Journal: Cell reports

Article Title: A therapeutically targetable positive feedback loop between lnc-HLX-2-7, HLX, and MYC that promotes group 3 medulloblastoma.

doi: 10.1016/j.celrep.2024.113938

Figure Lengend Snippet: Figure 4. HLX rescues G3 MB cell phenotypic changes caused by lnc-HLX-2-7 inhibition (A) Western blot analysis for HLX expression in D425 Med cells containing CTRL-KD, lnc-HLX-2-7-KD, lnc-HLX-2-7-KD + EX-NC, and lnc-HLX-2-7-KD + EX-HLX. Quantified band densities are presented below the images as relative values (HLX/GAPDH). (B) Cell viability assays performed with D425 Med cells containing CTRL-KD, lnc-HLX-2-7-KD, lnc-HLX-2-7-KD + EX-NC, and lnc-HLX-2-7-KD + EX-HLX. Points represent the mean and standard deviation of three biological replicates. n = 4, *p < 0.01 by Kruskal-Wallis analysis. (C) Luciferase-expressing D425 Med cells containing CTRL-KD, lnc-HLX-2-7-KD, lnc-HLX-2-7-KD + EX-NC, and lnc-HLX-2-7-KD + EX-HLX were implanted into the cerebellums of NOD-SCID mice; tumor formation was assessed by bioluminescence imaging. Changes in bioluminescent signal were examined weekly after tumor implantation. (D) Quantification of total photon counts from mice implanted with D425 Med cells containing CTRL-KD, lnc-HLX-2-7-KD, lnc-HLX-2-7-KD + EX-NC, and lnc-HLX-2-7-KD + EX-HLX. n = 5; results are presented as the mean ± SEM. *p < 0.05 by one-way ANOVA with Bonferroni post hoc tests. CTRL, control; EX, expressing; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; KD, knockdown; MB, medulloblastoma; NC, negative control.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deposited data RNA-seq data NCBI GEO GSE188746 ChIRP-seq data NCBI GEO GSE232749 Experimental models: Cell lines D425 Med Dr. Charles G. Eberhart lab RRID:CVCL_1275 MED211 Dr. Charles G. Eberhart lab N/A DAOY ATCC Cat# HTB-186 CHLA01 Med ATCC Cat# CRL-3021 Experimental models: Organisms/strains NOD-SCID mice Jackson Laboratory Strain # 001303 Oligonucleotides qPCR primers (see Table S2) This study N/A DNA-RNA-FISH probe sets (see Table S3) This study N/A ASO targeting lnc-HLX-2-7 (ASO-lnc-HLX-2-7) This study 50 to 3’ +T*+G*+A*G*A*G*A*T*T*A* A*T*C*T*A*G*A*T*+T*+G*+C The PS linkages are indicated with asterisks (*), and LNA-modified oligonucleotides are indicated with plus signs (+).

Techniques: Inhibition, Western Blot, Expressing, Standard Deviation, Luciferase, Imaging, Tumor Implantation, Control, Knockdown, Negative Control

Journal: Cell reports

Article Title: A therapeutically targetable positive feedback loop between lnc-HLX-2-7, HLX, and MYC that promotes group 3 medulloblastoma.

doi: 10.1016/j.celrep.2024.113938

Figure Lengend Snippet: Figure 5. A lnc-HLX-2-7, HLX, and MYC positive feedback loop strongly promotes G3 MB (A and B) mRNA expression of HLX (A) and MYC (B) in D425 Med and MED211 cells treated with small interfering RNAs (siRNAs) targeting the genes indicated in the x axis. Relative expression level to siRNA-NC (si-NC) is indicated on the y axis. n = 3, *p < 0.01 by Kruskal-Wallis analysis. (C) Protein expression of HLX and MYC in D425 Med and MED211 cells treated with si-NC, si-HLX, or si-MYC. Quantified band densities are presented below the images as relative values (HLX/GAPDH and MYC/GAPDH). (D) Schematic showing E-box motifs around the transcription start site of lnc-HLX-2-7. Open circles indicate E-box motifs. Black arrows indicate the position of the sgRNAs designed to target the E-box. Green arrows show the location of primers used for PCR to determine E-box deletion. Agarose gel electrophoresis revealed E-box loss in D425 Med and MED211 cell lines after addition of doxycycline (Dox). (E) Expression of MYC, HLX, and lnc-HLX-2-7 in D425 Med and MED211 cells with (Dox (+)) and without Dox (Dox ()). Values are indicated relative to abundance in Dox () cells. *p < 0.01 by Kruskal-Wallis analysis. (F) Cell viability assays performed with D425 Med and MED211 cells with and without Dox. Points represent the mean and standard deviation of three biological replicates. *p < 0.01 by Kruskal-Wallis analysis.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deposited data RNA-seq data NCBI GEO GSE188746 ChIRP-seq data NCBI GEO GSE232749 Experimental models: Cell lines D425 Med Dr. Charles G. Eberhart lab RRID:CVCL_1275 MED211 Dr. Charles G. Eberhart lab N/A DAOY ATCC Cat# HTB-186 CHLA01 Med ATCC Cat# CRL-3021 Experimental models: Organisms/strains NOD-SCID mice Jackson Laboratory Strain # 001303 Oligonucleotides qPCR primers (see Table S2) This study N/A DNA-RNA-FISH probe sets (see Table S3) This study N/A ASO targeting lnc-HLX-2-7 (ASO-lnc-HLX-2-7) This study 50 to 3’ +T*+G*+A*G*A*G*A*T*T*A* A*T*C*T*A*G*A*T*+T*+G*+C The PS linkages are indicated with asterisks (*), and LNA-modified oligonucleotides are indicated with plus signs (+).

