Journal: iScience

Article Title: Distinct but interchangeable subpopulations of colorectal cancer cells with different growth fates and drug sensitivity

doi: 10.1016/j.isci.2023.105962

Figure Lengend Snippet:

Article Snippet: For MSI1 overexpression, human MSI1 cDNA was amplified with the primers of MSI1_BamHI-F and MSI1_stopdead_XbaI-R ( ) using the PrimeScriptTM first strand cDNA Synthesis (Takara, 6210A) and PrimeSTAR Ⓡ Max DNA Polymerase kits according to manufacturer instructions (Takara, R045A).

Techniques: Recombinant, Concentration Assay, Membrane, RNAscope, Cell Viability Assay, SYBR Green Assay, Reverse Transcription, Expressing, Microarray, Derivative Assay, Software

Journal: Disease Models & Mechanisms

Article Title: Zebrafish models of skeletal dysplasia induced by cholesterol biosynthesis deficiency

doi: 10.1242/dmm.042549

Figure Lengend Snippet: Late-onset skeletal defects observed in the koliber nu7 ( kol nu7 ) mutant are the result of downregulation of msmo1 expression. (A) Compared to wild-type (wt) siblings (top), adult kol nu7 mutants display a reduced body length and small head size. wt n =300, kol nu7 n =300. (B) Whole-mount skeletal preparations reveal gross malformations and hyperossification throughout the adult kol nu7 craniofacial and axial skeleton after Alcian Blue (cartilage) and Alizarin Red (ossified bone and mineralized tissues) staining. wt n =100, kol nu7 n =100. (C-F) Early larval mutants do not display patterning defects or premature ossification. Whole-mount Alizarin Red staining of 4.7 mm (∼8 dpf) wt (C,E) and kol nu7 . (D,F). Ventral view (C,D) and lateral view (E,F). wt n =3, kol nu7 n =4. (G) Positional cloning reveals that the kol nu7 locus is located to the ∼457 kb critical region flanked by polymorphic markers with one or two recombinants out of 1844 meioses, corresponding to a genetic distance of 0.16 cM. (H) Screen of gene expression using quantitative RT-PCR from RNA extracted from hypural complex of ∼18 mm SL kol nu7 and wt siblings. wt n =2, kol nu7 =2. Only msmo1 level was significantly different out of 11 tested genes, located in the ∼1.2 Mb region encompassing the kol nu7 locus. Initial screen results: grhprb not detected (ND); uba6 mean difference 0.64, s.d. 0.36; abpp2 −1.62, s.d. 1.00; mettl14 1.16, s.d. 0.68; prss12 1.20, s.d. 0.72; ndst3 1.51, s.d. 1.50; ugt8 −1.03, s.d. 0.07; spock3 ND; tll1 0.67, s.d. 0.27; cpe 0.90, s.d. 0.42; msmo1 −10.07, s.d. 4.32. Confirmation test of msmo1 expression ( msmo1 −11.47, s.d. 8.49; P =0.0012), wt n =3, kol nu7 =5. Three technical replicates were included for all assays. Gene expression was normalized to the reference gene eefla1 . Fold change was calculated using Livak method . P -value calculated using unpaired Student's t -test on dCt values. (I) The msmo1 nu81 mutant allele is not able to complement the kol nu7 mutation. Adult kol nu7/+ :msmol nu81/+ transheterozygotes phenocopy the kol nu7 mutant. wt n =100, kol nu7/+ :msmol nu81/+ n =100. (J) Whole-mount skeletal preparations reveal gross malformations throughout the kol nu7/+ :msmol nu81/+ craniofacial and axial skeleton, similar to those observed in kol nu7 . wt n =10, kol nu7/+ :msmo1 nu81/+ n =10. (K) The msmo1 nu81 allele is the result of a 37 bp insertion, allowing for allele-specific expression analysis between msmo1 nu81 and kol nu7 . PAM, protospacer adjacent motif (underlined in red). (L) Strong downregulation of the kol nu7 -linked allele (asterisks) in kol nu7/+ :msmo1 nu81/+ compared to the wt allele in msmo1 nu81/+ suggests that the kol nu7 mutation is cis -acting. kol nu7/+ :msmo1 nu81/+ n =3, msmo1 nu81/+ n =3. The top band is a heterodimer of wt and mutant strands.

