- GFP (Neumann- Giesen et al., 2004), pEGFP- N1 ∆N- Flot2- GFP (Neumann- Giesen et al., 2004), 7×TRE SuperTOPFlash- NLS- mCherry (Moro et al., 2012), JNK KTR- mCherry (Regot et al., 2014), pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7- eGFP (from Rüdiger Rudolf), pEGFP- N1 LAMP1- mTurq2 (Addgene #98828), IRSp534K- mCherry/GFP (Stanganello et al., 2015), pCS2 + Wnt8a- mCherry (Stanganello et al., 2015), pCS2 + Ror2- mCherry (cloned in with ClaI and XbaI), pEGFP- N3 mRor2-∆CRD- GFP (XhoI- XhoI mRor2 insert taken from pcDNA- mRor2, subcloned into SalI site of pEGFP- N3 vector), pCS2 + Ror2- eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3- mCherry (cloned from Addgene plasmid pcDNA3.2- Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Membrane, Construct, Transfection, Control

Journal: eLife

Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer

doi: 10.7554/elife.77376

Figure Lengend Snippet: Figure 6. Flotillin-2 promotes cytoneme formation and Wnt8a signalling in zebrafish development. (A) Experimental setting to generate small clones expressing indicated constructs in the zebrafish embryo. (B, C) Confocal images of zebrafish epiblast cells injected with Flot2-GFP / memCherry and Flot2-GFP / Wnt8a-mCherry and imaged at 8 hpf. Scale bars represent 10 µm (D) Representative images of zebrafish epiblast cells injected with memCherry indicated constructs. Scale bar 10 µm. (E), (F), Quantification of filopodia from epiblast cells injected. Significance is calculated by

Article Snippet: The following plasmids were used in transfections and/or microinjections: pCS2 + membrane- mCherry (Mattes et al., 2018), pCAG- mGFP membrane- bound GFP (Addgene 14757), pEGFP- N1 Flot2- GFP (Neumann- Giesen et al., 2004), pEGFP- N1 ∆N- Flot2- GFP (Neumann- Giesen et al., 2004), 7×TRE SuperTOPFlash- NLS- mCherry (Moro et al., 2012), JNK KTR- mCherry (Regot et al., 2014), pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7- eGFP (from Rüdiger Rudolf), pEGFP- N1 LAMP1- mTurq2 (Addgene #98828), IRSp534K- mCherry/GFP (Stanganello et al., 2015), pCS2 + Wnt8a- mCherry (Stanganello et al., 2015), pCS2 + Ror2- mCherry (cloned in with ClaI and XbaI), pEGFP- N3 mRor2-∆CRD- GFP (XhoI- XhoI mRor2 insert taken from pcDNA- mRor2, subcloned into SalI site of pEGFP- N3 vector), pCS2 + Ror2- eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3- mCherry (cloned from Addgene plasmid pcDNA3.2- Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI).

Techniques: Clone Assay, Expressing, Construct, Injection

Journal: The Journal of pathology

Article Title: PAX9 regulates squamous cell differentiation and carcinogenesis in the oro-esophageal epithelium

doi: 10.1002/path.4998

Figure Lengend Snippet: Control and mutant esophagus were clustered based on differential expression of genes identified by SAM (A). IHC showed expression of SOX2 (B, C: Scale bar=50 µm), FOXA3 (D, E: Scale bar=50 µm), WNT3 (F, G: Scale bar=50 µm), SFRP5 (H, I: Scale bar=100 µm), KRT35 (J, K: Scale bar=50 µm), KRT5 (L, M: Scale bar=50 µm), P63 (N, O: Scale bar=50 µm), filaggrin (P, Q: Scale bar=50 µm), involucrin (R, S: Scale bar=50 µm) and loricrin (T, U: Scale bar=50 µm) in the mutant esophagus as compared with the wild-type esophagus. Bar represents mean ± SD.

Article Snippet: For IHC staining, deparaffinized sections were pre-treated to retrieve antigens using a Tris-based Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA, USA), prior to incubation with either a rabbit monoclonal anti-PAX9 (1:400, Cell Signaling Technology), a rat monoclonal anti-BrdU (1:1000, ab6326, Abcam), a rabbit polyclonal anti-loricrin (1:500, ab24722, Abcam), a mouse monoclonal anti-proliferating cell nuclear antigen (PCNA; 1:3000, P8825, Sigma-Aldrich), a rabbit monoclonal anti-SOX2 (1:100, ab92494, Abcam), a rabbit polyclonal anti-FOXA3 (1:100, PA1-17038, Affinity Bioreagents, Golden, CO, USA), a rabbit polyclonal anti-WNT3 (1:100, ab32249, Abcam), a rabbit polyclonal anti-SFRP5 (1:50, LS-C169017, LifeSpan Biosciences, Seattle, WA), a guinea pig polyclonal anti-KRT35 (1:200, LS- {"type":"entrez-nucleotide","attrs":{"text":"C20268","term_id":"1632539","term_text":"C20268"}} C20268 , LifeSpan Biosciences), a rabbit monoclonal anti-KRT5 (1:200, RM-2106-S0, Thermo Scientific, Rockford, IL), a rabbit polyclonal anti-P63 (1:100, GTX102425, GeneTex, Irvine, CA, USA), a rabbit polyclonal anti-involucrin (1:500, 924401, BioLegend, San Diego, CA, USA) or a rabbit polyclonal anti-filaggrin (1:100, GTX37695, GeneTex) overnight at 4 °C.

Techniques: Mutagenesis, Expressing