Fig 1: ITSN1 expression is downregulated in BC cell lines. (A and B) ITSN1 protein was expressed less in BC cell lines by Western blot assay; (C) ITSN1 mRNA was expressed less in BC cell lines by RT-PCR assay; (D and E) ITSN1 was expressed less in BT-549 cells transfected with siRNA-ITSN1 and highly expressed in MCF-7 cells transfected with ITSN1-overexpressing plasmid by Western blot analysis; (F) ITSN1 was expressed less in BT-549 cells transfected with siRNA-ITSN1 and highly expressed in MCF-7 cells transfected with ITSN1-overexpressing plasmid by RT-PCR assay. GAPDH was used as a load control. Data are presented as the mean ± standard deviation. **P<0.01 versus MCF-10A group/or Ctrl group/or Vector group.
Fig 2: Human Protein Atlas, UALCAN, and GEPIA database analyses of ITSN1. (A and B) Representative immunohistochemistry images of ITSN1 in BC tissues and normal breast tissues (Human Protein Atlas). ITSN1 protein’ medium expressions were observed in normal breast tissues, whereas their low expressions were observed in BC tissues; (C) mRNA expression of ITSN1 in BC tissues and adjacent normal liver tissues (UALCAN). mRNA expressions of ITSN1 were found to be downexpressed in primary BC tissues compared to normal samples, (D) ITSN1 is associated with clinical stages (UALCAN) in BC; (E) ITSN1 is not associated with poor survival rate in BC tissues (UALCAN); (F) mRNA expression of ITSN1 in BC tissues and adjacent normal liver tissues (GEPIA). mRNA expressions of ITSN1 were found to be downxpressed in primary BC tissues compared to normal samples; (G) ITSN1 is not associated with clinical stages in BC tissues (GEPIA); (H) ITSN1 is not associated with poor survival rate in BC tissues (GEPIA). **P<0.01 and *P<0.05.
Fig 3: Gene products of MAGI2, TNS2, DLC1, CDK20, and CAV1 physically and functionally interact to regulate RhoA/Rac1/Cdc42 activation. a Identification of six novel monogenic causes of NS reveals a regulatory network of RhoA activation. The large rounded square symbolizes a podocyte. All six proteins MAGI2, TNS2, DLC1, CDK20, ITSN1, and ITSN2, in which recessive defects were detected herein as novel causes of pTSNS, interact physically or functionally to regulate RhoA/Rac1/Cdc42. Yellow labels highlight proteins encoded by genes, which if mutated give rise to monogenic nephrosis as shown in this study (MAGI2, TNS2, DLC1, CDK20, ITSN1, and ITSN2) or as published (EMP2, ARHGDIA). Blue frame around yellow labels indicate that there is also a monogenic mouse model of NS or a zebrafish model known, as shown in this study for ITSN2 or as published for MAGI2, TNS2, EMP2, and RhoGDI-a. For each of the proteins, MAGI2, TNS2, DLC1, CDK20, TLN1, or ITSN1, the protein domains are shown. Red circles denote positions of mutations that we found in patients. Truncation mutations are represented by “x”. By identifying novel monogenic causes of NS we discovered a cluster proteins that regulate Rho/Rac/Cdc42 activation as being central for the pathogenesis of these patients. b MAGI2 interacts with TNS2 upon co-overexpression and coimmunoprecipitation (coIP) in HEK293T cells. Both mutant MAGI2 clones, Gly39* and Tyr746* (underlined) that reflect alleles of NS patients A5146-21 and B91 respectively, abrogate this interaction. c MAGI2 interacts with DLC1 upon co-overexpression and coIP in HEK293T cells. One mutant MAGI2 clone Gly39* (underlined) reflecting a mutation of NS patient A5146-21 abrogates this interaction. d DLC1 interacts with CDK20 upon co-overexpression in HEK293T cells. e DLC1 interacts with CAV1. Two mutant DLC1 c-DNA clones, reflecting Trp10* and Lys1358Thr alleles of NS patients A548-21 and A4967-21 respectively, lack this interaction. f In IMCD3 cells, migration rate is induced in the presence of serum as compared to scrambled control. Knockdown of Dlc1 in IMCD3 cells using mouse Dlc1 siRNA #1 impairs cell migration rate (red vs. black curve with serum). The decrease in migration is rescued by transfection with full-length human DLC1 cDNA (green curve). Transfection with four out ot six mutants (Trp10*, Ala456Val, Ala1352Val, and Lys1358Thr) failed to rescue this migratory phenotype
Fig 4: Interactions with the PSD, but not with the membrane or actin cytoskeleton are required for positioning of the EZ.(A) Example images of dendrites expressing Homer1c-ALFA and endocytic proteins fused to GFP co-expressed with control or mirShank-mCherry construct. Scale bar: 2 µm. (B) Fraction of PSDs associated with an EZ after Shank-KD relative to control plotted as mean ± SEM. GFP-CLCa (N = 8, p < 0.001), ß2-adaptin (N = 10, p < 0.01), Eps15 (N = 12, p < 0.05), Itsn1L (N = 10, p < 0.05), HIP1R (N = 11 p > 0.05), Dyn2 (N = 12, p > 0.05), CPG2 (N = 13, p > 0.05). (C) Illustration of possible mechanisms that could maintain the EZ adjacent to the PSD. (D) Example images of dendrites co-expressing Homer1c-ALFA and GFP-CLCa in dendrites treated with LatB, CK666, or Jasp. Scale bar: 2 µm. (E) Example images of dendrites expressing control constructs, CLCb-EED/QQN (left panel) and AP2m2-P1 (middle panel), or Itsn1 KD construct (mirItsn1; right panel). Scale bar: 2 µm. (F) Fraction of EZ-associated PSDs relative to control, plotted as mean ± SEM. Jasp (N = 4), LatB (N = 8), CK666 (N = 12), CLCb-EED/QQN (N = 7), AP2m2-P1 (N = 5), mirItsn1 (N = 11), shCPG2 (N = 8). Figure 7—source data 1.Excel sheet with numerical data represented as plots Figure 7D and F.
Fig 5: ITSN1 regulated proliferation and apoptosis of BC cells by affecting the expression of Ki67 and caspase-3 protein. (A and B) CCK-8 assay was used to detect the viability of BT-549 cells transfected with siRNA-ITSN1 or MCF-7 cells transfected with ITSN1-overexpressing plasmid. (C and D) Colony formation was used to detect the proliferation of BT-549 cells transfected with siRNA-ITSN1 or MCF-7 cells transfected with ITSN1-overexpressing plasmid. (E and F) Flow cytometry assay was used to detect the apoptosis level of BT-549 cells transfected with siRNA-ITSN1 or MCF-7 cells transfected with ITSN1-overexpressing plasmid. (G–I) Western blot assay was used to detect the protein expression level of Ki67 and cleaved caspase-3 in BT-549 cells transfected with siRNA-ITSN1 or MCF-7 cells transfected with ITSN1-overexpressing plasmid. GAPDH was used as a load control. Data are presented as the mean ± standard deviation. **P<0.01 and *P<0.05 versus Ctrl group/or Vector group.
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