Article Snippet: The following Made to Order Taqman probes (Thermo Fisher Scientific) were used: mettl14 (Assay ID: Dr03438758_g1), tll1 (Assay ID: Dr03138096_m1), cpe (Assay ID: Dr03188311_s1), grhprb (Assay ID: Dr03088390_g1), ugt8 (Assay ID: Dr03427025) msmo1 (Assay ID: Dr03133463_m1) and eef1a1l1 (Assay ID: Dr03432748_m1).

Techniques: Mutagenesis, Expressing, Staining, Cloning, Gene Expression, Quantitative RT-PCR

Journal: Disease Models & Mechanisms

Article Title: Zebrafish models of skeletal dysplasia induced by cholesterol biosynthesis deficiency

doi: 10.1242/dmm.042549

Figure Lengend Snippet: The suppression of early lethality of the msmo1 nu81 mutation yields adult fish with kol nu7 -like phenotype. (A) Survival of larvae from msmo1 nu81/+ in-crosses, from 7 dpf to 70 dpf. Most msmo1 nu81 mutants die by 9 dpf. [7 dpf wild type (wt; black) n =19, heterozygotes (het; pale gray) n =49, knockout mutants (KO; dark gray) n =16, P =0.02794; 8 dpf wt n =21, het n =39, KO n =17, P =0.8065; 9 dpf wt n =12, het n =40, KO n =3, *** P =0.0008; 10 dpf wt n =37, het n =44, KO n =3, **** P <0.0001; 15 dpf wt n =26, het n =51, KO n =3, **** P <0.0001; 70 dpf wt n =18, het n =35, KO n =0, *** P =0.001.] (B) Overexpression of msmo1 driven by daily heat shock of the transgenic line Tg(hsp70l:msmo1:IRESnlsGFP) nu99 rescued the lethality of msmo1 nu8 mutants. Transgenic screening based on cardiac GFP. (Control non-transgenic siblings 14 dpf wt n =14, het n =32, KO n =2, *** P =0.0035; transgenic siblings 14 dpf wt n =18, het n =44, KO n =28, P =0.3214.) cont, control; hs, heat shock. (C) Dietary cholesterol supplementation does not improve the survivability of msmo1 nu81 mutants. Clutches from msmo1 nu81/+ in-crosses were fed either a high-cholesterol diet (hcd) or a control standardized diet (cont) beginning at 5 dpf until collection at 10 dpf. (HCD wt n =26, het n =50, KO n =9, P =0.0089; control diet wt n =22, het n =66, KO n =5, P <0.0001.) All two-tailed P -values were calculated using chi-squared test. (D-F′) Whole-mount in situ hybridization during the first 5 days of development shows msmo1 expression predominately in the yolk syncytial layer (YSL) and liver. Expression is first detected during early somitogenesis in the YSL (D) and continues there at 3 dpf (E). At 4 dpf, strong expression is observed in the differentiated liver (F,F′). (G-I) Generation of msmo1 nu81 /wild-type chimeras using endoderm replacement rescues early lethality of msmo1 nu81 mutants and reveals a strong kol nu7 -like phenotype. (G) Schematic of the procedure. Wild-type Tg(ubi:Zebrabow-M) a131 donor embryos were injected with sox32 RNA to force an endodermal fate. At high stage (∼3 hpf), cells were transplanted from donor to host embryos collected from msmo1 nu81/+ in-crosses. (H) Surviving msmo1 nu81 mutants display a strong kol nu7 -like phenotype. msmo1 nu81 n =4. (I) The majority of organs of endodermal origin displayed a high enrichment in transplanted Tg(ubi:Zebrabow-M) a131 cells (red). msmo1 nu81 n =4. (J-L) Liver-specific msmo1 expression in msmo1 nu81 mutants rescues early lethality and produces juvenile msmo1 nu81 mutants with strong kol nu7 -like phenotype. (J) Liver-specific regulatory element fabp10a was used to drive msmo1 expression in msmo1 nu81 mutants. (K) Adult Tg(fabp10a:msmo1:pA) nu100 ; msmo1 nu81 mutants phenocopy kol nu7 mutant. Tg(fabp10:msmo1:pA) nu100 n =50, Tg(fabp10a:msmo1:pA) nu100 ; msmo1 nu81 n =50. (L) Whole-mount skeletal preparations reveal gross malformations throughout Tg(fabp10a:msmo1:pA) nu100 ; msmo1 nu81 craniofacial and axial skeleton, similar to those observed in kol nu7 . Tg(fabp10:msmo1:pA) nu100 n =3, Tg(fabp10a:msmo1:pA) nu100 ; msmo1 nu81 n =7.