Techniques: Expressing, Agarose Gel Electrophoresis, Standard Deviation

Journal: Cell reports

Article Title: A therapeutically targetable positive feedback loop between lnc-HLX-2-7, HLX, and MYC that promotes group 3 medulloblastoma.

doi: 10.1016/j.celrep.2024.113938

Figure Lengend Snippet: Figure 6. CNP-lnc-HLX-2-7 efficiently re- presses tumor growth in an intracranial MB xenograft mouse model (A) Schematic showing the treatment paradigm in the MB xenograft mouse model. D425 Med cells were injected into the brains of NOD-SCID mice. After 14 days, mice were treated with CNP-CTRL or CNP-lnc-HLX-2-7 every 3 days for 24 days. (B) Tumor formation was assessed by biolumines- cence imaging every 3 days after treatment. (C) Quantification of total photon counts from mice treated with CNP-CTRL or CNP-lnc-HLX-2-7. n = 10; results are presented as the mean ± SEM. *p < 0.05 and **p < 0.01 by one-way ANOVA with Bonferroni post hoc test. (D and E) Quantitative PCR analysis for lnc-HLX-2-7 and HLX (D) and western blot analysis for HLX and MYC (E) expression in mouse xenografts; results are presented as mean ± SEM. n = 4, *p < 0.05 by one- way ANOVA with Bonferroni post hoc test. (F) Overall survival was determined by Kaplan-Meier analysis, and the log-rank test was applied to assess the differences between groups. *p < 0.05 by Mantel- Cox log-rank test. CNP, cerium oxide nanoparticle; CTRL, control; IVIS, in vivo spectral imaging system; MB, medulloblastoma.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deposited data RNA-seq data NCBI GEO GSE188746 ChIRP-seq data NCBI GEO GSE232749 Experimental models: Cell lines D425 Med Dr. Charles G. Eberhart lab RRID:CVCL_1275 MED211 Dr. Charles G. Eberhart lab N/A DAOY ATCC Cat# HTB-186 CHLA01 Med ATCC Cat# CRL-3021 Experimental models: Organisms/strains NOD-SCID mice Jackson Laboratory Strain # 001303 Oligonucleotides qPCR primers (see Table S2) This study N/A DNA-RNA-FISH probe sets (see Table S3) This study N/A ASO targeting lnc-HLX-2-7 (ASO-lnc-HLX-2-7) This study 50 to 3’ +T*+G*+A*G*A*G*A*T*T*A* A*T*C*T*A*G*A*T*+T*+G*+C The PS linkages are indicated with asterisks (*), and LNA-modified oligonucleotides are indicated with plus signs (+).

Techniques: Injection, Imaging, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Control, In Vivo

Journal: Cell reports

Article Title: A therapeutically targetable positive feedback loop between lnc-HLX-2-7, HLX, and MYC that promotes group 3 medulloblastoma.

doi: 10.1016/j.celrep.2024.113938

Figure Lengend Snippet: Figure 7. Combination therapy with CNP-lnc- HLX-2-7 and cisplatin inhibits tumor growth and prolongs mouse survival (A) Schematic showing the treatment paradigm in the MB xenograft mouse model. D425 Med cells were implanted into the brains of NOD-SCID mice. After 14 days, mice were treated with CNP-CTRL + DMSO, CNP-lnc-HLX-2-7 + DMSO, CNP-CTRL + cisplatin, or CNP-lnc-HLX-2-7 + cisplatin every 3 days (CNP-CTRL or CNP-lnc-HLX-2-7) or 4 days (DMSO or cisplatin) for 24 days. (B) Tumor formation was assessed by biolumines- cence imaging every 3 days after treatments. (C) Quantification of total photon counts from mice treated with CNP-CTRL + DMSO, CNP-lnc-HLX-2- 7 + DMSO, CNP-CTRL + cisplatin, or CNP-lnc-HLX- 2-7 + cisplatin. n = 10; results are presented as the mean ± SEM. *p < 0.05 and **p < 0.01 by two-way ANOVA with Bonferroni post hoc test. (D) Overall survival was determined by Kaplan-Meier analysis, and the log-rank test was applied to assess the differences between groups. *p < 0.05 and **p < 0.01 by Mantel-Cox log-rank test. CNP, cerium oxide nanoparticle; CTRL, control; DMSO, dimethyl sulfoxide; MB, medulloblastoma.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Deposited data RNA-seq data NCBI GEO GSE188746 ChIRP-seq data NCBI GEO GSE232749 Experimental models: Cell lines D425 Med Dr. Charles G. Eberhart lab RRID:CVCL_1275 MED211 Dr. Charles G. Eberhart lab N/A DAOY ATCC Cat# HTB-186 CHLA01 Med ATCC Cat# CRL-3021 Experimental models: Organisms/strains NOD-SCID mice Jackson Laboratory Strain # 001303 Oligonucleotides qPCR primers (see Table S2) This study N/A DNA-RNA-FISH probe sets (see Table S3) This study N/A ASO targeting lnc-HLX-2-7 (ASO-lnc-HLX-2-7) This study 50 to 3’ +T*+G*+A*G*A*G*A*T*T*A* A*T*C*T*A*G*A*T*+T*+G*+C The PS linkages are indicated with asterisks (*), and LNA-modified oligonucleotides are indicated with plus signs (+).

Techniques: Imaging, Control