Article Snippet: The following Made to Order Taqman probes (Thermo Fisher Scientific) were used: mettl14 (Assay ID: Dr03438758_g1), tll1 (Assay ID: Dr03138096_m1), cpe (Assay ID: Dr03188311_s1), grhprb (Assay ID: Dr03088390_g1), ugt8 (Assay ID: Dr03427025) msmo1 (Assay ID: Dr03133463_m1) and eef1a1l1 (Assay ID: Dr03432748_m1).

Techniques: Mutagenesis, Knock-Out, Over Expression, Transgenic Assay, Control, Two Tailed Test, In Situ Hybridization, Expressing, Injection

Journal: Disease Models & Mechanisms

Article Title: Zebrafish models of skeletal dysplasia induced by cholesterol biosynthesis deficiency

doi: 10.1242/dmm.042549

Figure Lengend Snippet: Loss of Lss activity phenotype is epistatic to msmo1 nu81 mutation. (A) The lss nu60 allele. PAM, protospacer adjacent motif (underlined in red). (B) Survival analysis of clutches of lss nu60/+ in-crosses, from 9 dpf to 23 dpf, reveals that most lss nu60 mutants die by 12 dpf. [9 dpf wild type (wt; black) n =21, heterozygotes (het; pale gray) n =30, knockout mutants (KO; dark gray) n =15, P =0.4412; 10 dpf wt n =15, het n =27, KO n =16, P =0.8563; 11 dpf wt n =20, het n =44, KO n =14, P =0.3320; 12 dpf wt n =24, het n =48, KO n =10, * P =0.0277; 13 dpf wt n =33, het n =51, KO n =10, ** P =0.0026; 23 dpf wt n =8, het n =27, KO n =0, *** P =0.0009.] (C) Survival analysis of clutches of msmo1 nu81 /+ ;lss nu60/+ in-crosses reveals that loss of Lss activity increases survivability of msmo1 nu81 mutants at 10 dpf. [wt observed (O) n =11, expected (E) n =11; msmo1 nu81 O n =4, E n =11; msmo1 nu81 ;lss nu60/+ O n =9, E n =22; msmo1 nu81 ;lss nu60 O n =10, E n =11; lss nu60 O n =12, E n =11; msmo1 nu81/+ ;lss nu60 O n =30, E n =22; msmo1 nu81/+ ;lss nu60/+ O n =40, E n =44; msmo1 nu81/+ O n =18, E n =22; lss nu60 /+ O n =18, E n =22; * P =0.0431.] Two-tailed P -values were calculated using chi-squared test. (D,E) Suppression of early lethality of lss nu60 mutants by liver-specific expression of lss . (D) Adult Tg(fabp10:lss:pA) nu101 ;lss nu60 mutants phenocopy kol nu7 . Tg(fabp10:lss:pA) nu101 n =12, Tg(fabp10:lss:pA) nu101 ;lss nu60 n =12. (E) Whole-mount skeletal preparations reveal gross malformations throughout Tg(fabp10:lss:pA) nu101 ;lss nu60 craniofacial and axial skeleton, similar to those observed in kol nu7 . Tg(fabp10:lss:pA) nu101 n =3, Tg(fabp10:lss:pA) nu101 ;lss nu60 n =5.

Article Snippet: The following Made to Order Taqman probes (Thermo Fisher Scientific) were used: mettl14 (Assay ID: Dr03438758_g1), tll1 (Assay ID: Dr03138096_m1), cpe (Assay ID: Dr03188311_s1), grhprb (Assay ID: Dr03088390_g1), ugt8 (Assay ID: Dr03427025) msmo1 (Assay ID: Dr03133463_m1) and eef1a1l1 (Assay ID: Dr03432748_m1).

Techniques: Activity Assay, Mutagenesis, Knock-Out, Two Tailed Test, Expressing

Journal: Disease Models & Mechanisms

Article Title: Zebrafish models of skeletal dysplasia induced by cholesterol biosynthesis deficiency

doi: 10.1242/dmm.042549

Figure Lengend Snippet: Loss of Msmo1 and Lss activity is associated with abnormal endochondral bone formation. (A-T) The hyomandibular (A,E,I,M,Q), ceratohyal (B,F,J,N,R) and hypural complex (C,D,G,H,K,L,O,P,S,T) were compared between wild type (A-D), kol nu7 (E-H), kol nu7/+ :msmol nu81/+ (I-L), Tg(fabp10a:msmo1:pA) nu100 ; msmo1 nu81 (M-P) and Tg(fabp10:lss:pA) nu101 ;lss nu60 (Q-T). (E-T) The endochondral bones of the mutants are compressed and irregularly shaped with ectopic ossification and fusions throughout the growth plates, marked with arrows. Images represent disarticulated whole-mount adult skeletal preparations at ∼4 months of age. Scale bars: 100μm. Tg *, Tg(fabp10:msmo1:pA) nu100 ; Tg **, Tg(fabp10:lss:pA) nu101 . wt n =4, kol nu7 n =4, kol nu7/+ :msmol nu81/+ n =4, Tg(fabp10a:msmo1:pA) nu100 ; msmo1 nu81 n =4, Tg(fabp10:lss:pA) nu101 ;lss nu60 n =4.

Article Snippet: The following Made to Order Taqman probes (Thermo Fisher Scientific) were used: mettl14 (Assay ID: Dr03438758_g1), tll1 (Assay ID: Dr03138096_m1), cpe (Assay ID: Dr03188311_s1), grhprb (Assay ID: Dr03088390_g1), ugt8 (Assay ID: Dr03427025) msmo1 (Assay ID: Dr03133463_m1) and eef1a1l1 (Assay ID: Dr03432748_m1).

Techniques: Activity Assay

Journal: Disease Models & Mechanisms

Article Title: Zebrafish models of skeletal dysplasia induced by cholesterol biosynthesis deficiency

doi: 10.1242/dmm.042549

Figure Lengend Snippet: Loss of Msmo1 and Lss activities disrupts growth plate patterning. (A-P) Expression domains of col2a1a (A,D,G,J), col10a1a (B,E,H,K,M-P) and msmo1 (C,F,I,L) were analyzed within growth plates of wild type (A-C), kol nu7 (D-F), Tg(fabp10a:msmo1:pA) nu100 ; msmo1 nu81 (G-I) and Tg(fabp10:lss:pA) nu101 ;lss nu60 (J-L) using RNAscope in situ hybridization. (A) Strong col2a1a expression is observed in round and stacked chondrocytes in wild type, corresponding to presumed resting and proliferating chondrocytes. (B) Expression of col10a1a in wild type is observed complementary to col2a1a expression in presumed hypertrophic chondrocytes. The expression of col2a1a is expanded in kol nu7 (D) and Tg(fabp10a:msmo1:pA) nu100 ; msmo1 nu81 (G) growth plates, with a near-complete loss of col10a1a expression in kol nu7 (E) and Tg(fabp10a:msmo1:pA) nu100 ; msmo1 nu81 (H). Expression of col2a1a (J) and col10a1a (K) in Tg(fabp10:lss:pA) nu101 ;lss nu60 appears less affected when compared to kol nu 7 (D,E) and Tg(fabp10a:msmo1:pA) nu100 ; msmo1 nu81 (G,H). Expression of msmo1 is detected primarily in pre-hypertrophic chondrocytes of wild type (C); msmo1 expression is undetectable in kol nu7 growth plates (F), but is upregulated in transgenically rescued msmo1 nu81 (I) and lss nu60 (L) mutants. (M-P) Expression of col10a1a in osteoblasts is unchanged between wild type and mutants. Images represent paraffin sections of the pterotic at 2 months of age (∼15 mm SL). Boxed areas in A-L are shown at higher magnification in A′-L′. Scale bars: 100 μm. Tg *, Tg(fabp10:msmo1:pA) nu100 ; Tg **, Tg(fabp10:lss:pA) nu101 . wt n =6, kol nu7 n =6, Tg(fabp10a:msmo1:pA) nu100 ; msmo1 nu81 n =3, Tg(fabp10:lss:pA) nu101 ;lss nu60 n =3.

Article Snippet: The following Made to Order Taqman probes (Thermo Fisher Scientific) were used: mettl14 (Assay ID: Dr03438758_g1), tll1 (Assay ID: Dr03138096_m1), cpe (Assay ID: Dr03188311_s1), grhprb (Assay ID: Dr03088390_g1), ugt8 (Assay ID: Dr03427025) msmo1 (Assay ID: Dr03133463_m1) and eef1a1l1 (Assay ID: Dr03432748_m1).

Techniques: Expressing, RNAscope, In Situ Hybridization

Journal: Disease Models & Mechanisms

Article Title: Zebrafish models of skeletal dysplasia induced by cholesterol biosynthesis deficiency

doi: 10.1242/dmm.042549

Figure Lengend Snippet: Ihh signaling within endochondral growth plates is present despite loss of Msmo1 and Lss activity. (A-L) Expression domains of ihha (A,D,G,J), ihhb (B,E,H,K) and ptch1 (C,F,I,L) were analyzed within growth plates of wild type (A-C), kol nu7 (D-F), Tg(fabp10a:msmo1:pA) nu100 ; msmo1 nu81 (G-I) and Tg(fabp10:lss:pA) nu101 ;lss nu60 (J-L) using RNAscope in situ hybridization. (A-C) In wild type, expression of ihha and ihhb is observed in the pre-hypertrophic chondrocyte zone, with ptch1 expression seen to either side of these domains. (D-L) Expression of ihha , ihhb and ptch1 is present within mutant growth plates and similar to domains observed in wild type. Images represent paraffin sections of the pterotic at 2 months of age (∼15 mm SL). Scale bars: 100 μm. Tg *, Tg(fabp10:msmo1:pA) nu100 ; Tg **, Tg(fabp10:lss:pA) nu101 . wt n =3, kol nu7 n =3, kol nu7/+ :msmol nu81/+ n =3, Tg(fabp10a:msmo1:pA) nu100 ; msmo1 nu81 n =3, Tg(fabp10:lss:pA) nu101 ;lss nu60 n =3.

Article Snippet: The following Made to Order Taqman probes (Thermo Fisher Scientific) were used: mettl14 (Assay ID: Dr03438758_g1), tll1 (Assay ID: Dr03138096_m1), cpe (Assay ID: Dr03188311_s1), grhprb (Assay ID: Dr03088390_g1), ugt8 (Assay ID: Dr03427025) msmo1 (Assay ID: Dr03133463_m1) and eef1a1l1 (Assay ID: Dr03432748_m1).

Techniques: Activity Assay, Expressing, RNAscope, In Situ Hybridization, Mutagenesis

Journal: Disease Models & Mechanisms

Article Title: Zebrafish models of skeletal dysplasia induced by cholesterol biosynthesis deficiency

doi: 10.1242/dmm.042549

Figure Lengend Snippet: New zebrafish models for CDP. (A-D) Abnormal ossification, marked with arrows, within endochondral growth plates of kol nu7 (B), kol nu7/+ :msmol nu81/+ (C) and Tg(fabp10a:msmo1:pA) nu100 ; msmo1 nu81 (D) mutant phenotypes resembles defects observed in patients with chondrodysplasia punctata. Images represent lateral views of palatoquadrate growth plates of craniofacial bones from disarticulated whole-mount adult skeletal preparations at ∼4 months of age. Scale bars: 100 μm. Tg *, Tg(fabp10:msmo1:pA) nu100 . wt n =4, kol nu7 n =6, kol nu7/+ :msmol nu81/+ n =4, Tg(fabp10a:msmo1:pA) nu100 ; msmo1 nu81 n =4.

Article Snippet: The following Made to Order Taqman probes (Thermo Fisher Scientific) were used: mettl14 (Assay ID: Dr03438758_g1), tll1 (Assay ID: Dr03138096_m1), cpe (Assay ID: Dr03188311_s1), grhprb (Assay ID: Dr03088390_g1), ugt8 (Assay ID: Dr03427025) msmo1 (Assay ID: Dr03133463_m1) and eef1a1l1 (Assay ID: Dr03432748_m1).

Techniques: Mutagenesis

Journal: iScience

Article Title: Gene-environment interactions mediate stress susceptibility and resilience through the CaMKIIβ/TARPγ-8/AMPAR pathway

doi: 10.1016/j.isci.2021.102504

Figure Lengend Snippet: Disruption of GluA1 expression in PSD fraction in susceptible mice is mediated by CaMKIIβ dysfunction (A–D) Immunoblot estimation of GluA1 level in the whole cell extract (A), synaptosomal fraction (B), postsynaptic membrane (PSD) fraction (C), and non-PSD fraction (D) of the vCA1 in BALB mice exposed to chronic ultra-mild stress (CUMS) or non-stress (NS), treated with imipramine (IMI) or water. PSD95 and synaptophysin (Syn) were used as PSD and non-PSD markers, respectively. n = 9–10 mice per group. (E) Immunoblot estimation of GluA1 levels in the PSD fraction of the vCA1 in B6 mice exposed to CUMS. n = 7–8 mice per group. (F–H) Immunoblot estimation of GluA1 levels in the synaptosomal fraction (F), PSD fraction (G), and non-PSD fraction (H) of the vCA1 in BALB and B6 mice exposed to subchronic and mild social defeat stress (smSDS) or NS. n = 8–10 mice per group. (I) Experimental timeline. BALB mice were injected with AAV-CaMKIIβ-CA or AAV-GFP into the vCA1, subjected to smSDS, and euthanized to measure GluA1 levels. (J and K) Immunoblot estimation of GluA1 levels in the PSD fraction (J) and synaptosomal fraction (K). n = 8–10 mice per group. L. Experimental paradigm. B6 mice were injected with AAV-CaMKIIβ-DN or AAV-GFP into the vCA1, subjected to smSDS, and euthanized to measure GluA1 levels. (M and N) Immunoblot estimation of GluA1 levels in the PSD (M) and synaptosomal (N) fractions. n = 10 mice per group. One-way ANOVA followed by a Tukey's post hoc test (in A-D), two-way ANOVA followed by a Tukey's post hoc test (in F-H), and two-tailed Student's t-test (in E, J, K, M, and N) were used for statistical analyses. ∗p < 0.05. Bar graphs show mean ± SEM. See also Figure S4 . Complete statistical summaries are provided in .

Article Snippet: Anti-GluA1 antibodies (mixture of rabbit polyclonal anti-GluA1, Millipore, MAB2263 and rabbit polyclonal anti-GluA1, Cell Signaling Technology, #13185) were bound and crosslinked to Protein G Dynabeads (Thermo Fisher Scientific) according to the manufacturer's instructions.

Techniques: Disruption, Expressing, Western Blot, Membrane, Injection, Two Tailed Test

Journal: iScience

Article Title: Gene-environment interactions mediate stress susceptibility and resilience through the CaMKIIβ/TARPγ-8/AMPAR pathway

doi: 10.1016/j.isci.2021.102504

Figure Lengend Snippet: GluA1 function is crucial for behavioral responses to chronic stress (A) Experimental paradigm. BALB mice were subjected to 5-day subchronic and mild social defeat stress (smSDS). An AMPA receptor potentiator was infused into their vCA1 1 hr before smSDS exposure (day 1 to day 5, once per day). SIT, social interaction test; SPT, sucrose preference test. (B and C) Treatment with AMPAR potentiator rescues smSDS-elicited reduction of the time in the interaction zone in the SIT (B) and prevents smSDS-induced anhedonia in the SPT (C). n = 13–14 mice per group. (D) AAV vectors engineered to overexpress a GluA1 CT (AAV-GluA1 CT -T2A-mCherry) or a control construct (AAV-mCherry). P Camk2a , Camk2a promoter. (E) Experimental paradigm. B6 mice were injected with AAV-GluA1 CT -T2A-mCherry or AAV-mCherry and subjected to 5-day exposure to smSDS. (F) AAV microinjection into the vCA1. Region-specific expression of mCherry in the vCA1 is shown. Scale bar, 100 μm. (G) Overexpression of GluA1 CT decreases Fos levels in the vCA1 of B6 mice following smSDS exposure. n = 6 mice per group. (H) Representative examples of behavior traces of stressed mice given either AAV-mCherry or AAV-GluA1 CT -T2A-mCherry during the SIT (target session). (I and J) GluA1 CT overexpression reduces time in the interaction zone in the SIT (I) and induces anhedonia in the SPT (J) after smSDS. n = 14–15 mice per group. two-way ANOVA followed by a Tukey's post hoc test (in B, C, I, and J) and two-tailed Student's t-test (in G) were used for statistical analyses. ∗p < 0.05. Bar graphs show mean ± SEM. See also Figure S5 . Complete statistical summaries are provided in .

Article Snippet: Anti-GluA1 antibodies (mixture of rabbit polyclonal anti-GluA1, Millipore, MAB2263 and rabbit polyclonal anti-GluA1, Cell Signaling Technology, #13185) were bound and crosslinked to Protein G Dynabeads (Thermo Fisher Scientific) according to the manufacturer's instructions.

Techniques: Control, Construct, Injection, Microinjection, Expressing, Over Expression, Two Tailed Test

Journal: iScience

Article Title: Gene-environment interactions mediate stress susceptibility and resilience through the CaMKIIβ/TARPγ-8/AMPAR pathway

doi: 10.1016/j.isci.2021.102504

Figure Lengend Snippet: TARPγ-8-mediated GluA1 expression in the PSD fraction is associated with behavioral resilience to chronic stress (A) Expression of the Cacng8 gene (encoding TARPγ-8) by RNA FISH (RNAscope) in a coronal section of the mouse brain. vCA1, ventral part of CA1 in the hippocampus. Scale bar, 50 μm. (B and C) Immunoblot estimation of phosphorylated TARPγ-8 (pTARPγ-8) and total TARPγ-8 levels in the vCA1 of BALB and B6 mice exposed to subchronic and mild social defeat stress (smSDS) or no stress (NS). n = 8–10 mice per group. (D) Linear correlation between pTARPγ-8 levels and postsynaptic membrane (PSD) GluA1 levels in the vCA1 of BALB and B6 mice. (E) Linear correlation between pTARPγ-8 levels in the vCA1 and social interaction in BALB and B6 mice. (F) Schematic representation of the AAV vectors engineered to overexpress a constitutively active form of TARPγ-8 (AAV-TARPγ-8 D/D -T2A-mCherry) or a control construct (AAV-mCherry). (G) Experimental paradigm for behavioral testing. BALB mice were injected with AAV-TARPγ-8 D/D -T2A-mCherry or AAV-mCherry and subjected to 5-day smSDS exposure. SIT, social interaction test; SPT, sucrose preference test. (H) Coronal view of mCherry signals in the vCA1 of BALB mice injected with AAV-TARPγ-8 D/D -T2A-mCherry. Scale bar, 100 μm. (I and J) No change in synaptosomal GluA1 levels was observed between mice injected with AAV-TARPγ-8 D/D -T2A-mCherry and AAV-mCherry (I). BALB mice overexpressing TARPγ-8 D/D show increased PSD GluA1 levels in the PSD fraction under stress condition (J). n = 8 mice per group. (K and L) TARPγ-8 D/D overexpression leads to a significant increase of time in the interaction zone in the SIT (K) and sucrose preference in the SPT (L). n = 14–16 mice per group. (M) Overexpression of TARPγ-8 D/D increases Fos levels in the vCA1 of BALB mice following smSDS exposure. n = 6 mice per group. (N) Experimental paradigm for stress exposure and behavioral testing. (O) SIT occupancy heat maps during target session obtained by averaging the location of testing animals in each group. (P) GluA1 CT overexpression prevents the increase in social interaction time induced by the activation of TARPγ-8 under the smSDS condition. n = 12 mice per group. (Q) Experimental paradigm for stress exposure, drug treatment, and behavioral testing. (R) B6 mice given vehicle control showed normal social interaction following smSDS exposure, but intra-vCA1 JNJ55511118 injection decreased time in the interaction zone. One-way ANOVA followed by a Tukey's post hoc test (in P), two-way ANOVA followed by a Tukey's post hoc test (in C and R), two-tailed Student's t-test (in I-M), and Pearson's correlation (in D and E) were used for statistical analyses. ∗p < 0.05. Bar graphs show mean ± SEM. See also Figure S6 . Complete statistical summaries are provided in .

Article Snippet: Anti-GluA1 antibodies (mixture of rabbit polyclonal anti-GluA1, Millipore, MAB2263 and rabbit polyclonal anti-GluA1, Cell Signaling Technology, #13185) were bound and crosslinked to Protein G Dynabeads (Thermo Fisher Scientific) according to the manufacturer's instructions.

Techniques: Expressing, RNAscope, Western Blot, Membrane, Control, Construct, Injection, Over Expression, Activation Assay, Two Tailed Test

Journal: iScience

Article Title: Gene-environment interactions mediate stress susceptibility and resilience through the CaMKIIβ/TARPγ-8/AMPAR pathway

doi: 10.1016/j.isci.2021.102504

Figure Lengend Snippet: TARPγ-8 function is required for CaMKIIβ-mediated enhancement of GluA1 expression in the PSD and behavioral response to chronic stress (A) Experimental paradigm for AAV injection, stress exposure, and behavioral testing. BALB mice were injected with AAV- CaMKIIβ-CA and subjected to 5-day exposure to subchronic and mild social defeat stress (smSDS). (B) Immunoblot estimation of phosphorylated TARPγ-8 (pTARPγ-8) and total TARPγ-8 levels in the vCA1 of BALB mice injected with AAV-CaMKIIβ-CA or AAV-GFP. n = 8 mice per group. (C) Experimental paradigm for AAV injection, stress exposure, and behavioral testing. B6 mice were injected with AAV-CaMKIIβ-DN and subjected to 5-day smSDS exposure. (D) Immunoblot estimation of pTARPγ-8 and total TARPγ-8 levels in the vCA1 of B6 mice injected with AAV-CaMKIIβ-DN or AAV-GFP. n = 8 mice per group. (E) Experimental paradigm for AAV injection, stress exposure, drug treatment, and behavioral testing. (F–H) Immunoblot estimation of the PSD (G) and synaptosomal (H) GluA1 levels in the vCA1 of BALB mice exposed to smSDS. JNJ55511118 or vehicle was infused into the vCA1 region during the 5-day smSDS session. n = 8 mice per group. (I) Increased time in the interaction zone after CaMKIIβ-CA overexpression is disrupted by treatment with JNJ55511118. n = 14–15 mice per group. (J) Experimental paradigm for AAV injection, stress exposure, drug treatment, and behavioral testing. (K–M) Immunoblot estimation of the PSD (L) and synaptosomal (M) GluA1 levels in the vCA1 of smSDS-exposed B6 mice. Mice were injected with AAV-CaMKIIβ-DN or AAV-GFP and AAV-TARPγ-8 D/D -T2A-mCherry or AAV-mCherry. n = 10 mice per group. (N) Decreased time in the interaction zone after CaMKIIβ-DN overexpression is prevented by TARPγ-8 activation. n = 14–15 mice per group. (O) Proposed model of stress-induced depression based on vCA1 CaMKIIβ: In stress-resilient animals, the Camk2b transcript is normal but CaMKIIβ activity is enhanced following stress episodes. Increased CaMKIIβ activity stimulates the phosphorylation of TARPγ-8, which promotes GluA1 expression in synaptic sites, leading to stress adaptation. In stress-susceptible animals, the Camk2b transcript and CaMKIIβ function are downregulated, leading to reduced TARPγ-8 activity and GluA1 insertion. As a result, vCA1 dysfunction may cause depression-like behavioral alterations such as social impairment and anhedonia. One-way ANOVA followed by a Tukey's post hoc test (in I and N) and two-tailed Student's t-test (in B, D, G, H, L, and M) were used for statistical analyses. ∗p < 0.05. Bar graphs show mean ± SEM. Complete statistical summaries are provided in .

Article Snippet: Anti-GluA1 antibodies (mixture of rabbit polyclonal anti-GluA1, Millipore, MAB2263 and rabbit polyclonal anti-GluA1, Cell Signaling Technology, #13185) were bound and crosslinked to Protein G Dynabeads (Thermo Fisher Scientific) according to the manufacturer's instructions.

Techniques: Expressing, Injection, Western Blot, Over Expression, Activation Assay, Activity Assay, Phospho-proteomics, Two Tailed Test

Journal: iScience

Article Title: Gene-environment interactions mediate stress susceptibility and resilience through the CaMKIIβ/TARPγ-8/AMPAR pathway

doi: 10.1016/j.isci.2021.102504

Figure Lengend Snippet:

Article Snippet: Anti-GluA1 antibodies (mixture of rabbit polyclonal anti-GluA1, Millipore, MAB2263 and rabbit polyclonal anti-GluA1, Cell Signaling Technology, #13185) were bound and crosslinked to Protein G Dynabeads (Thermo Fisher Scientific) according to the manufacturer's instructions.

Techniques: Produced, Virus, Plasmid Preparation, Recombinant, SYBR Green Assay, RNAscope, Multiplex Assay, Mutagenesis, Staining, Western Blot, Real-time Polymerase Chain Reaction, Software

Journal: Cell host & microbe

Article Title: Commensal Microbes and Hair Follicle Morphogenesis Coordinately Drive Treg Migration into Neonatal Skin

doi: 10.1016/j.chom.2017.03.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Alternatively for confirmatory qRT-PCR, 1 μg of RNA was converted to cDNA using iScript cDNA synthesis kit (Bio-Rad) and mixed with appropriate primers and SYBR® Green Supermix.

Techniques: Virus, Plasmid Preparation, Recombinant, cDNA Synthesis, SYBR Green Assay, Cell Isolation, Selection, RNAscope, Staining, RNA Sequencing, Software